Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The article by M. Grzonka and H. Bazzi entitled: Mouse Sas-6 is required for centriole formation in embryos and integrity in embryonic stem cells, describes new findings in novel mouse models of Sas-6 knockouts (KO). This is an interesting study that reports two different mouse Sas6 KO models and the depletion of Sas6 from mouse embryonic stem cells (mESCs). This type of analysis has never been done before and so it reveals and describes a role for Sas-6 in centriole biogenesis in mouse.

The authors compare their analysis with Sas-4 KO and overall found similar results when compared to previous work from H. Bazzi, when Sas-4 was depleted in mouse embryos. Due to the mitotic stopwatch pathway, Sas6KO embryos die during development at extremely early stages and this can be rescued by depletion in p53 and other members of the pathway. Perhaps, not so surprisingly, these embryos do not contain centrioles, showing that in vivo, Sas6 is absolutely required for centriole duplication. More surprisingly, however, in cultures of mESCs, established and propagated in vitro, Sas-6 crispr induced KO, does not result in lack of centrioles. Instead, abnormal structures that show aberrant morphologies, length, and incapacity to assemble cilia were detected. In principle, this means that centrioles can be assembled independently of Sas-6, even if not in the correct manner.

The authors interpret these differences as possible differences in the pathways involved in centriole assembly and propose different requirements in different cell types, within the same species. I have problems with this interpretation. To me is very difficult to understand, how the "protein" absolutely required for cartwheel assembly at the early stages of centriole biogenesis, can be essential and dispensable at the same time. Although, I may be wrong, I think the authors have not envisage other possibilities to interpret their data, which should be taken into consideration.

1) I do not know anything about ESC and ESC cultures. So maybe this is a stupid suggestion. But can't they be derived exactly from the same genetic background of SAS-6KO embryos? Because the way the two (or even 3 as there are 2 mouse KO lines) are generated is different. Why is that?

2) Still on mESCs, are the authors sure that there are no WT Sas-6mRNAs still present in their ESC cells? Because tiny amounts are maybe sufficient to allow the initial cartwheel structure. In FigS2B, I can see a really faint band, very faint but it is there.

3) This last point goes also with the western-blot of Figure S2C- there is still a band, very tiny between the two very tick bands (marked with *). Maybe separating proteins better will help visualizing the real Sas-6 band? Have they used the Sas6 ab in other WBs from the KO embryos, for example? Can they use the Sas6 ab in immunostaining to show if the assembled abnormal centrioles completely lack Sas6. This will allow to distinguish between the hypothesis of having some (even if not much) sas6 left?

4) Then a more theoretical point? Have the authors considered that the difference is more in the stability of the abnormal structures. Let's say, without a cartwheel and maybe enough PLK4 activity and high level of other centriolar components, the centrioles are abnormally assembled- they have no cartwheel, but they are disassembled very fast in the embryo but not in ESCs?

5) Even if there is a real difference and without Sas-6 ESCs can make centrioles that are abnormal in structure and function (at least at the cilia assemble level), the choice of words "strictly required", I am not sure it is correct. Because, since Sas-6 is described by many studies as the factor required for cartwheel assembly, which occurs very early in the pathway, this means that in mESCs centrioles can assembled without forming a cartwheel. And so that the cartwheel is actually not required for the initial building block, but more as a structure that maintains the whole centriole in an intact manner?

6) The authors mentioned that in flies, abnormal Sas-6 structures have been described in certain cell types. Are these mutants, null mutants? In other words, do these structures assembled in a context of no Sas6 or abnormal Sas-6 protein or even low levels of Sas-6?

Other points:

• I think the 1st sentence of the abstract appears disconnected from the rest. The same goes for the 1st sentence of the introduction. And also, what is the evidence that pluripotent stem cells rely primarily on the proper assembly of a mitotic spindle? They relly on many other things, not sure this is the first one.
• The authors mention that centrioles are lost in Sas6-/- after "differentiation" of mESCs. The term differentiation is not appropriate, and confusing here. Differentiation normally refer to cells that stopped proliferating and exited the cell cycle, which is not the case here, as NPCs are progenitor cells that keep cycling.
• Figure S1: Percent of cells with centrosomes was assessed by a co-staining of tubulin and Cep164, which mark the mother centrioles. As Cep164 may be absent from centrosomes after lack of centriole maturation in sas6-/- embryos, another combination of staining should be performed to evaluate the percent of cells without centrosomes. tubulin staining can be seen in Sas6 em5/em5 embryos, while the quantification claims total absence of centrosomes. The authors use the CENT2-eGFP transgenic line to count the number of centrioles in Figure 3, they should do the same in Figure S1.

Significance

This study shows with a novel mouse model the requirement of centrioles during mouse development. It will be relevnat to centrosome labs, the novel mouse lines will be useful to many labs working on centrioles, cilia and centrosomes. My expertise: centrosome biology

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

We thank the Reviewers for their useful feedback on our manuscript. We have addressed the Reviewers’ comments and revised our manuscript accordingly. A point-by-point response is provided below.

Reviewer comments:

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Gopalan et al use quantitative, comprehensive lipid mass spectrometry of mouse brain tissue isolated at various time points in embryonic and postnatal development. They then go on to use the same quantitative analysis of mouse and human stem cells differentiated in vitro into neurons to define the lipid composition of these cultures.

Major Comments:

1. As mentioned above, it is difficult to assess whether the discrepancy in the lipotype acquisition between in vivo mouse brain development and stem cell differentiation is due to metabolic differences in the in vitro differentiation as the authors state or is due to a lack of the stem cells to actually acquire a neuronal phenotype. Perhaps showing more clearly that the protocols for neuronal differentiation work efficiently and/or how they compare to brains dissected would be helpful in stating that the lipotype is different. The protocol referenced here (Bogetofte et al) only gives ~30% TH+ positive DA neurons in their manuscript. What cell type the other 70% of the cells are is something that could be discussed as means of "diluting" out the lipotype seen in these cultures. Perhaps the 30% TH+ DA neurons do attain the "correct" lipotype, but the lipidomic analysis can not detect this due to the contaminating effects of the non-differentiated cells. In this work, it would be nice to see what percentage of the cells differentiate into the expected cell type rather than referencing previous manuscripts. As differentiation protocols and originating cell sources are highly variable and error-prone, it's difficult to know what the lipotype results are actually reporting on. Furthermore, discussion about these differentiation techniques and how well they represent functional neurons is warranted. The papers referenced here don't show 100% differentiation into the phenotypes that are described in this work such that the lipotype finding is not the only suggestion of a "general failure of in vitro neuronal differentiation models". Maybe a discussion of how the lack of ability to attain the neuronal lipotype due to the metabolic deficiencies discussed here could be causative to the inability to full recapitulate the neuronal phenotype is useful for the reader.

We thank the reviewer for this question and suggested experiments. Following the advice, we have now show immunofluorescence data of pan-neuronal markers (i.e., b-tub III or MAP2) in mESC and iPSCs. In agreement with previously published datasets from the Noh and Meyer labs (Gehre et al., 2020; Bogetofte et al.,2019), we show that the protocols we use generate a very high percentage of neurons. We have now included these images and quantifications in our manuscript as Figs. 2B and S6A,B.

From the discussion and work here is unclear why the stearate feeding of the stem cells did not result in an increase in the 18:0-containing sphingolipids. The authors state that the appropriate metabolic pathways are not fully established and go on to look at the CerS expression levels across the differentiation timeline. It appears that the results presented in Fig. S7 counter the authors' interpretation of the lipotype and more discussion here would be nice to clarify this discrepancy.

We thank the reviewer for highlighting this seemingly counterintuitive observation. We have now included a quantification of CerS mRNA from commercially available mouse tissues analysed in Sladitschek and Neveu, 2019 and compared this to the data from Gehre et al, 2020. In the mouse brain tissue, CerS1 expression is upregulated dramatically, while CerS5 and 6 are downregulated (see new panel A in Fig. S8). In contrast, during in vitro differentiation of mESCs, CerS5 is not downregulated and CerS6 is upregulated (Fig. S8B). Accordingly, we have expanded our discussion in the revised manuscript as follows:

“On the other hand, supplementing the cells with stearic acid (18:0) does not result in high levels of 18:0-sphingolipids. It is known that the Cer synthase CerS1 is specific for stearoyl-CoA (Venkataraman et al., 2002), which results in the production of 18:0-sphingolipids, while the synthases CerS5 and CerS6 are responsible for 16:0-sphingolipid production. During brain development, one observes a 35-fold increase in the expression of CerS1 and a downregulation of CerS5 and CerS6 compared to embryonic tissue (Sladitschek & Neveu, 2019) (Fig. S8A). In contrast, during in vitro neuronal differentiation, between day 8 and 12, CerS1 expression increases only by 5-fold and, contrary to expectation, CerS6 expression is upregulated and CerS5 expression is unchanged (Gehre et al., 2020) (Fig. S8B). This could underpin the observation that 16:0-sphingolipids remain elevated whereas brain-specific 18:0-sphingolipids only increase marginally, despite supplementation with stearic acid. Overall, this suggests that appropriate programming of the sphingolipid metabolic machinery is not fully established in stem cell-derived neurons.”

Minor comments:

1. I find the data presentation of the LENA analysis to be difficult to follow (Fig. 1E). In my opinion, the p-value is not the most important bit of information in this graph, though having it on the y-axis with other pertinent information encoded by colors or arrows being disguised. I would rather see the data on the x-axis that is above a certain p-value (denoted in the figure legend) plotted with the direction and magnitude of change shown.

We thank the reviewer for this suggestion. In the revised manuscript, we now plot log2(odds ratio) on the y-axis instead of the p-value. Moreover, we have dimensioned the size and color intensity of each point as function of the p-value (Fig. 1E and 2E, shown below).

In the PCA in Fig 1, what are the loadings that define the variable PC1 and PC2? What is predominantly changing the P21 samples that lead to such a large shift if most of the data shown in the subsequent panels are not changing much between P2 and P21.

In the revised manuscript, we now include a plot of the PCA loadings of the lipids majorly influencing principal components 1 and 2 as supplemental Fig. S3.

Reviewer #1 (Significance (Required)):

This work provides a nice reference for the complex lipidomes in embryonic and postnatal murine brain development. The details of the lipotype changes during development are well laid out and will of no doubt be of great use across a variety of scientific fields. While I found the in vivo data to be compelling, interesting, and useful, the lack of controls for the in vitro stem cell differentiation work makes this particular data set and comparison less useful. Further work to identify the limitations of the stem cell differentiation protocols as a valid comparison to in vivo brain development need to be done and/or the discussion of the direct comparisons between the two toned down.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The study used a quantitative lipidomics approach which I am very familiar with. The results should be highly reproducible.

Reviewer #2 (Significance (Required)):

The manuscript submitted by Gopalan et al. reported a quantitative and comparative lipidomics study between mouse brain samples from early embryonic to postnatal stages, and rodent and human stem cell-derived neurons. The authors found a couple of very unique characters only existing in brain samples, but not in stem cell-derived neurons, including 22:6-containing glycerophospholipids and 18:0-containing sphingolipids. The authors further found the brain-like lipotypes can only be partially established in stem cell-derived neurons after supplementing brain lipid precursors. These findings clearly suggest that stem cell-derived neurons might not be appropriately used to mechanistically study lipid biochemistry, membrane biology, and biophysics in brains. The study was well designed. and the manuscript was very informative and resourceful. I would suggest to accept the manuscript for publication.

We thank the Reviewer for the positive assessment of our work.

References

Gehre, M., Bunina, D., Sidoli, S., Lübke, M. J., Diaz, N., Trovato, M., Garcia, B. A., Zaugg, J. B., & Noh, K. M. (2020). Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy. Nature Genetics, 52(3), 273–282. https://doi.org/10.1038/s41588-020-0586-5

Levy, M., & Futerman, A. H. (2010). Mammalian ceramide synthases. IUBMB Life, 62(5), 347–356. https://doi.org/10.1002/iub.319

Sladitschek, H. L., & Neveu, P. A. (2019). A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit. Molecular Systems Biology, 15(12), 1–13. https://doi.org/10.15252/msb.20199043

Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Cameron Sullards, M., Merrill, A. H., & Futerman, A. H. (2002). Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells. Journal of Biological Chemistry, 277(38), 35642–35649. https://doi.org/10.1074/jbc.M205211200

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The study used a quantitative lipidomics approach which I am very familiar with. The results should be highly reproducible.

Significance

The manuscript submitted by Gopalan et al. reported a quantitative and comparative lipidomics study between mouse brain samples from early embryonic to postnatal stages, and rodent and human stem cell-derived neurons. The authors found a couple of very unique characters only existing in brain samples, but not in stem cell-derived neurons, including 22:6-containing glycerophospholipids and 18:0-containing sphingolipids. The authors further found the brain-like lipotypes can only be partially established in stem cell-derived neurons after supplementing brain lipid precursors. These findings clearly suggest that stem cell-derived neurons might not be appropriately used to mechanistically study lipid biochemistry, membrane biology, and biophysics in brains. The study was well designed. and the manuscript was very informative and resourceful. I would suggest to accept the manuscript for publication.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Gopalan et al use quantitative, comprehensive lipid mass spectrometry of mouse brain tissue isolated at various time points in embryonic and postnatal development. They then go on to use the same quantitative analysis of mouse and human stem cells differentiated in vitro into neurons to define the lipid composition of these cultures.

Major Comments:

1. As mentioned above, it is difficult to assess whether the discrepancy in the lipotype acquisition between in vivo mouse brain development and stem cell differentiation is due to metabolic differences in the in vitro differentiation as the authors state or is due to a lack of the stem cells to actually acquire a neuronal phenotype. Perhaps showing more clearly that the protocols for neuronal differentiation work efficiently and/or how they compare to brains dissected would be helpful in stating that the lipotype is different. The protocol referenced here (Bogetofte et al) only gives ~30% TH+ positive DA neurons in their manuscript. What cell type the other 70% of the cells are is something that could be discussed as means of "diluting" out the lipotype seen in these cultures. Perhaps the 30% TH+ DA neurons do attain the "correct" lipotype, but the lipidomic analysis can not detect this due to the contaminating effects of the non-differentiated cells. In this work, it would be nice to see what percentage of the cells differentiate into the expected cell type rather than referencing previous manuscripts. As differentiation protocols and originating cell sources are highly variable and error-prone, it's difficult to know what the lipotype results are actually reporting on.

Furthermore, discussion about these differentiation techniques and how well they represent functional neurons is warranted. The papers referenced here don't show 100% differentiation into the phenotypes that are described in this work such that the lipotype finding is not the only suggestion of a "general failure of in vitro neuronal differentiation models". Maybe a discussion of how the lack of ability to attain the neuronal lipotype due to the metabolic deficiencies discussed here could be causative to the inability to full recapitulate the neuronal phenotype is useful for the reader. 2. From the discussion and work here is unclear why the stearate feeding of the stem cells did not result in an increase in the 18:0-containing sphingolipids. The authors state that the appropriate metabolic pathways are not fully established and go on to look at the CerS expression levels across the differentiation timeline. It appears that the results presented in Fig S7 counter the authors' interpretation of the lipotype and more discussion here would be nice to clarify this discrepancy.

Minor comments:

1. I find the data presentation of the LENA analysis to be difficult to follow (Fig 1E). In my opinion, the p-value is not the most important bit of information in this graph, though having it on the y-axis with other pertinent information encoded by colors or arrows being disguised. I would rather see the data on the x-axis that is above a certain p-value (denoted in the figure legend) plotted with the direction and magnitude of change shown.
2. In the PCA in Fig 1, what are the loadings that define the variable PC1 and PC2? What is predominantly changing the P21 samples that lead to such a large shift if most of the data shown in the subsequent panels are not changing much between P2 and P21.

Significance

This work provides a nice reference for the complex lipidomes in embryonic and postnatal murine brain development. The details of the lipotype changes during development are well laid out and will of no doubt be of great use across a variety of scientific fields. While I found the in vivo data to be compelling, interesting, and useful, the lack of controls for the in vitro stem cell differentiation work makes this particular data set and comparison less useful. Further work to identify the limitations of the stem cell differentiation protocols as a valid comparison to in vivo brain development need to be done and/or the discussion of the direct comparisons between the two toned down.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

We thank the reviewers for their constructive comments, which greatly helped us in steering the manuscript in the right direction. Below is our point to point response to their concerns:

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The Abeliovich lab has discovered differential degradation rates for mitochondrial proteins by mitophagy, a selective form of autophagy of mitochondria. In previous publications, they identified two protein kinases Pkp1/2 and the protein phosphatase Aup1(Pct6), which regulate phosphorylation patterns of mitochondrial proteins and affect their degradation rates by mitophagy. In the current manuscript, the authors now analyze the role of the pyruvate dehydrogenase complex (PDC) in mitochondria in the regulation of these regulatory link between Pkp1/2 and Aup1 and their substrate proteins. They find that the deletion of pda1, a core subunit of PDC, inhibits the degradation of previously identified mitophagy substrates Mdh1, Aco2, Qcr2, Aco1, and Idp1, while the turnover of mtDHFR-GFP, a model substrate diffusely localized in the mitochondrial matrix, is not affected. These data indicate that tested mitochondrial proteins are excluded from mitochondrial turnover by mitophagy in the absence of Pda1 by as yet unknown sorting mechanisms. The authors next show that the mutation of a known Pkp1/2 and Aup1 phosphorylation site within Pda1 (S313A) drastically reduces mitophagic turnover of Mdh1, which is completely blocked by the additional deletion of AUP1. A mutation blocking Pda1 activity (R322C) also almost completely blocked Mdh1 degradation. However, PDC activity does not generally affect mitophagy, since an inactivating mutation in Lat1 (K75R) promotes mitophagy of Mdh1. Published work has shown that Pkp1/2 and Aup1, which affect the phosphorylation and degradation of Mdh1, physically interact with the PDC. Thus, the authors tested whether the absence of Pda1 affected the phosphorylation state of Mdh1 and thus its turnover. Indeed, Mdh1 showed globally reduced phosphorylation in the absence of Pda1, which was rescued by the overexpression of Pkp1/2 promoting degradation of Mdh1. Consistent with the effects on mitophagic turnover, Mdh1 was phosphorylated at higher level in the presence of Lat1-K75R. The authors analyzed the mitochondrial distribution of Mdh1 in dependence of Pda1. They provide cytological data suggesting that Mdh1 might segregate from a diffuse matrix localized mtRFP reporter, which is suppressed upon Pkp1/2 overexpression. Finally, the authors perform a mitochondrial phospho-proteome analysis and found that the absence of Pda1 affected phosphorylation sites in 8 mitochondrial matrix proteins. From these data the authors propose a model in which the PDC complex controls the activity of the associated protein kinase Pkp1/2 and the phosphates Aup1 by allosteric changes, which in turn regulates the phosphorylation and differential turnover of mitochondrial proteins by mitophagy. They speculate that the PDC and associated factors could resemble large protein kinase complexes as TORC.

(1) The authors postulate a structural role for the PDC in regulating Pkp1/2 and Aup1 for controllingmitophagy turnover of certain substrates. While the physical association of Pkp1/2 and Aup1 with PDC has been shown previously, the authors need to critically test their model and assess (a) whether this physicalinteraction occurs under their experimental conditions and (b) whether the different mutants that affectmitophagy of Mdh1 also affect the physical interaction of Pkp1/2 and Aup1 with PDC. Otherwise, their model, although consistent, remains purely speculative.

Response: We fully agree with the reviewer that this is an important point and we are currently trying to verify these interactions under our working conditions. Naturally, we will then test whether the documented Pdh1-Aup1 and the Pdh1-Pkp1/2 interactions are affected by mutants such as the lat1K75R mutation and the lat1Δ mutation, as insightfully suggested by the reviewer.

(2) It is important to test every mutant that affects Mdh1 turnover for effects on mitophagy in general using the mtDHFR-GFP reporter to be able to conclude specific effects on Mdh1 mitophagy. For example, the deletion of Lat1 has been shown to induce mitophagy of a mitochondrial matrix reporter during nitrogen starvation (Bockler and Westermann, 2013). While the authors observe a complete block of Mdh1 turnover in lat1 deletion cells, they do observe increased Mdh1 degradation in Lat1-K75R cells. In line with this reasoning, the authors have identified a number of Mdh1 variants in a previous publication (Kolitsida et al. 2019) that can be used to further explore the observed phenomenon. For example, the Mdh1- T199A could be used to test whether the expression of Pkp1/2 in pda1 deficient cells has specific or general effects on mitophagy. Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?*

Response: We completely agree with the reviewer, and we have tested the effect of the lat1Δ mutations on mtDHFR-GFP (Supplemental Figure 2). It is important to note, however, that while the Bockler and Westermann paper does report an increased red/green fluorescence ratio in lat1Δ cells using mtRosella as a mitophagy reporter, this is part of a general screen, and was never further investigated or validated with additional, more rigorous methods (that paper focused on the role of the HERMES complex in mitophagy). In vivo fluorescence can be affected by a multitude of physical intracellular and intra-organellar factors including local pH, ionic strength, and molecular crowding. Therefore, the result from the screen conducted by Bockler and Westermann - although intriguing- is somewhat preliminary and requires more scrutiny (e.g. validation with a GFP release assay, complementation, etc) before we can make conclusions regarding the effect of the lat1Δ mutation on mitophagy, under their experimental conditions.

Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?*

This is now addressed in Figure 3E and 3F. The results are consistent with our hypothesis, namely showing complete or near complete suppression of the phenotype when tested using the phosphomimetic reporter.

(3) The cytological data are not convincing. The imaging quality is low and it is very difficult for the reader to appreciate the suggested segregation of Mdh1-GFP and mtRFP. Thus, it is important to provide cortical sections in order to visualize mitochondrial tubules/networks and Mdh1 distribution. This is particularly important, because the authors mention effects of Pda1 on mitochondrial morphology, which appears to be rescued by Pkp1/2 overexpression.

We are sorry to hear that the reviewer is not convinced by the microscopy data. We agree with the comment that it is not trivial to observe the segregation by eye. We do not intend to say that the segregation is absolute, and the differences we observe in the distributions of the two signals are relative. By that, we mean that a prominent green dot in the cell, which is brighter than other green dots in the same cell, has a red channel counterpart which is not brighter than the other red dots in the cell, or may even be dimmer than the other red dots in the same cell. However, we do provide illustrative examples of segregation with specific arrows pointing to loci of segregation between the two channels. This was not pointed out in the original figure legend and this oversight has now been corrected. More importantly, we performed a mathematical analysis of the overlap between the two signals, and demonstrate a statistically (very) significant difference between the endogenous mitochondrial protein and the artificial mitochondrially-targeted GFP chimera. We would also like to stress that under our conditions (gluconeogenic medium, stationary phase) yeast mitochondria (at least in our genetic background) are not normally found in tubules (this pattern is specifically observed in cells growing in glucose-based medium). Nonetheless, we will attempt to carry out a 3-d reconstruction using our available z-sections, as per the reviewer’s request.

(3 continued) In this context it would be very informative to follow the localization of PDC in mitochondria. Previous work has shown that Pda1 forms punctate structures in mitochondria in proximity to ER-mitochondria contact sites marked by ERMES (Cohen et al. 2014). Is Pda1 itself degraded by mitophagy or is it excluded? Could the physical interaction of Mdh1 with Pda1 explain mitophagy phenotypes if Pda1 is excluded from mitophagy? In other words, could the PDC form a physical unit to segregate and prevent proteins from mitophagy turnover?

Response: It was shown by others that Pda1-GFP localizes to mitochondrial puncta, and we also observe this in our system. We have also briefly looked at the mitophagic efficiency of Pda1-GFP. While it is inefficiently delivered to the vacuole (5% free GFP after 5 days, vs 20% for Mdh1), it is not excluded from mitophagic targeting. We are not sure what the reviewer is referring to as a “physical interaction between Pdh1 and Mdh1”. We do not demonstrate such an interaction, and to our knowledge such an interaction has not been reported and validated in any publication. Even if such an interaction existed, it cannot explain the rescue of the pda1Δ phenotype by the co-overexpression of Pkp1 and 2, nor the effect of the pda1Δ mutation on Mdh1 phosphorylation, or the effect of the pda1Δ mutation on other mitophagy reporters which we used, such as Aco1, Qcr2, Idp1, and Aco2. We agree with the reviewer that there is a possibility that an organizing center, be it the PDC or another complex, may regulate intra-matrix segregation and mitophagic trafficking. However, addressing this question would require significant additional time and resources, and we would beg the referee to defer this question to future investigations, as it is not central to the claims made in the current manuscript.

(4) The conclusions that can be drawn from the analysis of the mitochondrial phospho-proteome independence of Pda1 are rather limited. First, the experimental setup does not distinguish between directeffects of PDC on Pkp1/2 or Aup1 activity and the effects of simply PDC dysfunction on mitochondrial proteins. Along those lines, it is unclear whether the few identified proteins with altered phosphorylation states are indeed targets of Pkp1/2 or Aup1. Thus, to be able to support their conclusion, the authors need to include a number of additional controls/strains.

Response: We agree with the reviewer that the phosphoproteomic analysis is not comprehensive. However, it was the best that we were able to do at the time, and it is unlikely that we could distinguish direct from indirect effects using this approach. The purpose of the experiment was to test whether we could identify more global effects of Pda1 on mitochondrial protein phosphorylation. This was in no way intended to imply a direct effect. Rather, it provides an unbiased map for potentially identifying the signaling network(s) involved. This may also include downstream events outside the mitochondrial matrix and even in the cytoplasm. We previously showed a connection of the Aup1-dependent signaling pathway to the RTG retrograde signaling pathway (Journo et al, 2009), and such a global analysis may allow a future understanding of how intra-matrix events can signal to the cytoplasm. However, we do not make any specific claims regarding which effects are direct and which are not. To address the reviewer’s concern, we have now modified the text to clarify these points (Page 9 line 28-Page 10 line 1).In an effort to improve the evidence for PDC- dependent mitochondrial matrix phosphorylation, we will carry out a phosphoproteomic analysis comparing WT cells to cells expressing the lat1K75R mutation. Since this mutation is expected to increase kinase activity, we expect to obtain a clearer picture that will complement the results obtained with the pda1Δ deletion mutant.

Reviewer #1 (Significance (Required)):

The authors continue to explore the mechanisms underlying their initial observation of differential turnover rates for mitochondrial proteins by mitophagy. This current work specifically builds on the previous publication identifying Pkp1/2 and Aup1 as regulators of the phosphorylation state of specific substrate proteins (Kolitsida et al. 2019). Here the authors now explore how these regulators might be organized and controlled by PDC. Thus, the study adds another layer of regulation. However, the molecular mechanisms of how PDC might regulate Pkp1/2 and Aup1 is not directly addressed. In conclusion, the current study opens up new lines of research that need to be explored to critically test the proposed model. A key question to me is of course how mitochondrial proteins can be excluded from mitophagy and whether the proteins in this study may play a direct role in these mechanisms.

To further address the mechanism by which the PDC affects mitophagy via Aup1, Pkp1, and Pkp2, we will carry out the following experiments: 1) We will verify the Pda1-Pkp1/2 interaction and test for effects of PDC mutants on this interaction, with an emphasis on the lat1K75R mutation 3) We will analyze the effect of the lat1K75R mutation on mitochondrial phosphoproteomics 4) we will analyze the effect of the pda1Δ mutation on the phosphorylation state of additional proteins which exhibit defective mitophagy in the pda1Δ background (Figure 1B). Our basic hypothesis regarding the reviewers’ question, namely the molecular mechanism behind the observed regulation is based on mitochondrial heterogeneity. We suggest that mitochondrial heterogeneity underlies mitophagic selectivity and that factors which affect heterogeneity also affect mitophagic selectivity.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The manuscript by Kolitsida et al. unravels an interesting link between mitochondrial pyruvatedehydrogenase complex (PDC) and protein sorting for mitophagy-mediated clearance. Using yeast genetics combined with immunoblotting-based mitophagy protein sorting reporter assay, authors show that targeting components of PDC and associated regulatory factors (kinases and phosphatases) interferes with phosphorylation status of at least some mitochondrial matrix proteins and prevents their degradation by autophagic machinery. Experimental design is sound, and statistic evaluation seems appropriate. Although I find this work important and overall valuable, some of the conclusions made by the authors are still preliminary and could require additional experimental testing.

We thank the reviewer for these supportive comments

*In this regard, some of my major suggestions include: *

1. Authors propose that the interaction of dedicated kinases and phosphatases with pyruvate dehydrogenase complex changes their affinity/activity toward the non-PDC substrates (possibly by allosteric regulation). Yet, alternatively, it can also be considered that the PDC complex acts as a docking station that stabilizes the associated regulatory factors inside the mitochondria and allows their additional functions. Indeed, simultaneous overexpression of Pkp1/2 increases the free GFP signal and Mdh1 phosphorylation even in the absence of core E1 PDC component (pda1, figure 3 C, D and figure 4 A, B), suggesting that the interaction of phosphatases and kinases with pda1 is not essential to mediate the phosphorylation of other mitoproteins. Are the protein levels of regulatory PDC factors (Pkp1, Pkp2, Aup1) somewhat altered by depletion of their target E1a subunit (pda1)? Could depletion of pda1 trigger destabilization of these factors and decrease their half-life? This can be tested by western-blotting and combined with cycloheximide chase assay.

We agree with the reviewer that, theoretically, the PDC could be necessary for the stability of Pkp1, Pkp2 and Aup1. However, have now tested this, and found that deletion of PDA1 has no effect on the expression levels of Aup1, Pkp1 and Pkp2 under our conditions (see Supplementary Figure 2).

2. Allosteric regulation of specificity of PDC kinases and phosphatases is an intriguing yet still slightly unbacked hypothesis. To provide further experimental evidence for this explanation, authors could develop the classical in vitro kinase assay for Pkp1/2 on their model substrate (Mdh1) in the absence or presence of the Pda1 subunit. Even more excitingly, authors could isolate the Pkp1/2 from wild-type and Pda1- compromised cells and see whether they are able to phosphorylate their model substrate in vitro. Additionally, does specific inhibition of pyruvate dehydrogenase kinases (e.g., with dichloroacetate) affect the Mdh1 phosphorylation levels?

We present the hypothesis that the PDC allosterically regulates Aup1 and Pkp1/2 , as an explanation for the data. We agree with the reviewer that further experimental work will be necessary to verify this hypothesis. While the Pkp1/2 kinase assays could be useful in this regard, several issues temper our enthusiasm for this experiment:

i) This assay is far from “classical” with respect to this study. Neither of the two yeast mitochondrial kinases (Pkp1 and Pkp2) was previously characterized using in vitro kinase assays. While the mammalian assay conditions may work here, this is not guaranteed.

ii) A negative result will have no value

iii) A positive result (e.g. phosphorylation of recombinant Mdh1 by recombinant Pkp1/2) could arise due to low stringency conditions in the assay

iv) The nature of the kinase activity is unclear. We do not know if it is Pkp1, Pkp2 or a heterodimer of Pkp1 and Pkp2, or whether currently unknown ancillary factors are necessary for activity. This greatly complicates the analysis.

In lieu of the direct in vitro kinase assay, we have carried out an experiment demonstrating that an Mdh1-GFP variant with a phosphomimetic threonine to aspartate mutation at position 199, which we previously demonstrated to suppress both the aup1Δ and Pkp2Δ phenotypes (Kolitsida et al, 2019), can also suppress the pda1Δ phenotype (New Figure 3E, F). We suggest that this result provides sufficient evidence of a phosphorylation cascade linking the PDC, Pkp1/2 and Mdh1 mitophagic trafficking.

In addition, we will also attempt to further support the allosteric regulation hypothesis, by testing whether mutations in LAT1 such as lat1K75R modulate the known Aup1-Pda1 and Pkp1/2-Pda1 interactions (also see response to referee #1).

We greatly appreciate the suggestion to test the effect of DCA on our assays, and this experiment will be carried out in the near future. However DCA has not been shown to affect Pda1 phosphorylation in yeast or to modulate kinase activity of Pkp1/2, and it is not clear whether this approach will work. Many reagents and inhibitors which act in mammalian cells, are unable to cross the yeast cell wall.

3. Are the Pkp1/Pkp2 changing their interactome upon PDC alternation (e.g., Pda1 depletion)? Do their interactome alters upon stimulation of mitophagy? Co-immunoprecipitation or bioID assay could shed light on how the partitioning of the Pkp1/Pkp2/Aup1 functions between the metabolic regulation and protein quality control is maintained.

We agree with the referee that this is an excellent question. Our current hypothesis posits that the established physical interaction between Pkp1/2 and Pda1 is crucial for activity of the kinases towards "third party" clients. To test this we will assay whether the catalytically inactive lat1K75R mutation affects the Pkp1/2 - Pda1 interaction. If we cannot find evidence for such a direct mechanism using these straightforward hypothesis-driven experiments, then we will also analyze effects the pda1Δ mutation on the general interactomes of Pkp1 and Pkp2.

Minor points:

1. The paper would benefit from a brief explanation of the principles of the mitophagy reporter assay used in this study.

Thank you for the suggestion. We have now expanded our explanation of the GFP release assay (see Methods section, Page 13 lines 8-16).

2. MS phosphoproteomics is indeed a great approach to tackle many questions in this study. Surprisingly,the authors did not discuss these data extensively. Although changes in the phosphorylation status of some proposed targets are apparent (as QCR2), many others were not detected (including Aco1/2, Idp1, and model substrate - Mdh1; Figure 1).

In our experiment, the coverage of the mitochondrial phosphoproteome was not complete. Some proteins, such as Mdh1, were observed only in a subset of the replicates, and therefore are not included in the figure. In the revision, we plan to add an analysis of the mitochondrial phosphoproteome in the lat1K75R mutation, and hopefully this will provide further insights.

Furthermore, many significant changes occur in the proteins that do not belong to the mitochondrial matrix compartment (as TOMM20 or YAT1), questioning the direct involvement of Pkp1/Pkp2/Aup1. How do authors interpret these data?

We previously showed that the Aup1 signaling pathway converges with the RTG retrograde signaling pathway, which signals from the mitochondrial matrix to the nucleus (Journo et al, 2009). We would like to suggest that these effects on non-matrix proteins may indicate a possible role in transducing this signal. We have now added this comment to the discussion on Page 9 lines 28- Page 10 line 1.

3. Minor spelling mistakes should be reviewed (e.g., "mutophagic" pg. 7).

Thank you. Fixed.

*Reviewer #2 (Significance (Required)): *

*This work further expands on the previous observation by the authors (Kolitsida et al., 2019) and provides a novel and interesting hypothesis that may potentially impact our understanding of mitophagy selectivity toward particular mito proteome content. Furthermore, it unravels the additional function of PDC kinases and phosphatases behind the well-established regulation of energy metabolism. Therefore, it could interest the broad field of researchers interested in mitochondrial quality control and its interplay with cellular metabolism. *

We thank the reviewer for these encouraging comments.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this elegant and creative study, Dr. Abeliovich and his collaborators describe a novel pathway for the selective elimination of specific proteins. The flow of the experiments is logical and the writing is clear. The topic is very timely as the subject of mitochondrial quality control is of great interest. The main strength of this study is the creative ideas and the very out-of-the-box concept of selective elimination of specific proteins. The main shortcoming of this study is the incomplete direct evidence for a mechanism and conclusions that exceed the results. I believe these are fixable and I am sure this study will be well received after these issues are addressed.

We thank the reviewer for this positive assessment of the manuscript.

Specific comments:

Major comments:

1. It is not clear what is the mechanism for the recovery of pkp1 and pkp2 shown in figure 4. Supposedly, it is downstream of mitophagy, by how? Is there any data on that?

We presume the referee is referring to Figure 4A and B, where we demonstrate suppression of the pda1Δ phosphorylation phenotype by co-overexpression of Pkp1 and Pkp2. As indicated in the manuscript, we interpret these results as indicating that Pkp1 and Pkp2 function downstream of the PDC in the regulation of mitophagic trafficking. To further bolster this conclusion we now also show that a phosphomimetic mutation in Mdh1 that suppresses the pkp2Δ mutation as well as the aup1Δ mutation (Kolitsida et al, 2019), is also able to suppress the pda1Δ mutation. In addition, we will use co-IPs to test whether mutations in Lat1, and specifically the lat1K75R mutation which increases mitophagy and Mdh1 phosphorylation, can affect the known Pkp1/2-Pda1 interaction.

2. Pertaining to the experiment shown in figure 4, it is not clear if this was conducted in the presence of low glucose and if this was required for the effect.

As pointed out in the legend to Figure 4, the cells were grown in SL medium. As per the “Methods” section, SL contains, in addition to 2% lactate, 0.1% glucose. This small amount of glucose is always added to gluconeogenic media because initial growth in the total absence of glucose is very sluggish. 0.1% glucose does not induce the crabtree effect (does not inhibit respiration) and is consumed during the initial growth phase.

3. It is not clear if the effect is directly mediated by pct 5&7 acting on MDH1 or if this is an indirect effect. Can this be stated and then addressed experimentally? Are pct5 and pct7 the only phosphatases in the matrix?

The purpose of this experiment was to untangle the apparent paradox wherein Aup1/Ptc6 is the opposing phosphatase countering Pkp1/2 on Pda1, but this is clearly not the case for the signaling pathway uncovered in this study. This finding further differentiates our signaling pathway from the conventional PDC regulation cascade, and provides a previously unidentified function for Ptc5. We do not claim that these effects are direct. There are 3 documented phosphatases in yeast mitochondria: Ptc5, Ptc7 and Ptc6/Aup1. Since we have previously shown that the aup1Δ phenotype is very similar to that of pkp2Δ, it disqualifies Aup1 from being a candidate for the opposing phosphatase acting on Mdh1-GFP. The experiment shown in Figure 4D and E indicates that Ptc5 is the prime candidate for a phosphatase that targets Mdh1-GFP. We now clarify this point in the discussion (Page 10, lines 14-24), and we suggest that a more definitive identification will be made in future studies.

4. Figure 6 uses deletion of PDA1 as a loss of function experiment. However in this case, this is a rather crude approach since the point the authors are trying to make is that it is the regulation rather than the expression levels that is critical. If possible, the authors should try and affect the regulatory site by mutation rather than delete the gene.

We fully agree with this suggestion. We will now test the effect of the lat1K75R mutation on the mitochondrial phosphoproteome, in order to address this deficiency.

Minor comments:

1. The title should include that this was done in yeast. Similarly, yeast should be mentioned in the abstract too.

We have now added this information to the abstract. We do not wish to do the same for the title, as most journals have strict limits on the number of characters in the title.

2. In the introduction, it is stated that mitophagy is a process degrading dysfunctional mitochondria. It will be more accurate to say that it is degrading dysfunctional mitochondria as well as removing mitochondria in cells that shrink or rebuild their mitochondrial proteome.

Thank you. We have now made these changes (Page 3, lines 8-11)

3.Are pkp1 and pkp2 found only in the mitochondria?

As per the literature, Pkp1 and 2 are exclusively mitochondrial

4. In figure 1A/B only the blots of MDH1 are shown. It will be informative to show the blots of the other

proteins (currently in the supplementary)

We have added these blots to Figure 1

5. It is not clear how the reporter in fig 1C is different from the reporter in 1A.

This has now been clarified in the text (Page 4 line 28- Page 5 line 2)

Figure 4C. Please add an image of the colonies.

Thank you. This will be added to Figure 4.

6. It is not clear what the author defines as "increased segregation of Mdh1-GFP relative to generic mtRFP". Please clarify "generic mtRFP" and explain how the relativeness was deducted.

Thank you. We have now dropped the qualification “generic” and explain the difference. We now also explain the protocol for quantifying the overlap between the two signals (Page 8 lines 9-12, Page 16 lines 19-21 and 27-31).

Reviewer #3 (Significance (Required)):

This is a very hot topic and this lab is at the front of it It is for broad cell biology and biochemistry audience

We thank the referee for his/her generous and supportive comments

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In this elegant and creative study, Dr. Abeliovich and his collaborators describe a novel pathway for the selective elimination of specific proteins. The flow of the experiments is logical and the writing is clear. The topic is very timely as the subject of mitochondrial quality control is of great interest. The main strength of this study is the creative ideas and the very out-of-the-box concept of selective elimination of specific proteins. The main shortcoming of this study is the incomplete direct evidence for a mechanism and conclusions that exceed the results. I believe these are fixable and I am sure this study will be well received after these issues are addressed.

Specific comments:

Major comments:

1. It is not clear what is the mechanism for the recovery of pkp1 and pkp2 shown in figure 4. Supposedly, it is downstream of mitophagy, by how? Is there any data on that?
2. Pertaining to the experiment shown in figure 4, it is not clear if this was conducted in the presence of low glucose and if this was required for the effect.
3. It is not clear if the effect is directly mediated by pct 5&7 acting on MDH1 or if this is an indirect effect. Can this be stated and then addressed experimentally? Are pct5 and pct7 the only phosphatases in the matrix?
4. Figure 6 uses deletion of PDA1 as a loss of function experiment. However in this case, this is a rather crude approach since the point the authors are trying to make is that it is the regulation rather than the expression levels that is critical. If possible, the authors should try and affect the regulatory site by mutation rather than delete the gene.

Minor comments:

1. The title should include that this was done in yeast. Similarly, yeast should be mentioned in the abstract too.
2. In the introduction, it is stated that mitophagy is a process degrading dysfunctional mitochondria. It will be more accurate to say that it is degrading dysfunctional mitochondria as well as removing mitochondria in cells that shrink or rebuild their mitochondrial proteome. 3.Are pkp1 and pkp2 found only in the mitochondria?
3. In figure 1A/B only the blots of MDH1 are shown. It will be informative to show the blots of the other proteins (currently in the supplementary)
4. It is not clear how the reporter in fig 1C is different from the reporter in 1A. Figure 4C. Please add an image of the colonies.
5. It is not clear what the author defines as "increased segregation of Mdh1-GFP relative to generic mtRFP". Please clarify "generic mtRFP" and explain how the relativeness was deducted.

Significance

This is a very hot topic and this lab is at the front of it It is for broad cell biology and biochemistry audience

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The manuscript by Kolitsida et al. unravels an interesting link between mitochondrial pyruvate dehydrogenase complex (PDC) and protein sorting for mitophagy-mediated clearance. Using yeast genetics combined with immunoblotting-based mitophagy protein sorting reporter assay, authors show that targeting components of PDC and associated regulatory factors (kinases and phosphatases) interferes with phosphorylation status of at least some mitochondrial matrix proteins and prevents their degradation by autophagic machinery. Experimental design is sound, and statistic evaluation seems appropriate. Although I find this work important and overall valuable, some of the conclusions made by the authors are still preliminary and could require additional experimental testing.

In this regard, some of my major suggestions include:

1. Authors propose that the interaction of dedicated kinases and phosphatases with pyruvate dehydrogenase complex changes their affinity/activity toward the non-PDC substrates (possibly by allosteric regulation). Yet, alternatively, it can also be considered that the PDC complex acts as a docking station that stabilizes the associated regulatory factors inside the mitochondria and allows their additional functions. Indeed, simultaneous overexpression of Pkp1/2 increases the free GFP signal and Mdh1 phosphorylation even in the absence of core E1 PDC component (pda1, figure 3 C, D and figure 4 A, B), suggesting that the interaction of phosphatases and kinases with pda1 is not essential to mediate the phosphorylation of other mitoproteins. Are the protein levels of regulatory PDC factors (Pkp1, Pkp2, Aup1) somewhat altered by depletion of their target E1a subunit (pda1)? Could depletion of pda1 trigger destabilization of these factors and decrease their half-life? This can be tested by western-blotting and combined with cycloheximide chase assay.
2. Allosteric regulation of specificity of PDC kinases and phosphatases is an intriguing yet still slightly unbacked hypothesis. To provide further experimental evidence for this explanation, authors could develop the classical in vitro kinase assay for Pkp1/2 on their model substrate (Mdh1) in the absence or presence of the Pda1 subunit. Even more excitingly, authors could isolate the Pkp1/2 from wild-type and Pda1-compromised cells and see whether they are able to phosphorylate their model substrate in vitro. Additionally, does specific inhibition of pyruvate dehydrogenase kinases (e.g., with dichloroacetate) affect the Mdh1 phosphorylation levels?
3. Are the Pkp1/Pkp2 changing their interactome upon PDC alternation (e.g., Pda1 depletion)? Do their interactome alters upon stimulation of mitophagy? Co-immunoprecipitation or bioID assay could shed light on how the partitioning of the Pkp1/Pkp2/Aup1 functions between the metabolic regulation and protein quality control is maintained.

Minor points:

1. The paper would benefit from a brief explanation of the principles of the mitophagy reporter assay used in this study.
2. MS phosphoproteomics is indeed a great approach to tackle many questions in this study. Surprisingly, the authors did not discuss these data extensively. Although changes in the phosphorylation status of some proposed targets are apparent (as QCR2), many others were not detected (including Aco1/2, Idp1, and model substrate - Mdh1; Figure 1). Furthermore, many significant changes occur in the proteins that do not belong to the mitochondrial matrix compartment (as TOMM20 or YAT1), questioning the direct involvement of Pkp1/Pkp2/Aup1. How do authors interpret these data?
3. Minor spelling mistakes should be reviewed (e.g., "mutophagic" pg. 7).

Significance

This work further expands on the previous observation by the authors (Kolitsida et al., 2019) and provides a novel and interesting hypothesis that may potentially impact our understanding of mitophagy selectivity toward particular mito proteome content. Furthermore, it unravels the additional function of PDC kinases and phosphatases behind the well-established regulation of energy metabolism. Therefore, it could interest the broad field of researchers interested in mitochondrial quality control and its interplay with cellular metabolism.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The Abeliovich lab has discovered differential degradation rates for mitochondrial proteins by mitophagy, a selective form of autophagy of mitochondria. In previous publications, they identified two protein kinases Pkp1/2 and the protein phosphatase Aup1(Pct6), which regulate phosphorylation patterns of mitochondrial proteins and affect their degradation rates by mitophagy. In the current manuscript, the authors now analyze the role of the pyruvate dehydrogenase complex (PDC) in mitochondria in the regulation of these regulatory link between Pkp1/2 and Aup1 and their substrate proteins. They find that the deletion of pda1, a core subunit of PDC, inhibits the degradation of previously identified mitophagy substrates Mdh1, Aco2, Qcr2, Aco1, and Idp1, while the turnover of mtDHFR-GFP, a model substrate diffusely localized in the mitochondrial matrix, is not affected. These data indicate that tested mitochondrial proteins are excluded from mitochondrial turnover by mitophagy in the absence of Pda1 by as yet unknown sorting mechanisms.

The authors next show that the mutation of a known Pkp1/2 and Aup1 phosphorylation site within Pda1 (S313A) drastically reduces mitophagic turnover of Mdh1, which is completely blocked by the additional deletion of AUP1. A mutation blocking Pda1 activity (R322C) also almost completely blocked Mdh1 degradation. However, PDC activity does not generally affect mitophagy, since an inactivating mutation in Lat1 (K75R) promotes mitophagy of Mdh1. Published work has shown that Pkp1/2 and Aup1, which affect the phosphorylation and degradation of Mdh1, physically interact with the PDC. Thus, the authors tested whether the absence of Pda1 affected the phosphorylation state of Mdh1 and thus its turnover. Indeed, Mdh1 showed globally reduced phosphorylation in the absence of Pda1, which was rescued by the overexpression of Pkp1/2 promoting degradation of Mdh1. Consistent with the effects on mitophagic turnover, Mdh1 was phosphorylated at higher level in the presence of Lat1-K75R. The authors analyzed the mitochondrial distribution of Mdh1 in dependence of Pda1. They provide cytological data suggesting that Mdh1 might segregate from a diffuse matrix localized mtRFP reporter, which is suppressed upon Pkp1/2 overexpression.

Finally, the authors perform a mitochondrial phospho-proteome analysis and found that the absence of Pda1 affected phosphorylation sites in 8 mitochondrial matrix proteins.

From these data the authors propose a model in which the PDC complex controls the activity of the associated protein kinase Pkp1/2 and the phosphates Aup1 by allosteric changes, which in turn regulates the phosphorylation and differential turnover of mitochondrial proteins by mitophagy. They speculate that the PDC and associated factors could resemble large protein kinase complexes as TORC.

Critical points:

1. The authors postulate a structural role for the PDC in regulating Pkp1/2 and Aup1 for controlling mitophagy turnover of certain substrates. While the physical association of Pkp1/2 and Aup1 with PDC has been shown previously, the authors need to critically test their model and assess (a) whether this physical interaction occurs under their experimental conditions and (b) whether the different mutants that affect mitophagy of Mdh1 also affect the physical interaction of Pkp1/2 and Aup1 with PDC. Otherwise, their model, although consistent, remains purely speculative.
2. It is important to test every mutant that affects Mdh1 turnover for effects on mitophagy in general using the mtDHFR-GFP reporter to be able to conclude specific effects on Mdh1 mitophagy. For example, the deletion of Lat1 has been shown to induce mitophagy of a mitochondrial matrix reporter during nitrogen starvation (Bockler and Westermann, 2013). While the authors observe a complete block of Mdh1 turnover in lat1 deletion cells, they do observe increased Mdh1 degradation in Lat1-K75R cells. In line with this reasoning, the authors have identified a number of Mdh1 variants in a previous publication (Kolitsida et al. 2019) that can be used to further explore the observed phenomenon. For example, the Mdh1-T199A could be used to test whether the expression of Pkp1/2 in pda1 deficient cells has specific or general effects on mitophagy. Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?
3. The cytological data are not convincing. The imaging quality is low and it is very difficult for the reader to appreciate the suggested segregation of Mdh1-GFP and mtRFP. Thus, it is important to provide cortical sections in order to visualize mitochondrial tubules/networks and Mdh1 distribution. This is particularly important, because the authors mention effects of Pda1 on mitochondrial morphology, which appears to be rescued by Pkp1/2 overexpression. In this context it would be very informative to follow the localization of PDC in mitochondria. Previous work has shown that Pda1 forms punctate structures in mitochondria in proximity to ER-mitochondria contact sites marked by ERMES (Cohen et al. 2014). Is Pda1 itself degraded by mitophagy or is it excluded? Could the physical interaction of Mdh1 with Pda1 explain mitophagy phenotypes if Pda1 is excluded from mitophagy? In other words, could the PDC form a physical unit to segregate and prevent proteins from mitophagy turnover?
4. The conclusions that can be drawn from the analysis of the mitochondrial phospho-proteome in dependence of Pda1 are rather limited. First, the experimental setup does not distinguish between direct effects of PDC on Pkp1/2 or Aup1 activity and the effects of simply PDC dysfunction on mitochondrial proteins. Along those lines, it is unclear whether the few identified proteins with altered phosphorylation states are indeed targets of Pkp1/2 or Aup1. Thus, to be able to support their conclusion, the authors need to include a number of additional controls/strains.

Significance

The authors continue to explore the mechanisms underlying their initial observation of differential turnover rates for mitochondrial proteins by mitophagy. This current work specifically builds on the previous publication identifying Pkp1/2 and Aup1 as regulators of the phosphorylation state of specific substrate proteins (Kolitsida et al. 2019). Here the authors now explore how these regulators might be organized and controlled by PDC. Thus, the study adds another layer of regulation. However, the molecular mechanisms of how PDC might regulate Pkp1/2 and Aup1 is not directly addressed. In conclusion, the current study opens up new lines of research that need to be explored to critically test the proposed model. A key question to me is of course how mitochondrial proteins can be excluded from mitophagy and whether the proteins in this study may play a direct role in these mechanisms.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1

SUMMARY

The manuscript by Smoak et al., provides an analysis of the Hyr/Iff-like (Hil) genes in Candida species with a strong focus on C. auris. The authors demonstrate a repeated expansion of these genes in unique lineages of fungal species, many of which are associated with stronger clinical disease. There is evidence of selection operating on the gene family in the primary domain used for identification. These genes include a repeat just downstream of that core domain that changes frequently in copy number and composition. The location of these genes tends to cluster at chromosome ends, which may explain some aspects of their expansion. The study is entirely in silico in nature and does not include experimental data.

MAJOR POINTS

Altogether, many of the general findings could be convincing but there are some aspects of the analysis that need further explanation to ensure they were performed correctly. To start, a single Hil protein from C. auris was used as bait in the query to find all Hil proteins in yeast pathogens. Would you get the same outcome if you started with a different Hil protein? What is the basis for using Hil1 as the starting point? It also doesn't make sense to me to remove species just because there are already related species in the list. This may exclude certain evolutionary trends. Furthermore, it would be helpful to know how using domain presence and the conservation of position changes the abundance of the gene family across species? (beginning of results).

We appreciate the reviewer’s criticisms on our strategy for identifying Hil proteins. In response, we have significantly revised our pipeline. In particular, we now combine the search results from three queries: in addition to C. auris Hil1’s Hyphal_reg_CWP domain (XP_028889033), we added the Hyphal_reg_CWP sequences from C. albicans Hyr1 and C. glabrata Hyr1. They were chosen as representatives in the two phylogenetic groups distinct from the one containing C. auris in order to avoid the bias due to the query’s phylogenetic position. Using the same criteria as we did for the original search, we identified three additional hits compared with the original 104 homologs list. In response to the criticism of the arbitrary exclusion of some species, we now include any species from the BLASTP search results as long as it is part of the 332 yeast species studied by Shen et al. 2018 (PMID: 30415838). The reason for this criterion is so that we can use the high-quality species phylogeny generated by Shen et al. 2018 to properly study the gene family evolution by reconciling the gene tree with the species tree. We additionally include the species in the MDR clade closely related to C. auris and used Muñoz et al. 2018 (PMID: 30559369) as the basis for the species phylogeny in the clade. Lastly, we no longer require the particular domain organization in classifying Hil family members. All BLASTP hits satisfying the E-value cutoff of 1x10-5 and query coverage > 50% are included.

A major challenge in the analysis like this one is in dealing with repetitive sequences present in amplified gene families. For example, testing modes of selection on non-conserved sites is fraught. It's not clear if all sites used for these tests are positionally conserved and this should be clarified. Alignments at repeat edges will need to maintain this conservation and relatively good alignments as stated in lines 241-242 are concerning that this includes sequence that does not retain this structure necessary for making predictions of selection.

We appreciate the reviewer’s comment. In the original manuscript, we performed two different types of analyses, one on the conserved and well-aligned Hyphal_reg_CWP domain and another on the rapidly evolving repeat region. For the former, we performed phylogenetic dN/dS analyses using maximum-likelihood, for which a reliable alignment is crucial and is the case here. The Hyphal_reg_CWP domain alignment for C. auris Hil1-Hil8 is shown below and also included as Fig. S7 in the revised manuscript: (figure in the response file)

In the text, we added this sentence to emphasize this point: “We chose to focus on the Hyphal_reg_CWP domain because of its potential importance in mediating adhesion and also because the high-quality alignment in this domain allowed us to confidently infer the evolutionary rates (Fig. S7).”

For the repeat domain, what we did in the original version was to calculate the pairwise dN/dS between individual repeat units found in Hil1 and Hil2. This didn’t require aligning the entire repeat regions in the two proteins, but instead relied on the alignment of the individual ~44 aa repeat units, which were highly conserved (see below). In the revised manuscript, however, we decided to focus our analyses on the Hyphal_reg_CWP domain because of a different concern, namely gene conversions between paralogs could distort the evolutionary history of the repeats (the same concern was addressed for the effector domain using an additional step of detecting recombination breakpoints, but the same analysis would be challenging for the repeat region due to alignment issues).

(figure in the response file)

It's also unclear to me why Figure S12 is here. The parameters for this analysis should be tested ahead of building models so only one set of parameters should be necessary to run the test. The evolutionary tests within single genes and across strains is really nice!

We appreciate the reviewer’s suggestion. Based on the reviewer’s suggestion, we removed Fig. S12 and describe the model set up in the Materials and Methods section. We were not sure if the last point was a comment or a suggestion. We didn’t perform a population level selective sweep scan in C. auris. Such an analysis has in fact been attempted by Muñoz et al. 2021, who identified several members of the Hil family as the top candidates for positive selection (PMID: 33769478). We cited this in our Discussion:

“Lastly, scans for selective sweep in C. auris identified Hil and Als family members as being among the top 5% of all genes, suggesting that adhesins are targets of natural selection in the recent evolutionary history of this newly emerged pathogen (Muñoz et al. 2021).”

A major challenge for expanded gene families is rooting based on the inability to identify a strong similarity match for the full length sequence. The full alignment mentioned would certainly include significant gaps. If those gaps are removed and conserved sites only are used, does it produce the same tree? Inclusion of unalignable sequences would be expected to significantly alter the outcomes of those analysis and may produce some spurious relationships in reconciling with the species trees. Whether or not there are similar issues in the alignment of PF11765 need to be addressed as well. There's nothing in the methods that clarifies site selection.

We appreciate reviewer’s comment and agree with the concern about alignment quality affecting phylogenetic reconstruction. To clarify, all phylogenetic analyses in this work are based on the alignment of the Hyphal_reg_CWP domain, which is well aligned (shown above for the subset of eight homologs in C. auris). Alignment of all 215 homologs is provided for readers to review (shorturl.at/kDEJ3). To clarify this choice, we now include the following in Results:

“To further characterize the evolutionary history of the Hil family, including among closely related Candida lineages, we reconstructed a species tree-aware maximum likelihood phylogeny for the Hil family based on the Hyphal_reg_CWP domain alignment (Fig. 1C, Fig. S2).”

We also included detailed steps for reconstructing the gene tree in Materials and Methods.

To test the effect of gaps in the alignment on phylogenetic tree inference, we used two trimming programs, ClipKit (PMID: 33264284) and BMGE (PMID: 20626897), with author-recommended modes. They resulted in consistent gene tree results. We present the tree based on the ClipKit trimmed alignment in the main results. The root of the gene tree was inferred by jointly maximizing the likelihood scores for the gene tree based on the alignment and the evolution of the gene family within the species tree, using GeneRax (Morel et al. 2020, PMID: 32502238).

Figure 1A: the placement of evolved pathogenesis is a little arbitrary. It's just as feasible that a single event increased pathogenesis in the LCA of C. albicans and C. parapsilosis that was subsequently lost in L. elongisporus. These should be justified or I'd suggest removing. The assignment of Candida species here also seems incomplete. The Butler paper notes both D. hansineii and C. lusitaniae as Candida species whereas they are excluded here.

We removed Figure 1 entirely based on this and another reviewer’s comment. We note that there is broad consensus that opportunistic yeast pathogens have independently arisen multiple times, such as C. auris, C. albicans and C. glabrata. Whether Candida pathogens that are more closely related evolved separately or not are subjects of ongoing research (PMID: 24034898).

It is tricky to include scaffolds in analysis of chromosomal location of the HIL genes. The break in the scaffold may be due to the assc repeats of these proteins alone or other, nearby repeats. Any statistics would be best done to include only known chromosomes or those that are strongly inferred by Munoz, 2021. This will change the display of Figure 7, but is unlikely to change the take home message.

We agree with the reviewer’s concern. In the revised manuscript and with more species included, we now only analyze genomes assembled to a chromosomal level, with the exception of C. auris B8441, which is supported by Muñoz et al. 2021 as having chromosome-length sequences. The revised Figure 7 now only includes these results. We also removed the accompanying supplementary figure that showed results based on scaffold-level assemblies.

MINOR POINTS

Line 18: "spp." Should be "spps."

Addressed throughout the revised manuscript.

Line 41: I might rephrase this as "how pathogenesis arose in yeast..."

Accepted (line 43 in revised manuscript).

I might use a yeast-centric example around line 40 for duplication and divergence. This could include genes for metabolism of different carbon sources in S. cerevisiae.

Accepted (lines 47-48)

The Butler paper referenced on line 51 compared seven Candida species and 9 Saccharomyces species

Changed (line 48)

The autors state no other evolutionary analysis of adhesins has been performed but do not acknowledge this study: https://academic.oup.com/mbe/article/28/11/3127/1047032

We appreciate the reviewer pointing this important reference to us. We now cite it in the introduction (line 64) and discussion (line 340)

The first paragraph of the Results could be condensed

Addressed.

How was the species tree in Figure 1A obtained?

The previous figure 1 is now removed. The species tree used throughout the manuscript is based on Shen et al. 2018 with MDR clade species added, based on Muñoz et al. 2018.

Figure 2: In panel A, "DH" and "SS" are not defined. I'd be careful with use of "non-albicans Candida" in Figure 2B. This usually includes C. tropicalis and C. dubliniensis and may confuse the reader.

We removed the DH and SS labels. Instead, we highlighted three clades, which were defined in previous studies. These are the Candida/Lodderomyces clade (based on NCBI taxonomy database), the MDR clade (e.g., Muñoz et al. 2018, PMID: 30559369) and the glabrata clade (e.g., Gabaldón et al. 2013, PMID: 24034898).

How was the binding domain defined to extract those sequences are produce a phylogeny? In building a ML model, how were parameters chosen?

We now provide the following details in the Materials and Methods section:

“To infer the evolutionary history of the Hil family, we reconstructed a maximum-likelihood tree based on the alignment of the conserved Hyphal_reg_CWP domain. First, we used hmmscan (HmmerWeb version 2.41.2) to identify the location of the Hyphal_reg_CWP domain in each Hil homolog. We used the “envelope boundaries” to define the domain in each sequence, and then aligned their amino acid sequences using Clustal Omega with the parameter {--iter=5}. We then trimmed the alignment using ClipKit with its default smart-gap trimming mode (Steenwyk et al. 2020). RAxML-NG v1.1.0 was compiled and run on the University of Iowa ARGON server with the following parameters on the alignment: raxml-ng-mpi --all --msa $align --model LG+G --seed 123 --bs-trees autoMRE.”

The parameters for the ML tree reconstruction is listed on the last line above. The main parameter was the evolutionary model (LG+G), which accounts for rate variations using a gamma distribution. Other protein evolution models, e.g., VT+I+G, were tested and resulted in nearly identical tree topologies.

Figure 3C/D could be just one panel.

The structure predictions are now reorganized and presented on their own in the new Figure 3.

Can you relate more the fungal hit to the Hil proteins conveyed in lines 152-154?

We appreciate the reviewer’s comment, which referred to CgAwp1 and CgAwp3, whose effector domain structures were reported in a recent study (Reithofer et al. 2021, PMID: 34962966). We now discuss them in relation to the predicted Hyphal_reg_CWP structure, by showing them in Figure 3 and describing them in the Results (lines 181) “crystal structures for the effector domains of two Adhesin-like Wall Proteins (Awp1 and Awp3b) in C. glabrata, which are distantly related to those in the Hil family were recently reported, and the predicted structure of one of C. glabrata’s Hil family members (Awp2) was found to be highly similar to the two solved structures (Reithofer et al. 2021)”

Line 168: Should read "Hence, ..."

The original sentence was removed, but this grammatical error was checked for and corrected.

Label proteins along the top of Figure 4 too.

Accepted (in new Figure 4).

Figure 5: for tests of selection, were sites conserved across the group? What does the black number at each node indicate? Dn and Ds are given as decimals. This is based on what attribute? For panel B, it is unclear what each tip denotes i.e., Hil1_tr6. Hil1 is the gene but what is "tr6"?

In the revised manuscript, we provide the multiple sequence alignment for the Hyphal_reg_CWP domain used for the selection analysis as Fig. S7 to illustrate the level of conservation. The black numbers at the internal nodes are numeric indices used to refer to those nodes. In the revised manuscript, we use some of them to refer to the internal branches, e.g., 12…14 in the legend. In the new Figure 5, we do not list the numeric values of Dn and Ds (aka Ka, Ks). Instead, we use a color gradient to represent the estimated dN/dS ratios. The raw estimates are available in the project github repository. Panel B in the original Figure 5 and other panels related to the evolution of the repeats are now removed.

It's unclear why comparison of the PF11765 domain includes the MRD proteins when those aren't included in the comparison to the repeats alone. Could that skew the comparison due to unequal sample numbers or changed variation frequencies in MDR relative to the other two groups?

These results pertaining to the evolution of the repeats are now removed.

Table 2 doesn't add much. This section could probably be reduced to a few sentences since it's highly speculative (intraspecies variation).

Table 2 is now Table S5. We also simplified the result section in the revised version. While the functional implications of the intraspecific variable number of tandem repeats (VNTR) is speculative, it is founded on two bases: 1) the identification of the VNTR is credible, as the copy number variation is consistent within clades but differ between clades, which is not expected if they are caused by assembly errors; 2) experimental studies in S. cerevisiae for the Flo family strongly supported a direct impact of adhesin length on the adhesive phenotype of the cells (PMID: 16086015).

Table 3 is not needed.

Table 3 is now removed.

Figure 6 - color coding in 6A needs to be explained. I'm assuming this is a taxonomical coding.

In the revised Figure 6A, the coloring scheme is consistent with what we used in Figure 1 based on the three clades, and a legend is provided.

Figure 1B is unnecessary. A Model of the protein indicating domains is sufficient here. Figure 1C needs labels for all termini, not just the pathogenic red branches. The figure doesn't provide clear association between adhesin families and the associated species. This could be omitted, especially since Flo is often associated with Saccharomyces species. Figure 1D is unnecessary.

We have removed the original Figure 1.

SIGNIFICANCE

The work here is sorely needed in expanded gene families and in fungi specifically. No analysis at this level has been performed, to the best of my knowledge, in any fungal associated gene family and certainly not in relationship to pathogenic potential. The authors do a good job in citing the foundational literature upon which their study builds in most cases (one exception is noted above). It would be of general interest to those interested in the evolution of virulence, but the analysis is tricky. This is the biggest drawback I currently have as some of the information to assess the results is missing.

We really appreciate the reviewer's positive comments. We agree and plan to explore the relationship between the adhesin family evolution and virulence phenotypes.

Expertise: gene families, evolution dynamics, human fungal pathogens

Reviewer #2

SUMMARY

Gene duplication and divergence of adhesin proteins are hypothesized to be linked with the emergence of pathogenic yeasts during evolution; however, evidence supporting this hypothesis is limited. Smoak et al. study the evolutionary history of Hil genes and show that expansion of this gene family is restricted to C. auris and other pathogenic yeasts. They identified eight paralogous Hil proteins in C. auris. All these proteins share characteristic domains of adhesin, and the structural prediction supports that their tertiary structures are adhesin-like. Evolutionary analysis of protein domains finds weak evidence of positive selection in the ligand-binding domain, and the central domain showed rapid changes in repeat copy number. However, performed tests cannot unambiguously distinguish between positive selection and relaxed selection of paralogs after gene duplication. Some alternative tests are suggested that may be able to provide more unambiguous evidence. Together with these additional tests, the detailed phylogenetic analyses of Hil genes in C. auris might be able to better support the hypothesis that the expansion and diversification of adhesin proteins could contribute to the evolution of pathogenicity in yeasts.

We appreciate the reviewer’s comments and will address specific points below.

MAJOR COMMENTS

The authors present extensive analyses on the evolution of Hil genes in C. auris. There is significant merit in these analyses. However, the analyses conducted so far are incomplete, lacking proper consideration of other confounding factors. Detailed explanations of our major comments are listed below.

1. First, the authors restricted genes in the Hil family to those only containing the Hyphal_reg_CWP domain. Yet, previous work included genes containing the ligand-binding domain or the repeat domain as Hil genes. More justification is needed whether the author's choice represents the natural evolutionary history of Hil genes appropriately. For instance, are the genes only containing the ligand-binding domain monophyletic or polyphyletic? We recommend including the phylogeny of all the Hil candidate genes, to discern whether evolutionary histories of the repeat domain and ligand-binding domain are congruent. Authors can use this phylogeny as justification to focus only on the ligand-binding domain containing genes.

Butler et al. 2009 (PMID: 19465905) defined the Als family and the Hyr/Iff family as having either the N-terminal effector domain or the intragenic tandem repeats (ITR). Their rationale for the latter was that the ITS sequences were often conserved across species. Upon close inspection (Fig. S19,20 in that paper), however, we found that the ITS tend to be conserved in closely related species, but diverged among more distantly related species. Moreover, proteins in those figures that only contain the ITS and not the ligand-binding domains are all missing either the signal peptide, the GPI-anchor or both. This raises questions as to whether these proteins sharing the ITS sequence alone act as adhesins.

More generally, defining the evolutionary history of proteins with multiple domains is complicated by recombination, which causes different parts (e.g., domains) of the protein to have distinct evolutionary histories. In fact, our study and others show that there exist “chimeras” that combine the effector domain from one adhesin family and the repeat sequence found in another (Zhao et al. 2011, PMID: 21208290, Oh et al. 2019, PMID: 31105652). In these cases, one phylogenetic tree is insufficient to describe the evolutionary history of the whole protein. We chose to define the Hil family by the Hyphal_reg_CWP domain and thus focus on the evolutionary history of this region because 1) while tandem repeat regions also contribute to adhesion in yeasts (Rauceo et al. 2006, PMID: 16936142), the effector domain likely plays a more important role in ligand binding and specificity. Therefore, we believe using the effector domain to define a protein family is more likely to group proteins with similar functional properties than if the repeat sequences were used. Also, while putative fungal adhesins lacking a recognizable ligand-binding domain exist, they are rare (Lipke 2018, PMID: 29772751); 2) The repeat region evolved much more rapidly than the effector domain, as we illustrate in Figures 2, 4 and 6 in our revised manuscript. While some repeat units are highly conserved, e.g., the ~44 aa unit found in Hil1-4 in C. auris and close relatives in the MDR clade, many others are short and degenerate, making it difficult to reliably identify homologs sharing the repeat. Besides, since each protein could contain many distinct repeats, it is not clear how one defines two sequences as belonging to the same family if they share one out of six types of repeats. We acknowledge that this definition leaves out the evolutionary history defined by the tandem repeats, which may reveal intriguing evolutionary dynamics, with functional implications. A recent review for the Als family discussed similar definition challenges and partly supported our choice (Hoyer and Cota, 2016, PMID: 27014205).

In the analysis of positive selection, the authors do not adequately control for the effect of recombination on the evolutionary histories of protein sequences, especially given that Hil genes are rich in repetitive sequences. To account for recombination, GARD, an algorithm detecting recombination, should be performed to detect any recombination breakpoints within a protein domain. If recombination did occur within a protein domain, the authors should treat the unrecombined part as a single unit and use the phylogenetic information of this part to proceed with PAML analysis, instead of using the phylogeny of the entire protein domain. The authors should consider doing GARD analysis for the ligand-binding and repeat domains. For the repeat domain, low BS values in Fig. 5C indicate recombination between repeat units. Thus, the authors should analyze each repeat unit with GARD and re-analyze dN/dS.

We deeply appreciate the reviewers’ criticism here. In the revised manuscript, we removed the analysis of the repeat units and followed the reviewers’ suggestion to carry out GARD analysis on the effector domain, which we now show reveals evidence of intra-domain recombination. Using the inferred breakpoints (Fig. S8), we identified two putatively non-recombining partitions and performed all downstream phylogenetic analyses on them separately. The results are presented in Fig. 5 and Table S6. Compared with the previous result based on the entire Hyphal_reg_CWP domain alignment, the new results reveal clearer patterns, including significantly elevated dN/dS on a subset of the branches. Newly added branch-site test results support a role of positive selection on the effector domain during the expansion of the Hil family in C. auris, suggesting functional diversification following gene duplications.

The authors concluded positive selection in the ligand-binding domain based on the branch-wise model of PAML. Yet, w values were not higher than one, and it's unclear whether the difference in selective pressures the authors claimed here is biologically significant. Overall, what the authors present so far seems to be weak evidence of positive selection but is much more consistent with variation in the degree of purifying selection or evolutionary constraint. Using the site-wise model (m7 vs. m8) in PAML would allow the authors to detect which residues of the ligand-binding domain underwent recurrent positive selection. Combining the evolutionary information of protein residues and the predicted 3D structure will provide molecular insights into the biological impact of rapidly evolving residues. This would be a significant addition and raise the significance of the study, besides providing potentially stronger evidence of positive selection.

We appreciate the reviewers’ criticism and suggestions. In the revised manuscript, we performed site tests comparing models M2a vs M1a, M8 vs M7 and M8a vs M8. For partition 1 (P1-414), all three tests were insignificant. For partition 2 (P697-987), the M2a vs M1a test was insignificant (P > 0.05) but M8 vs M7 and M8a vs M7a were both significant at a 0.01 level, and the omega estimate for the positively selected category was estimated to be ~15. The site tests require all branches to evolve under the same selection regime. To relax this constraint, we performed additional branch-site tests by designating the branches with an estimated dN/dS > 10 as the foreground (based on the free-ratio model estimates). This test was significant for both branches at a 0.01 level and the Bayes Empirical Bayes (BEB) procedure identified a total of 5 residues as having been under positive selection. Although three of the five residues, located in the C-terminus of the Hyphal_reg_CWP domain, are part of the α-crystallin domain, we refrain from drawing any functional conclusions because 1) the BEB procedure is known to be lacking power in identifying positively selected residues and 2) we still lack structure-function relationship for the α-crystallin domain. But we agree and believe that this line of analysis is promising in yielding functional insight into the evolution of the effector domain in the family.

MINOR COMMENTS

1. In Fig 1c, the figure legend should include more specific details: which adhesin proteins are shown here? Please specify species names on the species tree

Figure 1 is removed in the revised manuscript

In Fig 3E, secondary structures are labeled with the wrong colors. Sheet: purple, helix: yellow

In the revised manuscript, the structures of SRRP-BR (original 3E) is now colored in a single color.

What's the ligand-binding activity of the b-solenoid fold? How structurally similar are C. auris PF 11765 domains compared to C. glabrata Awp domains? This information will support the role of adhesin for the ligand-binding domain of Hil genes.

We discuss the ligand-binding activity of the β-solenoid as follows in Discussion:

“The elongated shape and rigid structure of the β-helix are consistent with the functional requirements of adhesins, including the need to protrude from the cell surface and the capacity for multiple binding sites along its length that facilitate adhesion. In some bacterial adhesins, such as the serine rich repeat protein (SRRP) from the Gram-positive bacterium, L. reuterii, a protruding, flexible loop in the β-helix was proposed to serve as a binding pocket for its ligand (Sequeira et al. 2018). Such a feature is not apparent in the predicted structure of the Hyphal_reg_CWP domain. Further studies are needed to elucidate the potential substrate for this domain and its mechanism of adhesion.”

We also compare the structures of the C. auris Hil1/Hil7 Hyphal_reg_CWP domain and the CgAwp1/3 in Figure 3, with this in the legend “(C) Crystal structure of the C. glabrata Awp1 effector domain, which is highly similar to C. auris Hil1 and Hil7, but with the disulfide bond in a different location.”

We added a section in the Discussion to comment on the structure-function relationship based on known β-helix (aka β-solenoid) structures. The main insight comes from similar structures identified through DALI searches, many of which are bacterial and viral surface proteins mediating adhesion. The ligand binding pocket and specificity would require additional structural studies to elucidate.

In lines 248-249, the authors should also consider the influence of evolutionary history. For instance, repeats within the same Hil protein appeared later in evolution, compared to Hil gene duplication, and therefore these repeats experience less time for sequence divergence.

In the revised manuscript, we removed the analyses pertaining to the evolution of the repeat regions due to multiple challenges including alignment, potential of gene conversion and recombination. This is an important and intriguing aspect of adhesin family evolution that we plan to follow up in future work.

Although the bioinformatic evidence of C. auris Hil genes acting as adhesins is strong, it is still worthwhile to discuss the experiments of confirming the function of adhesins.

We agree with the reviewer and acknowledge in the revised manuscript the limitation of our work:

“Future experimental tests of these hypotheses will be important biologically for improving our understanding of the fungal adhesin repertoire, important biotechnologically for inspiring additional nanomaterials, and important biomedically for advancing the development of C. auris-directed therapeutics.”

SIGNIFICANCE

Overall, this study is interesting to investigate the evolutionary history of a crucial virulent gene in C. auris. Such evolutionary understanding will help us identify critical molecular changes associated with the pathogenicity of an organism during evolution, providing insights into the emergence of pathogens and novel strategies to cure fungal infections. The research question is important; however, the current analyses on the positive selection are incomplete, so the conclusion is modest so far. We recommend that the authors re-do the PAML analysis with the above considerations. This work will bring more significance to the mycology field if the functional impact of rapid evolution in protein domains can be supported or inferred.

This manuscript is well-written, and the authors also did a great job specifying all the necessary details in the M&M.

We appreciate the reviewers’ positive comments.

Reviewer #3

Summary:

The manuscript by Smoak et al. provides considerable information gleaned from analysis of HYR/IFF genes in 19 fungal species. A specific focus is on Candida auris. The main conclusion is that this gene family repeatedly expanded in divergent pathogenic Candida lineages including C. auris. Analyses focus on the sequences encoding the protein's N-terminal domain and tracts of repeated sequences that follow. The authors conclude with the hypothesis that expansion and diversification of adhesin gene families underpin fungal pathogen evolution and that the variation among adhesin-encoding genes affects adhesion and virulence within and between species. The paper is easy to read, includes clear and attractive graphics, as well as a considerable number of supplementary data files that provide thorough documentation of the sources of information and their analysis.

We appreciate the positive comment.

MAJOR COMMENTS:

• Are the key conclusions convincing?

Overall, the authors' conclusions are supported by the information they present. However, the overall conclusion is stated as a hypothesis and that hypothesis is not particularly novel. The idea that expansion of gene families associated with pathogenesis occurs in the pathogenic species dates back at least to Butler et al. 2009, who first presented the genome sequences for many of the species considered here.

We appreciate the reviewer’s comment. Our main conclusions are 1) the Hil family is strongly enriched in distinct clades of pathogenic yeasts after accounting for phylogenetic relatedness. This enrichment results from independent duplications, which is ongoing between closely related species; 2) the protein sequence of the Hil family homologs diverged rapidly following gene duplication, driven largely by the evolution of the tandem repeat content, generating large variation in protein length and β-aggregation potentials; 3) there is strong evidence for varying levels of selective constraint and moderate evidence for positive selection acting on the N-terminal effector domain during the expansion of the family in C. auris as our focal species. Based on these observations, we propose that expansion of adhesin gene families is a key preliminary step towards the emergence of fungal pathogenesis.

Indeed, some version of this hypothesis has been proposed by several groups before us. We fully acknowledged this in our previous as well as the revised manuscript, by citing Butler et al. 2009 (PMID: 19465905), Gabaldón et al. 2013, 2016 (PMID: 24034898, 27493146). Our study built on these earlier efforts and extended them by addressing several limitations. First, we performed phylogenetic regression to test for the association between gene family size and the life history trait (pathogen or not) in order to properly account for the phylogenetic relatedness. This was not done in previous studies. Second, most earlier studies didn’t construct a family-wide gene tree to fully investigate the evolutionary history of the family. Gabaldón et al. 2013 did a phylogenetic analysis for the Epa family and a few others within the Nakaseomycetes, revealing highly dynamic expansions. In the present study, we expanded this effort by comprehensively identifying homologs within the Hil family in all yeasts and beyond. Third and perhaps the most important novelty in our study is our detailed analysis of sequence divergence and role of natural selection during the evolution of the family post duplication. This allowed us to present a complete picture of the family’s evolution, not just in its increase in copy number but also its diversification after the duplications, which is a key part of how gene duplications contribute to the evolution of novel traits. As such, we believe our study provides strong support for the above hypothesis.

One key issue with a manuscript of this type is whether genome sequence data are accurate. The authors are not the first research group to take draft genome sequence data at face value and attempt to draw major conclusions from it. The accuracy of public genome data continues to improve, especially with the emergence of PacBio sequencing. Because the IFF/HYR genes contain long tracts of repeated sequences, genome assemblies from short-read data are frequently inaccurate. For example, is it reasonable to have confidence that the number of copies of a tandemly repeated sequence in a specific ORF is exactly 21 (an example taken from Table 2) when each repeat is 40+ amino acids long and highly conserved? Table S6 would benefit from inclusion of the type of sequence data used to construct each draft genome sequence. It is also reasonable to question whether the genome of the type strain is used as a template to construct the draft genomes of the other strains. If that was standard practice, conservation of the repeat copy number among strains might be an artefact. Conservation of repeat sequences to the degree shown is not a feature of the ALS family, a point of contrast between gene families that could be explored in the Discussion.

We appreciate the reviewer’s comment and agree strongly that a key limitation in gene family evolution studies like ours is the quality of the genome assembly. In the original manuscript, we took several steps to ensure the completeness and accuracy of the Hil family homologs, primarily by basing our results on the high quality RefSeq collection of assemblies, and supplementing it with two fungi-specific databases. In the revised manuscript, we performed further quality analyses to assess and correct for inaccuracy in the BLASTP hits. Because RefSeq aims to provide a stable reference, it is often slow in replacing older assemblies with newer ones based on improved technologies. We thus compared the RefSeq hits for species in which a newer, long-read based assembly had become available. The results are documented in Text S1 and in summary, while we did find examples of missing homologs and inconsistent sequences, the problems were isolated to specific species and the inconsistency pertains only to the tandem repeat regions. Regarding the specific example of within-species variable number of tandem repeats (VNTR) in C. auris Hil1-Hil4, we are confident of both the copy number and the sequence variation for two reasons. First, all C. auris strain genomes analyzed in this study were assembled de novo rather than based on a reference genome, and all were long-read based (PacBio) (Table S4). Second, empirically, we found the VNTR identified in Hil1-Hil4 agree among strains within one of the four clades of C. auris while differing between clades (Table S5). Since assembly errors are not expected to produce clade-specific patterns, we believe this is strong evidence for the VNTR identified being real.

We also appreciate the reviewer’s suggestion on discussing the conservation of the repeats as an interesting trait for a group of Hil proteins in comparison to the Als family. We now added a section in Discussion focusing on the special properties of this group of Hil proteins.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

Due to the nature of my comments, this review will not be anonymous. I will include some of the data from my laboratory to further illustrate the point about the quality of draft genome sequences, especially for gene families that contain repeated sequences. My laboratory group has spent the past several years looking at the families of cell wall genes in these species and know that the C. tropicalis genome sequence used in the current analysis is highly flawed. There is even a manuscript from several years ago that documents problems in the assembly (doi: 10.1534/g3.115.017566). There is a new PacBio sequence available that has considerably improved data for this group of genes, but still is not perfect. We designed primers and amplified the various coding regions to verify whether the IFF/HYR were correct in the draft genome sequences. For C. tropicalis, we know that 7 of the genes listed in this paper are broken (i.e. prematurely terminated) giving a false impression of their construction. The current study did not verify any gene sequences, so broken/incomplete genes are a stumbling block for developing conclusions.

We deeply appreciate the reviewer pointing out the flaws in the C. tropicalis genome. Using the PacBio sequence-based new assembly, we were able to confirm the reviewer’s comment on the sequence and annotation error in the RefSeq assembly for C. tropicalis. We listed the comparisons between the two assemblies in Table S8. Because the differences reside outside of the Hyphal_reg_CWP domain, they don’t impact our phylogenetic analyses, which are based on the effector domain alignment. To determine if this is a widespread issue affecting all genome assemblies based on older technologies, and in response to the reviewer’s criticism, we systematically checked the sequences of BLASTP hits based on the RefSeq assemblies against newer, long-read based ones when available. As detailed in Text S1 in the revised manuscript, the problems seen in C. tropicalis were not observed in four other species. While the sample size is small, we believe the issues with C. tropicalis are likely due to a combination of specific issues with the original assembly and special properties of the genome.

Similarly, the recent work from Cormack's lab features a PacBio C. glabrata sequence (doi: 10.1111/mmi.14707). The paper details how the authors focused on accurate assembly of the types of genes studied here. Sequences from the current project should be compared to the PacBio assembly to determine if they provide the same results.

We compared the sequences of the three C. glabrata Hil homologs identified in the RefSeq assembly (GCF_000002545.3) to the best BLAST hits in one of the new Cormack lab assemblies for (BG2 strain, GCA_014217725.1). Two of the three proteins showed identical sequences between the assemblies. One of them is longer in the new assembly than in the RefSeq (1861 vs 1771 aa, XP_447567.2, QHS67215.1). The main difference, however, was the number of hits recovered. Performing BLASTP searches in the new assembly recovered 13 hits vs 3 from the RefSeq assembly, of which 12 were in the subtelomeric region. For this reason, we used the new assembly as the basis for the Hil homologs in our subsequent analyses. To determine if we missed homologs in other genomes due to incomplete subtelomeric regions in the RefSeq assemblies, we repeated the BLASTP search in four other genomes (Text S1). In one of the four species, C. nivariensis, we recovered one more homolog than in the RefSeq. In all other three, we identified the same number (S. cerevisiae: 0, K. lactis: 1, C. albicans: 12), suggesting that the issues seen in C. glabrata is likely specific to this species and its RefSeq assembly.

Another part of the study that deserves additional attention or perhaps altered presentation is the idea that the Iff/Hyr N-terminal domain binds ligands. The literature on the Iff/Hyr proteins is limited. In my opinion, though, the authors of this paper could more completely present the information that is known. The paper by Uppuluri et al. is cited (doi: 10.1371/journal.ppat.1007056), but I did not see any information about their data regarding interaction of C. albicans Hyr1 with bacterial proteins mentioned in the manuscript under review. It is formally possible that the N-terminal domain of Iff/Hyr proteins does not bind a ligand. The current manuscript includes a great deal of speculation on that point, suiting it better to a Hypothesis and Theory format rather than other types of publications.

We appreciate the reviewer’s criticism and suggestion. We made two revisions based on the comments. First, we no longer refer to the Hyphal_reg_CWP domain as ligand-binding. Instead, we refer to it as the effector domain, following existing practices in the field (Lipke 2018, PMID: 29772751, de Groot et al. 2013, PMID: 23397570). Second, during the description of the predicted structure for the domain, we mentioned that it lacks an apparent binding pocket as suggested/identified in other β-solenoid proteins with carbohydrate binding abilities. Therefore, we suggest that the potential substrate and mechanism of binding by this domain remain to be determined with further experiments. We do, however, believe that there is strong evidence for the domain being involved in adhesion. A recent study (Reithofer et al. 2021) presented structural and phenotypic characterization of three Adhesin wall-like proteins (Awp1,2,3) in C. glabrata. In particular, experimental studies of CgAwp2, a Hil family protein, showed that its deletion resulted in the reversion of the hyperadhesive phenotype in one of the C. glabrata strains. Plastic was one of the substrates being evaluated, although, as the reviewer’s work pointed out, adhesion to plastics doesn’t indicate ligand binding, as it can be mediated by non-specific hydrophobic interactions (Hoyer and Cota 2016, PMID: 27014205). Nonetheless, the results presented in Reithofer et al. 2021 and other lines of evidence presented in the current work strongly supported adhesin functions of the Hil family.

Table 1 attempts to offer evidence that the Iff/Hyr N-terminal domain has adhesive function but falls short of convincing the reader. One of the example structural templates is a sugar pyrophosphorylase that seems irrelevant to the current discussion. In the column called "Function", the word adhesin is found several times, but no detail is presented. The only entry that offers an example ligand indicates that the domain binds cellulose which is not likely relevant for mammalian pathogenesis, the main focus of the work. Other functions listed include self-association and cell aggregation--using the N-terminal domain. It is formally possible that Iff/Hyr proteins drive aggregation using the N-terminal domain and beta-aggregation sequences in the repeated region. The authors should develop these ideas further. Discussion of adhesive/aggregative function related to the ALS family can be found in Hoyer and Cota, 2016 (doi: 10.3389/fmicb.2016.00280).

We appreciate the reviewer’s comments. In the revised manuscript, we removed Table 1, which was based on I-TASSER identified templates. Instead, we identified similar structures in the PDB50 database to the AlphaFold2 prediction for the Hyphal_reg_CWP domain in C. auris Hil1 using DALI (Table S3). We described the functional implications based on this list as follows:

“We identified a number of bacterial adhesins with a highly similar β-helix fold but no α-crystallin domain (Table S3), e.g., Hmw1 from H. influenzae (PDB: 2ODL), Tāpirins from C. hydrothermalis (PDB: 6N2C), TibA from enterotoxigenic E. coli (PDB: 4Q1Q) and SRRP from L. reuteri (PDB: 5NY0). For comparison, the binding region of the Serine Rich Repeat Protein 100-23 (SRRP100-23) from L. reuteri was shown in Fig. 3F (Sequeira et al. 2018). Together, these results strongly suggest that the Hyphal_reg_CWP domain in the C. auris Hil family genes mediate adhesion.”

One line of evidence that suggest the Hyphal_reg_CWP domain may have ligand-binding activity is from the L. reuteri SRRP-BR, which is one of the bacterial adhesins identified as having a highly similar β-helical structure (but missing the α-crystallin domain). In Sequeira et al. 2018 (PMID: 29507249), the authors showed via both in-vitro and in-vivo experiments that this domain “bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH”. However, the predicted structure for the Hyphal_reg_CWP domain in C. auris Hil1 and Hil7 lack a protruding, flexible loop in the β-helix, which was proposed to serve as a binding pocket for the ligand in SRRP-BR. We therefore commented in the text “Such a feature is not apparent in the predicted structure of the Hyphal_reg_CWP domain. Further studies are needed to elucidate the potential substrate for this domain and its mechanism of adhesion.”

We also appreciate the reviewer’s suggestion to discuss the potential role of the Hil proteins in mediating adhesion vs cell aggregation. We now have a section in Discussion that focuses on the potential role of the β-aggregation sequences especially in the subset of Hil proteins led by C. auris Hil1-Hil4, which have an unusually large number of such sequences. We discuss the recent literature suggesting the potential of such features mediating cell-cell aggregation.

The incredibly large number of figures that focus on the repeated sequences in the genes does not appear to include mention of the idea that these regions are frequently highly glycosylated. Knowing how much carbohydrate is added to these sequences in the mature protein would also have bearing on whether the beta-aggregation potential is realized. The Iff/Hyr proteins could stick to other things based on ligand binding (adhesion), hydrophobicity, aggregative activity, etc. Not much is really known about protein function so the conclusions are only speculative. The authors are largely accurate in presenting their conclusions as speculative, but the conclusions are not developed fully and always land on the idea that the N-terminal domain has adhesive function when that aspect clearly is not known.

We appreciate the reviewer’s comment. We have performed N- and O-glycosylataion predictions for the Hil family proteins in C. auris as a focal example and presented the results in Figure 2 of the revised manuscript. Briefly, we found that all eight proteins are predicted to be heavily O-glycosylated (Fig. 2C). N-glycosylation is rare except in Hil5 and Hil6, in regions with a low Ser/Thr content (Fig. 2C). We also deemphasized the ligand-binding ability of the effector domain and its importance in assessing the adhesin function of the Hil family proteins. At the same time, we highlighted other mechanisms as the reviewer pointed out, such as aggregative activities, in our discussion on the potential importance of the large number of β-aggregation motifs.

Another aspect of the analysis that is not mentioned is that several of the species discussed are diploid. What effect does ploidy have on the conclusions? Most draft genomes for diploid species are presented in a haploid display, so are not completely representative of the species. Additionally, some species such as C. parapsilosis are known to vary between strains in their composition of gene families, with varying numbers of loci in different isolates.

We appreciate the reviewer raising this issue. The potential impact of having diploid genomes represented as haploids is twofold. First, if the genome sequencing was performed on a diploid cell sample with some highly polymorphic regions, that would present difficulties to the assembly and could result in poorly assembled sections. Second, either because of the first issue, or because the researchers used the haploid phase of the organism for sequencing, the representative haploid genome will not be “completely representative of the species” as the reviewer suggested. The second problem is not specific to diploids – even for haploids, any single or collection of genomes would represent just a slice of the genetic diversity in the species. We did two things to look into this. First, we analyzed multiple strains in C. auris to reveal both Hil family size variation and also sequence polymorphism, particularly in the tandem repeat region. We also, as part of the quality control, compared and searched assemblies for different strains of some species when available. We agree that characterizing multiple genomes in a species is important for fully revealing the gene pool diversity and could have important consequences on our understanding of the emergence of novel yeast pathogens.

Regarding the first issue, we checked the original publications for two large-scale yeast genome sequencing projects that included 10 of the 32 species in the present study (Dujon et al. 2004, PMID: 15229592 and Butler et al. 2009, PMID: 19465905). In Dujon et al. 2004, the authors stated that haploid cells were used in cases where the species has both haploid and diploid phases. In Butler et al. 2009, the authors said in the Methods that “for highly polymorphic regions of diploid genomes, initial sequence assemblies were iteratively re-assembled in regions of high polymorphism to minimize read disagreement from the two haplotypes while maximizing coverage.”. Therefore, the potential issue of heterozygosity is likely minimal. In addition, many diploid yeasts have large regions in their genomes being homozygous, both as a result of clonal expansion and also due to loss of heterozygosity (LOH), as documented in C. albicans and other Candida species (e.g., PMID: 28080987). Nonetheless, we acknowledge that this issue is yet another challenge to having high-quality, complete genome assemblies. In the discussion, we fully acknowledge the limitation of our study by genome assemblies, and believe that ongoing improvement thanks to the development of long-read technologies will allow more in-depth studies, particularly in the subtelomeric regions and for repeat-rich sequences.

The manuscript concludes that having more genes is better, that the gene family represents diversification that must be driven by its importance to pathogenesis, without recognizing that some species evolve toward lower pathogenesis. This concept could be explored in the Discussion. …My own experience makes me wonder if the authors found any examples of species that provide an exception to the idea that having more genes is better and positively associated with pathogenesis. The parallel between IFF/HYR and ALS genes is made many times in the manuscript. Spathaspora passalidarum, a species that is not pathogenic in humans, but clearly within the phylogenetic group examined here, has 29 loci with sequence similarity to ALS genes. How many IFF/HYR genes are in S. passalidarum?

We appreciate the reviewer’s comment. We will address the two comments above together as they are related. First of all, S. passalidarum is now included in our extended BLAST search list and we identified a total of 3 homologs in this species. When compared with the related Candida/Lodderomyces clade, which includes C. albicans, the Hil family in this species is relatively small (3 vs. >10). More generally, we observe a significant correlation between the Hil family size and the species’ pathogenic potential (Figure 1B and the phylogenetic regression result in the text).

Regarding the first comment, we did identify two species that had a large Hil family (>8 based on C. auris) and yet were not known to infect humans. One of them, M. bicuspidata, has 29 Hil homologs and is interestingly a parasite for freshwater animals, such as Daphnia. The other species, K. africana, has 10 homologs and its ecology is not well described in the literature. With respect to the relationship between adhesin family and pathogenicity, we would like to make two points. First, as mentioned above, we observed a strong correlation between the Hil family size and the pathogen status, after correcting for phylogenetic relatedness, suggesting that expansion of the Hil family is a shared trait among pathogenic species. This doesn’t rule out the possibility that some species may have an expanded adhesin family, such as the example the reviewer mentioned, for reasons other than infecting a human host. Second, a key point in our work is that expansion of the adhesin family is only the first step – the crucial contribution of gene duplications to adaptation is not just in the increase in copy number, but also in providing the raw materials for selection to generate novel phenotypes. On that front, we documented the rapid divergence of the central domains both between and within species, as well as signatures of relaxed selective constraint and positive selection acting on the effector domain following gene duplications in C. auris, both of which support the above theme.

There are several current taxonomies for the species in this region of the tree. The source of the names used in this paper could be specific more completely.

We appreciate the reviewer’s comment. We now gave the complete Latin names for all species in Figure 1 and only use abbreviated names, e.g., C. auris, after the first occurrence. For species with multiple names in the literature, we followed the species name and phylogenetic placement in Shen et al. 2018 (PMID: 30415838).

The Results and Discussion sections are largely redundant. The tone of the paper is conversational, making it easy to read, but there seems little left to say in the Discussion that has not already been mentioned as the background for the various types of analyses. The authors should revise the paper to eliminate discussions of published literature from the Results and expand the Discussion to include some of the themes that have not been mentioned yet.

We appreciate the reviewer’s comment. In the revised manuscript, we have moved discussion points from the Result to the Discussion section. We also overhauled the Discussion to focus on the implications based on, but not already covered, in the Result part, including the points the reviewer suggested, e.g., the implications of the structure on adhesion mechanism.

Another point that the authors do not mention is documented recombination between IFF and ALS genes (doi: 10.3389/fmicb.2019.00781) and the effect of that process on evolution among these gene families.

We appreciate the reviewer’s comment. We now mention this and related observations in Discussion as part of the discussion on the mutational mechanisms for the evolution of the family:

“Diversification of adhesin repertoire within a strain can arise from a variety of molecular mechanisms. For example, chimeric proteins generated through recombination between Als family members or between an Als protein’s N terminal effector domain and an Hyr/Iff protein’s repeat region have been shown (Butler et al. 2009; Zhao et al. 2011; Oh et al. 2019). Some of the adhesins with highly diverged central domains may have arisen in this manner (Fig. S10).”

My reading of the work by Xu et al. 2021 (doi: 10.1111/mmi.14707) does not match the direction of its presentation in the current paper. Oh et al., 2021 (doi: 10.3389/fcimb.2021.794529) discussed that point recently, providing another point for the Discussion in the current paper.

We appreciate the reviewer’s comment and agree that our original reading of Xu et al. 2021 was incorrect. Instead of suggesting a higher mutation rates in the subtelomeric region, the authors instead suggested the evolution of the Epa family in the subtelomere was driven by Break-Induced Replication. We now update our discussion in the following way, also citing Oh et al. 2021

“Finally, as reported by (Muñoz et al. 2021), we found that the Hil family genes are preferentially located near chromosomal ends in C. auris and also in other species examined (Fig 7), similar to previous findings for the Flo and Epa families (Teunissen and Steensma 1995; De Las Peñas et al. 2003; Xu et al. 2020; Xu et al. 2021) as well as the Als genes in certain species (Oh et al. 2021). This location bias of the Hil and other adhesin families is likely a key mechanism for their dynamic expansion and sequence evolution, either via ectopic recombination (Anderson et al. 2015) or by Break-Induced Replication (Bosco and Haber 1998; Sakofsky and Malkova 2017; Xu et al. 2021). Another potential consequence of the subtelomeric location of Hil family members is that the genes may be subject to epigenetic silencing, which can be derepressed in response to stress (Ai et al. 2002). Such epigenetic regulation of the adhesin genes was found to generate cell surface heterogeneity in S. cerevisiae (Halme et al. 2004) and leads to hyperadherent phenotypes in C. glabrata (Castaño et al. 2005).”

I might have missed it, but I could not find what constitutes a BLAST-excluded sequence (Table S7). Additional explanation (or making the explanation easier to find) would help the reader.

We apologize for the inadvertent mistake of leaving out Table S7. In the revised manuscript, we include all hits from species that are part of the 322 species phylogeny in Shen et al. 2018. Thus, we removed the original Table S7.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Ideally, validation of all sequences would provide a stronger foundation for the work. However, that request is not realistic in terms of time or resources.

We agree with the reviewer and appreciate the understanding. In the revised manuscript, we performed additional analyses to evaluate the accuracy and correct the sequences of the BLASTP hits from RefSeq database by comparing them to long-read based assemblies when possible. Please see previous replies to reviewers’ comments and Text S1 for details.

• Are the data and the methods presented in such a way that they can be reproduced?

Yes, the data and methods are documented clearly and perhaps too thoroughly in many places. A considerable amount of confidence is placed in sequences that might not be accurate and tracking details down to the amino acid residue may not be reasonable in this context. A disclaimer might help--everyone probably already knows that genome sequences are not perfect but stating that the analysis is only as good as the genome sequence acknowledges that fact.

We appreciate the reviewer’s comment. In the revised manuscript, we tried to strike a balance between providing enough methodological details for the readers to assess the conclusions and yet also keeping the flow of the paper. We also accepted the reviewer’s suggestion by adding a disclaimer in the Discussion:

“we acknowledge the possibility of missing homologs in some species and having inaccurate sequences in the tandem-repeat region. We believe the expected improvements in genome assemblies due to advances in long-read sequencing technologies will be crucial for future studies of the adhesin gene family in yeasts.”

• Are the experiments adequately replicated and statistical analysis adequate?

The idea of replicates does not really apply to this analysis. I think that the species sampled are reasonable to represent the region of the phylogenetic tree on which the analysis is focused. The authors clearly documented their computational methods in an admirable way.

We appreciate the reviewer’s comment.

MINOR COMMENTS:

Figure 1 has elements that would make a nice graphical summary, but most of it should not be part of the final manuscript. For example, Panel A is repeated in Figure 2. It is not clear what Panel C means until the reader gets to Figure 2. Panel D is unnecessary. The image in Panel B is a good graphic. Endothelial adhesion is not mentioned, though. It is also debatable whether the proteins bind directly to plastic or to the body fluids that coat the plastic.

Based on this and another reviewer’s comments, we removed Figure 1 from the revised manuscript.

Compared to Figure 1, the information in Figure 3 is inconsistent. The "central domain" in Panel A is not central to anything as drawn, located at the end of the protein. The figure should be revised to be consistent with the majority of the authors' results.

We appreciate the reviewer’s suggestion. The terminologies used to describe the different parts of a typical yeast adhesin vary in the literature. In the Als family literature, central domain refers to the region after the N-terminal effector domain and before the C-terminal Ser/Thr-rich stalk domain. In the Hil family proteins, there is not a clear distinction between a “central” and a “stalk” region. In Boisramé et al. 2011 (PMID: 21841123), the authors referred to the region between the Hyphal_reg_CWP domain and the GPI-anchor as the central domain. We adopted that use. We realize that this can lead to confusion especially for Als researchers. In some other literature, e.g., Reithofer et al. 2021, this part of the protein is referred to as the B-region. But we couldn’t find wide use of that term. We decided to stay with “central domain” in this work and hope that by defining the term in Figure 2A, we would avoid any confusion within the scope of this work.

Are the low-complexity repeats mentioned in the Figure 4 legend present anywhere else in the C. auris genome or elsewhere among the species used in this study? The answer to that question may also provide evolutionary clues.

We did find one other putative GPI-anchored cell wall protein containing this ~44aa repeat unit, but with a different effector domain (GLEYA, PF10528). This protein (PIS58185.1 in C. auris B8441), appears to be a hybrid between the repeat region of C. auris Hil1 and an N-terminal effector domain of a different family. This result fits the theme of the reviewer’s work in C. albicans and C. tropicalis on the chimeric adhesins formed between the Als and Hyr/Iff families. Due to the scope of the current work, we omitted this finding from the main result.

Figure S1 legend. How was the distance to C. glabrata measured to call it equal?

The original Figure S1 was removed in the revised manuscript. A consistent set of criteria was employed in deciding which BLASTP hits to include as Hil family members.

Figure S4 could be presented better. Both diagonals have the same information. One could be emptied or could alternatively present nucleotide identity.

The original Figure S4 was removed in the revised manuscript.

Italicize the species names in Panel C of Figure S8.

The original Figure S8C is now Figure S9 and we systematically checked to make sure that species Latin names are italicized. Thanks for pointing this out.

Lines 256-257: The paper selectively samples the Iff/Hyr family and does not examine the "entire" family. Please revise.

We appreciate the reviewer’s comment. In the revised manuscript, we no longer selectively sample species. Instead, we only exclude three species that are not part of the 322-yeast species phylogeny in Shen et al. 2018 and Muñoz et al. 2018, namely Diutina rugosa, Kazachstania barnettii and Artibeus jamaicensis. Our extensive BLASTP searches also indicated that the family as defined in this work is specific to the budding yeast subphylum. We therefore believe it is accurate to describe the work as examining the entire Hil family.

• Are prior studies referenced appropriately?

I was disappointed to see that the paper does not reference my laboratory's work at all. When ALS genes are featured so strongly in a report, it seems reasonable to include something we have done over 30+ years. Our most-recent ALS paper (Oh et al., 2021 doi: 10.3389/fcimb.2021.794529) would be a reasonable source for defending the gene numbers used in Figure 2A. Other examples of our work that directly relate to concepts in this paper were mentioned above.

We sincerely apologize for our negligence. We are new to the fungal adhesin field through an accidental finding, and despite our effort to digest the relevant literature, we did unfortunately overlook the extensive work done on the Als family, much of which came from the reviewer’s lab. We have carefully read the papers suggested by the reviewer as well as others, and now have better incorporated prior foundational and insightful work into our result and discussion sections (see previous replies to the reviewer’s comments).

• Are the text and figures clear and accurate?

Suggestions for improvement are incorporated into the comments above.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Please present Methods and Results in the past tense. I still make the same mistake when I try to get my ideas on the page but proofread one more time and ensure the verb tenses are accurate.

We appreciate the reviewer’s comments and have edited the Methods and Results sections accordingly.

SIGNIFICANCE

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The paper reads as if it is presenting preliminary data for a grant proposal. Perhaps Prof. He's lab wants to seek functional evidence for the role of the Iff/Hyr proteins. The current paper provides an exhaustive background for such a pursuit. As presented, there is little functional data for these proteins, genome sequences are not 100% accurate, but the trends noted are defendable.

We appreciate the reviewer’s comments. We acknowledge that experimental studies will be needed to prove and further establish the functional importance of our findings. However, we believe our gene family evolutionary studies provided important novel insights and serve as an example for adhesin family evolution.

• Place the work in the context of the existing literature (provide references, where appropriate).

The ideas presented here are similar to those pioneered in the Butler et al. Nature paper in 2009 (doi: 10.1038/nature08064). We now have the benefit of more genome sequences so the analysis can encompass more species. C. auris adds a newer focus on part of the phylogenetic tree that was not previously emphasized. The idea of "more is better" is very simplistic, though. Parallel work for the ALS family shows complexity in gene expression levels, suggesting that some adhesins are poised to make a large contribution while others are likely to have a scant presence on the cell surface. Those concepts are not really explored in the current paper, either. See Hoyer and Cota 2016 (doi: 10.3389/fmicb.2016.00280); Oh et al. (doi: 10.3389/fmicb.2020.594531).

We appreciate the reviewer’s comments and have included a discussion about the potential diversity of the duplicated Hil family proteins, in terms of function and their regulation in the Discussion. Also see our response to the first comment of the reviewer regarding the novelty of our hypothesis and the significance of our findings.

• State what audience might be interested in and influenced by the reported findings.

Potential readers would come from the fields of fungal adhesion and pathogenesis, as well as evolutionary biology.

We appreciate the reviewer’s comments.

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

I discovered and named the ALS gene family in C. albicans and have spent 30+ years characterizing it. Most recently, my lab has focused on providing an accurate gene census and validated gene sequences for the cell wall "adhesinome" in the pathogenic Candida species. Some families are expanded and some are not. Some proteins appear only in a few species and demonstrate key roles in host-fungus interactions. There are many nuances to interpretation of what these fungi are doing from the standpoint of cell-surface adhesins and we look forward to exploring these ideas across many genomes, using validated gene sequences. We have a tremendous dataset that might make good fuel for a collaboration with Prof. He, given his enthusiasm for this area of study, as well as his outstanding expertise and perspectives on evolutionary analyses.

We sincerely thank the reviewer for the critical analysis of our manuscript and appreciate the many suggestions for improving the manuscript.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary:

The manuscript by Smoak et al. provides considerable information gleaned from analysis of HYR/IFF genes in 19 fungal species. A specific focus is on Candida auris. The main conclusion is that this gene family repeatedly expanded in divergent pathogenic Candida lineages including C. auris. Analyses focus on the sequences encoding the protein's N-terminal domain and tracts of repeated sequences that follow. The authors conclude with the hypothesis that expansion and diversification of adhesin gene families underpin fungal pathogen evolution and that the variation among adhesin-encoding genes affects adhesion and virulence within and between species. The paper is easy to read, includes clear and attractive graphics, as well as a considerable number of supplementary data files that provide thorough documentation of the sources of information and their analysis.

Major comments:

• Are the key conclusions convincing?

Overall, the authors' conclusions are supported by the information they present. However, the overall conclusion is stated as a hypothesis and that hypothesis is not particularly novel. The idea that expansion of gene families associated with pathogenesis occurs in the pathogenic species dates back at least to Butler et al. (2009; doi: 10.1038/nature08064) who first presented the genome sequences for many of the species considered here.

One key issue with a manuscript of this type is whether genome sequence data are accurate. The authors are not the first research group to take draft genome sequence data at face value and attempt to draw major conclusions from it. The accuracy of public genome data continues to improve, especially with the emergence of PacBio sequencing. Because the IFF/HYR genes contain long tracts of repeated sequences, genome assemblies from short-read data are frequently inaccurate. For example, is it reasonable to have confidence that the number of copies of a tandemly repeated sequence in a specific ORF is exactly 21 (an example taken from Table 2) when each repeat is 40+ amino acids long and highly conserved? Table S6 would benefit from inclusion of the type of sequence data used to construct each draft genome sequence. It is also reasonable to question whether the genome of the type strain is used as a template to construct the draft genomes of the other strains. If that was standard practice, conservation of the repeat copy number among strains might be an artefact. Conservation of repeat sequences to the degree shown is not a feature of the ALS family, a point of contrast between gene families that could be explored in the Discussion. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

Due to the nature of my comments, this review will not be anonymous. I will include some of the data from my laboratory to further illustrate the point about the quality of draft genome sequences, especially for gene families that contain repeated sequences. My laboratory group has spent the past several years looking at the families of cell wall genes in these species and know that the C. tropicalis genome sequence used in the current analysis is highly flawed. There is even a manuscript from several years ago that documents problems in the assembly (doi: 10.1534/g3.115.017566). There is a new PacBio sequence available that has considerably improved data for this group of genes, but still is not perfect. We designed primers and amplified the various coding regions to verify whether the IFF/HYR were correct in the draft genome sequences. For C. tropicalis, we know that 7 of the genes listed in this paper are broken (i.e. prematurely terminated) giving a false impression of their construction. The current study did not verify any gene sequences, so broken/incomplete genes are a stumbling block for developing conclusions.

Similarly, the recent work from Cormack's lab features a PacBio C. glabrata sequence (doi: 10.1111/mmi.14707). The paper details how the authors focused on accurate assembly of the types of genes studied here. Sequences from the current project should be compared to the PacBio assembly to determine if they provide the same results.

Another part of the study that deserves additional attention or perhaps altered presentation is the idea that the Iff/Hyr N-terminal domain binds ligands. The literature on the Iff/Hyr proteins is limited. In my opinion, though, the authors of this paper could more completely present the information that is known. The paper by Uppuluri et al. is cited (doi: 10.1371/journal.ppat.1007056), but I did not see any information about their data regarding interaction of C. albicans Hyr1 with bacterial proteins mentioned in the manuscript under review. It is formally possible that the N-terminal domain of Iff/Hyr proteins does not bind a ligand. The current manuscript includes a great deal of speculation on that point, suiting it better to a Hypothesis and Theory format rather than other types of publications.

Table 1 attempts to offer evidence that the Iff/Hyr N-terminal domain has adhesive function but falls short of convincing the reader. One of the example structural templates is a sugar pyrophosphorylase that seems irrelevant to the current discussion. In the column called "Function", the word adhesin is found several times, but no detail is presented. The only entry that offers an example ligand indicates that the domain binds cellulose which is not likely relevant for mammalian pathogenesis, the main focus of the work. Other functions listed include self-association and cell aggregation--using the N-terminal domain. It is formally possible that Iff/Hyr proteins drive aggregation using the N-terminal domain and beta-aggregation sequences in the repeated region. The authors should develop these ideas further. Discussion of adhesive/aggregative function related to the ALS family can be found in Hoyer and Cota, 2016 (doi: 10.3389/fmicb.2016.00280).

The incredibly large number of figures that focus on the repeated sequences in the genes does not appear to include mention of the idea that these regions are frequently highly glycosylated. Knowing how much carbohydrate is added to these sequences in the mature protein would also have bearing on whether the beta-aggregation potential is realized. The Iff/Hyr proteins could stick to other things based on ligand binding (adhesion), hydrophobicity, aggregative activity, etc. Not much is really known about protein function so the conclusions are only speculative. The authors are largely accurate in presenting their conclusions as speculative, but the conclusions are not developed fully and always land on the idea that the N-terminal domain has adhesive function when that aspect clearly is not known.

Another aspect of the analysis that is not mentioned is that several of the species discussed are diploid. What effect does ploidy have on the conclusions? Most draft genomes for diploid species are presented in a haploid display, so are not completely representative of the species. Additionally, some species such as C. parapsilosis are known to vary between strains in their composition of gene families, with varying numbers of loci in different isolates.

The manuscript concludes that having more genes is better, that the gene family represents diversification that must be driven by its importance to pathogenesis, without recognizing that some species evolve toward lower pathogenesis. This concept could be explored in the Discussion.

The Results and Discussion sections are largely redundant. The tone of the paper is conversational, making it easy to read, but there seems little left to say in the Discussion that has not already been mentioned as the background for the various types of analyses. The authors should revise the paper to eliminate discussions of published literature from the Results and expand the Discussion to include some of the themes that have not been mentioned yet.

My own experience makes me wonder if the authors found any examples of species that provide and exception to the idea that having more genes is better and positively associated with pathogenesis. The parallel between IFF/HYR and ALS genes is made many times in the manuscript. Spathaspora passalidarum, a species that is not pathogenic in humans, but clearly within the phylogenetic group examined here, has 29 loci with sequence similarity to ALS genes. How many IFF/HYR genes are in S. passalidarum?

There are several current taxonomies for the species in this region of the tree. The source of the names used in this paper could be specific more completely.

Another point that the authors do not mention is documented recombination between IFF and ALS genes (doi: 10.3389/fmicb.2019.00781) and the effect of that process on evolution among these gene families.

My reading of the work by Xu et al. 2021 (doi: 10.1111/mmi.14707) does not match the direction of its presentation in the current paper. Oh et al., 2021 (doi: 10.3389/fcimb.2021.794529) discussed that point recently, providing another point for the Discussion in the current paper.

I might have missed it, but I could not find what constitutes a BLAST-excluded sequence (Table S7). Additional explanation (or making the explanation easier to find) would help the reader. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Ideally, validation of all sequences would provide a stronger foundation for the work. However, that request is not realistic in terms of time or resources. - Are the data and the methods presented in such a way that they can be reproduced?

Yes, the data and methods are documented clearly and perhaps too thoroughly in many places. A considerable amount of confidence is placed in sequences that might not be accurate and tracking details down to the amino acid residue may not be reasonable in this context. A disclaimer might help--everyone probably already knows that genome sequences are not perfect but stating that the analysis is only as good as the genome sequence acknowledges that fact. - Are the experiments adequately replicated and statistical analysis adequate?

The idea of replicates does not really apply to this analysis. I think that the species sampled are reasonable to represent the region of the phylogenetic tree on which the analysis is focused. The authors clearly documented their computational methods in an admirable way.

Minor comments:

• Specific experimental issues that are easily addressable.

Figure 1 has elements that would make a nice graphical summary, but most of it should not be part of the final manuscript. For example, Panel A is repeated in Figure 2. It is not clear what Panel C means until the reader gets to Figure 2. Panel D is unnecessary. The image in Panel B is a good graphic. Endothelial adhesion is not mentioned, though. It is also debatable whether the proteins bind directly to plastic or to the body fluids that coat the plastic.

Compared to Figure 1, the information in Figure 3 is inconsistent. The "central domain" in Panel A is not central to anything as drawn, located at the end of the protein. The figure should be revised to be consistent with the majority of the authors' results. Structures in Panels C to E would benefit from the "through the spiral" view that is featured in Figure S9. What experimental technique was used to solve the structure in Panel E? Adding that information to the legend would be helpful to the reader. Also, the secondary structure colors seem to be reversed between the legend and domain structure. Adding the coordinates of the domains shown would help the reader to understand their location in the mature protein.

Are the low-complexity repeats mentioned in the Figure 4 legend present anywhere else in the C. auris genome or elsewhere among the species used in this study? The answer to that question may also provide evolutionary clues.

Figure S1 legend. How was the distance to C. glabrata measured to call it equal?

Figure S4 could be presented better. Both diagonals have the same information. One could be emptied or could alternatively present nucleotide identity.

Italicize the species names in Panel C of Figure S8.

Lines 256-257: The paper selectively samples the Iff/Hyr family and does not examine the "entire" family. Please revise. - Are prior studies referenced appropriately?

I was disappointed to see that the paper does not reference my laboratory's work at all. When ALS genes are featured so strongly in a report, it seems reasonable to include something we have done over 30+ years. Our most-recent ALS paper (Oh et al., 2021 doi: 10.3389/fcimb.2021.794529) would be a reasonable source for defending the gene numbers used in Figure 2A. Other examples of our work that directly relate to concepts in this paper were mentioned above. - Are the text and figures clear and accurate?

Suggestions for improvement are incorporated into the comments above. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Please present Methods and Results in the past tense. I still make the same mistake when I try to get my ideas on the page but proofread one more time and ensure the verb tenses are accurate.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The paper reads as if it is presenting preliminary data for a grant proposal. Perhaps Prof. He's lab wants to seek functional evidence for the role of the Iff/Hyr proteins. The current paper provides an exhaustive background for such a pursuit. As presented, there is little functional data for these proteins, genome sequences are not 100% accurate, but the trends noted are defendable. - Place the work in the context of the existing literature (provide references, where appropriate).

The ideas presented here are similar to those pioneered in the Butler et al. Nature paper in 2009 (doi: 10.1038/nature08064). We now have the benefit of more genome sequences so the analysis can encompass more species. C. auris adds a newer focus on part of the phylogenetic tree that was not previously emphasized. The idea of "more is better" is very simplistic, though. Parallel work for the ALS family shows complexity in gene expression levels, suggesting that some adhesins are poised to make a large contribution while others are likely to have a scant presence on the cell surface. Those concepts are not really explored in the current paper, either. See Hoyer and Cota 2016 (doi: 10.3389/fmicb.2016.00280); Oh et al. (doi: 10.3389/fmicb.2020.594531). - State what audience might be interested in and influenced by the reported findings.

Potential readers would come from the fields of fungal adhesion and pathogenesis, as well as evolutionary biology. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

I discovered and named the ALS gene family in C. albicans and have spent 30+ years characterizing it. Most recently, my lab has focused on providing an accurate gene census and validated gene sequences for the cell wall "adhesinome" in the pathogenic Candida species. Some families are expanded and some are not. Some proteins appear only in a few species and demonstrate key roles in host-fungus interactions. There are many nuances to interpretation of what these fungi are doing from the standpoint of cell-surface adhesins and we look forward to exploring these ideas across many genomes, using validated gene sequences. We have a tremendous dataset that might make good fuel for a collaboration with Prof. He, given his enthusiasm for this area of study, as well as his outstanding expertise and perspectives on evolutionary analyses.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary

Gene duplication and divergence of adhesin proteins are hypothesized to be linked with the emergence of pathogenic yeasts during evolution; however, evidence supporting this hypothesis is limited. Smoak et al. study the evolutionary history of Hil genes and show that expansion of this gene family is restricted to C. auris and other pathogenic yeasts. They identified eight paralogous Hil proteins in C. auris. All these proteins share characteristic domains of adhesin, and the structural prediction supports that their tertiary structures are adhesin-like. Evolutionary analysis of protein domains finds weak evidence of positive selection in the ligand-binding domain, and the central domain showed rapid changes in repeat copy number. However, performed tests cannot unambiguously distinguish between positive selection and relaxed selection of paralogs after gene duplication. Some alternative tests are suggested that may be able to provide more unambiguous evidence. Together with these additional tests, the detailed phylogenetic analyses of Hil genes in C. auris might be able to better support the hypothesis that the expansion and diversification of adhesin proteins could contribute to the evolution of pathogenicity in yeasts.

Major Comments

The authors present extensive analyses on the evolution of Hil genes in C. auris. There is significant merit in these analyses. However, the analyses conducted so far are incomplete, lacking proper consideration of other confounding factors. Detailed explanations of our major comments are listed below.

1. First, the authors restricted genes in the Hil family to those only containing the Hyphal_reg_CWP domain. Yet, previous work included genes containing the ligand-binding domain or the repeat domain as Hil genes. More justification is needed whether the author's choice represents the natural evolutionary history of Hil genes appropriately. For instance, are the genes only containing the ligand-binding domain monophyletic or polyphyletic? We recommend including the phylogeny of all the Hil candidate genes, to discern whether evolutionary histories of the repeat domain and ligand-binding domain are congruent. Authors can use this phylogeny as justification to focus only on the ligand-binding domain containing genes.
2. In the analysis of positive selection, the authors do not adequately control for the effect of recombination on the evolutionary histories of protein sequences, especially given that Hil genes are rich in repetitive sequences. To account for recombination, GARD, an algorithm detecting recombination, should be performed to detect any recombination breakpoints within a protein domain. If recombination did occur within a protein domain, the authors should treat the unrecombined part as a single unit and use the phylogenetic information of this part to proceed with PAML analysis, instead of using the phylogeny of the entire protein domain. The authors should consider doing GARD analysis for the ligand-binding and repeat domains. For the repeat domain, low BS values in Fig. 5C indicate recombination between repeat units. Thus, the authors should analyze each repeat unit with GARD and re-analyze dN/dS.
3. The authors concluded positive selection in the ligand-binding domain based on the branch-wise model of PAML. Yet, w values were not higher than one, and it's unclear whether the difference in selective pressures the authors claimed here is biologically significant. Overall, what the authors present so far seems to be weak evidence of positive selection but is much more consistent with variation in the degree of purifying selection or evolutionary constraint. Using the site-wise model (m7 vs. m8) in PAML would allow the authors to detect which residues of the ligand-binding domain underwent recurrent positive selection. Combining the evolutionary information of protein residues and the predicted 3D structure will provide molecular insights into the biological impact of rapidly evolving residues. This would be a significant addition and raise the significance of the study, besides providing potentially stronger evidence of positive selection.

Minor Comments

1. In Fig 1c, the figure legend should include more specific details: which adhesin proteins are shown here? Please specify species names on the species tree
2. In Fig 3E, secondary structures are labeled with the wrong colors. Sheet: purple, helix: yellow
3. What's the ligand-binding activity of the b-solenoid fold? How structurally similar are C. auris PF 11765 domains compared to C. glabrata Awp domains? This information will support the role of adhesin for the ligand-binding domain of Hil genes.
4. In lines 248-249, the authors should also consider the influence of evolutionary history. For instance, repeats within the same Hil protein appeared later in evolution, compared to Hil gene duplication, and therefore these repeats experience less time for sequence divergence.
5. Although the bioinformatic evidence of C. auris Hil genes acting as adhesins is strong, it is still worthwhile to discuss the experiments of confirming the function of adhesins.

Significance

Overall, this study is interesting to investigate the evolutionary history of a crucial virulent gene in C. auris. Such evolutionary understanding will help us identify critical molecular changes associated with the pathogenicity of an organism during evolution, providing insights into the emergence of pathogens and novel strategies to cure fungal infections. The research question is important; however, the current analyses on the positive selection are incomplete, so the conclusion is modest so far. We recommend that the authors re-do the PAML analysis with the above considerations. This work will bring more significance to the mycology field if the functional impact of rapid evolution in protein domains can be supported or inferred.

This manuscript is well-written, and the authors also did a great job specifying all the necessary details in the M&M.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The manuscript by Smoak et al., provides an analysis of the Hyr/Iff-like (Hil) genes in Candida species with a strong focus on C. auris. The authors demonstrate a repeated expansion of these genes in unique lineages of fungal species, many of which are associated with stronger clinical disease. There is evidence of selection operating on the gene family in the primary domain used for identification. These genes include a repeat just downstream of that core domain that changes frequently in copy number and composition. The location of these genes tends to cluster at chromosome ends, which may explain some aspects of their expansion. The study is entirely in silico in nature and does not include experimental data.

Major points

Altogether, many of the general findings could be convincing but there are some aspects of the analysis that need further explanation to ensure they were performed correctly.

To start, a single Hil protein from C. auris was used as bait in the query to find all Hil proteins in yeast pathogens. Would you get the same outcome if you started with a different Hil protein? What is the basis for using Hil1 as the starting point? It also doesn't make sense to me to remove species just because there are already related species in the list. This may exclude certain evolutionary trends. Furthermore, it would be helpful to know how using domain presence and the conservation of position changes the abundance of the gene family across species? (beginning of results).

A major challenge in the analysis like this one is in dealing with repetitive sequences present in amplified gene families.

• For example, testing modes of selection on non-conserved sites is fraught. It's not clear if all sites used for these tests are positionally conserved and this should be clarified. Alignments at repeat edges will need to maintain this conservation and relatively good alignments as stated in lines 241-242 are concerning that this includes sequence that does not retain this structure necessary for making predictions of selection.
• It's also unclear to me why Figure S12 is here. The parameters for this analysis should be tested ahead of building models so only one set of parameters should be necessary to run the test. The evolutionary tests within single genes and across strains is really nice!
• A major challenge for expanded gene families is rooting based on the inability to identify a strong similarity match for the full length sequence. The full alignment mentioned would certainly include significant gaps. If those gaps are removed and conserved sites only are used, does it produce the same tree? Inclusion of unalignable sequences would be expected to significantly alter the outcomes of those analysis and may produce some spurious relationships in reconciling with the species trees.
• Whether or not there are similar issues in the alignment of PF11765 need to be addressed as well. There's nothing in the methods that clarifies site selection. Figure 1A: the placement of evolved pathogenesis is a little arbitrary. It's just as feasible that a single event increased pathogenesis in the LCA of C. albicans and C. parapsilosis that was subsequently lost in L. elongisporus. These should be justified or I'd suggest removing. The assignment of Candida species here also seems incomplete. The Butler paper notes both D. hansineii and C. lusitaniae as Candida species whereas they are excluded here. It is tricky to include scaffolds in analysis of chromosomal location of the HIL genes. The break in the scaffold may be due to the assc repeats of these proteins alone or other, nearby repeats. Any statistics would be best done to include only known chromosomes or those that are strongly inferred by Munoz, 2021. This will change the display of Figure 7, but is unlikely to change the take home message.

Minor points

Line 18: "spp." Should be "spps."

Line 41: I might rephrase this as "how pathogenesis arose in yeast..."

I might use a yeast-centric example around line 40 for duplication and divergence. This could include genes for metabolism of different carbon sources in S. cerevisiae.

The Butler paper referenced on line 51 compared seven Candida species and 9 Saccharomyces species The autors state no other evolutionary analysis of adhesins has been performed but do not acknowledge this study: https://academic.oup.com/mbe/article/28/11/3127/1047032

The first paragraph of the Results could be condensed

How was the species tree in Figure 1A obtained?

Figure 2: In panel A, "DH" and "SS" are not defined. I'd be careful with use of "non-albicans Candida" in Figure 2B. This usually includes C. tropicalis and C. dubliniensis and may confuse the reader. How was the binding domain defined to extract those sequences are produce a phylogeny? In building a ML model, how were parameters chosen?

Figure 3C/D could be just one panel.

Can you relate more the fungal hit to the Hil proteins conveyed in lines 152-154?

Line 168: Should read "Hence, ..." Label proteins along the top of Figure 4 too.

Figure 5: for tests of selection, were sites conserved across the group? What does the black number at each node indicate? Dn and Ds are given as decimals. This is based on what attribute? For panel B, it is unclear what each tip denotes i.e., Hil1_tr6. Hil1 is the gene but what is "tr6"?

It's unclear why comparison of the PF11765 domain includes the MRD proteins when those aren't included in the comparison to the repeats alone. Could that skew the comparison due to unequal sample numbers or changed variation frequencies in MDR relative to the other two groups?

Table 2 doesn't add much. This section could probably be reduced to a few sentences since it's highly speculative (intraspecies variation).

Table 3 is not needed.

Figure 6 - color coding in 6A needs to be explained. I'm assuming this is a taxonomical coding.

Figure 1B is unnecessary. A Model of the protein indicating domains is sufficient here. Figure 1C needs labels for all termini, not just the pathogenic red branches. The figure doesn't provide clear association between adhesin families and the associated species. This could be omitted, especially since Flo is often associated with Saccharomyces species. Figure 1D is unnecessary.

Significance

The work here is sorely needed in expanded gene families and in fungi specifically. No analysis at this level has been performed, to the best of my knowledge, in any fungal associated gene family and certainly not in relationship to pathogenic potential. The authors do a good job in citing the foundational literature upon which their study builds in most cases (one exception is noted above). It would be of general interest to those interested in the evolution of virulence, but the analysis is tricky. This is the biggest drawback I currently have as some of the information to assess the results is missing. Expertise: gene families, evolution dynamics, human fungal pathogens

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

We would like to thank the Reviewers for their valuable comments and constructive suggestions concerning our manuscript entitled " Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes" (RC-2022-01629).

Please find below our responses to the Reviewers' questions and comments. We have revised the Manuscript following the Reviewers' suggestions. The changes in the Manuscript are indicated in blue.

Reviewer #1 (Evidence, reproducibility and clarity (Required)): ____ This manuscript by Uhlirova and colleagues identified an unwanted off-target effect in the pVALIUM10 TRiP RNAi lines that are commonly used in the fly community. The pVALIUM10 lines use long double-stranded hairpins and are useful vectors for somatic gene knock-down, hence they are widely used.

Here the authors find that any pVALIUM10 TRiP RNAi line can create the silencing of any transgenes that were cloned with the commonly used Gateway system. this is caused by targeting attB1 and attB2 sequences, which are also present in other Drosophila stocks including the transgenic flyORF collection. Hence, this is an important and useful information for the fly community that should be published quickly. All experiments are well documented and well controlled. I only have a few minor comments.

1. I recommend to mention the number of 1800 pVALIUM10 lines in Bloomington in the abstract rather than 11% to make clear that this is an important number of lines. (1800 of 13,698 lines in Bloomiongton are 13 and not 11 per cent?)

We now include the absolute number of pVALIUM10 lines in the manuscript abstract. The percentages have been corrected. Furthermore, we updated/corrected the total number of RNAi lines available from various stock centers in the Discussion, L153-L156.

The status on 23.10.2022

VDRC - 23,411 in total (12,934 GD lines; 9,674 KK lines; 803 shRNA lines)

Bloomington - 13,410 TRiP lines based on pVALIUM vectors (13,674 in total, including 264 non-pVALIUM, and 48 non-fly genes targeting lines)

NIG - 12,365 in total (5,676 TRiP lines; 7,923 NIG RNAi lines)

The authors may consider to call the 'unspecific' silencing effect an 'off-target' effect compared to intended 'on-target'. Such a nomenclature would be more consensus.

We changed the wording in the manuscript as suggested by the reviewer.

Ideally, all the imaging results in Figure 2 and 3 would be quantified. The simple 'V10' label in the Figure 3L and 3M is not the most intuitive, at least it took me a while to figure out what the authors compare.

The labeling in the charts has been changed. We now provide quantifications for the data shown in Figure 2 and 3.

Does the silencing also affect attR sequences? These are present after cassette exchange in many transgenes, most of the time not in the mRNA though, so it might not be so relevant.

A 22 nucleotide stretch of the attB2 site indeed shows a 100% match to the attL2 site. See the example alignment below (availbale in word/PDF version of the Letter). While we did not assess this possibility experimentally, attL sites would likely be susceptible to the same undesirable off-target silencing effects if present in the nascent or mature transcript.

Reviewer #1 (Significance (Required)): This is an important and useful information for the fly community that should be published quickly.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): ____ Stankovic, Csordas, and Uhlirova show that a specific subset of the TRiP RNAi lines available, namely the pVALIUM10 subset, can cause a knockdown of certain co-expressed transgenes that contain attB1 and attB2 sites. The authors demonstrate that while pVALIUM20 or Vienna KK lines for BuGZ or myc RNAi do not affect RNase H1:GFP expression, pVALIUM10 RNAi lines against BuGZ or myc significantly decrease expression of the RNAseH1:GFP transgene. The authors propose that, due to how these RNAi lines were constructed, the siRNA products could be targeting to attB1 and attB2 sites in transgenes that were made using similar methodology. To support this idea, they ubiquitously express mCherry transgenes encoding mRNAs either containing or lacking attB sites. They find that the knockdown of mCherry seen with several different pVALIUM10 RNAi lines is observed with the reporter mRNA containing attB sites, but is suppressed when the attB sites are removed from mCherry mRNA. They also find that the pVALIUM10 RNAi lines reduce the expression of the FlyORF transgene SmD3:HA.

The paper is very clearly written and the data presented is convincing.

Minor suggestions:

1. Figure 3 L+M The labels for the ubi-mcherry and ubiΔattb-mcherry are switched in these graphs (i.e. ubiΔattb-mcherry should be the one with a higher intensity in the pouch compared to the notum).

Figure 3M the labels don't match the RNAi lines used in H-K.

We corrected the labelling in the charts.

Figure 2 and 3. For the images of the transgenes, it seems as if the BuGZ RNAi line has a more drastic effect on RNaseH1 than mCherry, and vice versa for the myc RNAi lines. Did the authors notice a pattern with the decreased expression. Do some of the RNAi lines have a more consistent/severe impact, or might different transgenes be impacted to different extents?

Throughout the study and multiple experimental trials, we did not observe that the BuGZRNAi and mycRNAi silencing efficiency would depend on whether the monitored reporter was RNase H1::GFP or mCherry. What has been reproducible is the differential impact of the three tested mycRNAi lines on ubi-RNaseH1::GFP transgene. While pVALIUM10-based mycRNAi[TRiP.JF01761] reduces RNaseH1::GFP signal Valium20 mycRNAi[TRiP.HMS01538] enhances it and GD mycRNAi[GD2948] has no effect, although the number of replicates for the latter is lower compared to the other tested lines. Why Valium20 mycRNAi[TRiP.HMS01538] increases RNaseH1::GFP signal remains unclear for now.

We would like to refrain from directly quantitatively comparing the effects of phenotypically different RNAi lines on differently tagged mRNAs/proteins. As the RNAseH1::GFP fusion protein is nuclear while the mCherry is cytoplasmic, their distinct subcellular localization and/or turnover rate may give a different overall impression on the change in fluorescence intensity (Boisvert et al, 2012; Mathieson et al, 2018). Another confounding factor is the described roles of Drosophila Myc in regulating transcription, translation, and cell growth (Gallant, 2007).

Line 150 unnecessary comma after Both Line 131 knockdown should be knocked down Line 133 should be "using an additional" Figure legend 1 wing disc should be at least written out when the abbreviation (WD) is first used.

We thank the reviewer for pointing these out, the relevant corrections were performed.

Reviewer #2 (Significance (Required)):

Overall, this manuscript is an informative reminder that RNAi lines can have weaknesses that have not yet been considered, and we appreciate the authors work to inform the fly community about this specific issue. These insights are crucial for fly labs to consider when planning experiments that will use the pVALIUM10 RNAi lines in combination with other transgenesis modalities. The manuscript also provides a cautionary note for the usage of similar resources in other model organisms.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): Summary: In their manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes", Stankovic et al. describe off-target silencing of transgenes expressed from Gateway systems when expressed in transgenic RNAi drosophila lines from the VALIUM10 collection. Using fluorescence microscopy and immunostaining, the authors show that this unintended silencing is specific to VALIUM20 lines and is not observed with VALIUM20, KK or GD lines that also allow gene-specific RNAi silencing. This pleiotropic silencing effect was observed in 10 different VALIUM20 lines and affected Gateway-based transgene expressed from an ubiquitous promoter (poly-ubiquitin, ubi) or from Gal4/UAS systems. Finally, the authors identify the molecular basis of VALIUM20 pleiotropic silencing on Gateway transgenes as being due to the presence of short sequences used for PhiC31-based recombination in the Gateway and the VALIUM systems, and could lead to the production of siRNAs against PhiC31 recombination sites in VALIUM10 lines. Using Gateway transgenes lacking the recombination sites (attB1 and attB2), the authors could abrogate silencing of the transgene in VALIUM10 lines, confirming the recombination as shared targets between the Gateway and the VALIUM systems.

Major comments: - The study is well designed and the key conclusions are convincing. - However, the authors provide only fluorescence microscopy data to show decreased transgene expression. To confirm pleiotropic RNAi effect on Gateway transgenes in VALIUM10, the authors should assess silencing with another technique. For instance, expression levels of proteins from Gateway transgenes could be measured by Western blot (e.g.: by assessing protein levels of GFP or other tags present in the Gateway transgenes).

In the manuscript, we present microscopy data as this is the typical use case for fluorescent reporters. The strength of the microscopy, in contrast to Western Blot or RT-qPCR approach, is that it allows us to directly compare the impact of RNAi silencing on cells that express the dsRNA transgene (cell-autonomous) to surrounding neighbor cells. The fluorescent imaging of WDs where all cells express the reporter construct, but only a subset of cells trigger RNAi-mediated silencing, provides spatial resolution and means for normalization while minimizing artifacts that can arise during tissue processing for WB and RT-qPCR. We provide data on GFP and HA-tagged transgenes, respectively, and untagged mCherry expressed from Gateway vectors under ubiquitin or UAS regulatory sequences with the explicit reason to show that the silencing effect is independent of the type of the protein tag or the expression regulator sequence.

In addition, the claim on line 141,"These results strongly indicate that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs" , should be modified. The authors only present fluorescence microscopy data to show decreased transgene expression and do not actually provide data on siRNA expression in the pVALUM20 lines. Therefore, with the current data, the authors should only say that their results suggest that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs.

In order to substantiate their claim about pleiotropic RNAi effects from VALIUM lines on Gateway transgenes due to the production of attB1- and attB2 -siRNAs, the authors should perform an experiment to show attB1- and attB2 -siRNAs production in VALIUM10 lines and not in VALIUM20, KK or GD lines. Deep-sequencing analysis of siRNA (i.e.: miRNA-seq) from tissue expressing the corresponding RNAi transgenes would be an excellent approach to assess siRNA production in multiple samples at once. Alternatively, the authors could search published miRNA-seq datasets from VALIUM10 and other RNAi lines to assess the presence of attB1- and attB2 -siRNAs only in VALIUM10 lines. This would be free and require only a few days of data mining and analysis, if such datasets exist already. Another cheaper and faster approach (if lacking easy access to sequencing platform or bioinformatics capability) would be to perform small RNA northern blots analysis from fly tissues expressing VALIUM10 vs VALIUM20 (or KK or GD lines) and should take only a few days to do as described in doi: 10.1038/nprot.2008.67.

If such experiments or analyses cannot be performed, then the authors can only conclude that their data suggest that the unintended silencing of Gateway transgenes in VALIUM10 is likely due to the production attB1- and attB2 -siRNAs production.

We thank the reviewer for the valuable suggestions on experimental approcahes to identify the exact interfering RNAs produced by the VALIUM10-based RNAi constructs, which can be useful for controlling the specificity of knockdown of transgenes in studies using the resources mentioned in this report.

We believe the fluorescence micrographs and quantifications demonstrate the off-target silencing effects of pVALIUM10-based RNAi lines on transgenic reporters generated using the Gateway LR cloning approach. Furthermore, we provide genetic evidence that removing the attB1 and attB2 sites from the reporter construct, which is otherwise identical to the original transgene (same promoter, same position of insertion, same genetic background), is sufficient to abolish the off-target effect. We would argue that the functional genetic experiments we performed with the original and mutated reporters represent the strongest possible evidence to confirm that silencing is taking effect via the attB sites.

As we do not attempt to detect siRNA complementary to attB1/attB2 sites directly, we have changed the statements in question as per the recommendation of the reviewer.

• The current data and methods are adequately detailed and presented, and the statistical analysis adequate.

Minor comments:

• The current manuscript does not have specific experimental issues.
• Prior studies are referenced appropriately
• Overall the text and figures are clear and accurate except for the following issues with Figure 3 and its legends On lines 396, 397, 399 and 403, the authors refer to "wild-type" ubi-mCherry. This transgene directs the ubiquitous expression of an heterologous reporter gene and thus can not as "wild type". It could instead be referred to as the "original" or "unmodified" transgene.

We removed "wild-type" from the text.

Fig.3 L: the x-axis labels are wrong. Decrease in the mCherry intensity ratio is observed with the ubi-mCherry construct and not in the ubi∆attB-mCherry, where the attB sequences thought to be targeted by the pVALIUM10 have been deleted.

More space should be added between the first row of images (B-G), the second (H-L) and also the third (M-P) to avoid confusion between the labeling of the figures. Finally, to help contextualize their findings and gauging the extent of the risk of using VALIUM10 lines in RNAi screen where a Gateway transgene is involved, the authors could provide information on the overlap between the VALIUM10 collection and VALIUM20, GD and KK collections. Knowing how many genes are uniquely targeted by VALIUM10, could be helpful.

We corrected the Figure panels according Reviewer 1 and 3’s observation.

Of the TRiP pVALIUM-based RNAi stocks currently available in BDSC, 686 genes are targeted exclusively by pVALIUM10 RNAi lines. Considering KK, GD and shRNA transgenic lines from VDRC and NIG RNAi collection, 17 genes remain unique targets for pVALIUM10 lines. The graphical overview of the availbale lines is availbale in the word/PDF file of the Response to Reviewers Letter.

Reviewer #3 (Significance (Required)):

• The manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes" by Stankovic et al. is a technical study that sheds light on potential limitations of using common RNAi drosophila lines, namely the VALIUM10 collection.
• The study provides information about very specific genetic screens conditions in Drosophila, that are likely to be rare. A rapid Pubmed search with the following terms: "drosophila TRiP screen" returns only 11 citations, while a similar search with "drosophila CRISPR screen" returns 99 citations. This suggests that in vivo RNAi screen in Drosophila using TRiP RNAi collections might not be as common or powerful as CRISPR-based screens.
• The reported findings might be of interest mostly to a small group of scientists working with Drosophila melanogaster that specifically rely on VALIUM10 lines to perform in vivo RNAi screen in combination with Gateway transgene expression. This very specific combination of parameters is rare, since other RNAi fly stock collections exist (e.g.: VALIUM20, 21, KK, GD...). Furthermore, the advent of CRISPR tools that allows tissue-specific gene knock-out has led to the rapid expansion of CRISPR fly stock collections (https://doi.org/10.7554/eLife.53865). Regardless of the limited scope of the study, this kind information is still valuable, albeit to a very limited audience.
• My relevant fields of expertise for this study are : insect RNAi, RNAi of RNAi screens and drosophila genetics.

We would like to raise some points concerning the above comments.

While TRiP-screen may not be an often-used keyword combination, the use of the TRiP lines is, in fact, ubiquitous in the Drosophila community. The tissue-specific RNA interference is still commonly utilized as a rapid, first-generation screening method that can be performed in a tissue-specific manner, representing one of the key advantages of the Drosophila model. To illustrate, since the submission of our manuscript a new study published by Rylee and co-workers investigated Drosophila pseudopupil formation by screening 3971 TRiP RNAi lines (Rylee et al, 2022). In contrast, genetic screens relying on mutant alleles usually require at least one additional cross, effectively doubling the time of the experiment. In addition, tissue-specific or temporarily restricted knockdown is sometimes required in screens, as full-body loss of function is often lethal or has developmental phenotypes incompatible with assessing gene function later in life.

The use of tissue-specifically driven Cas9 with integrated gRNA-expressing vectors is indeed becoming more common. However, this technique, much like RNA interference, is not without flaws. First, this produces knockout instead of knockdown, which means it has to be induced early in order for the resulting mutation to take effect. Otherwise, the remaining mRNA/protein may prevent the development of a phenotype. Second, the Cas9 must be titrated as high Cas9 levels have adverse phenotypes (Huynh et al, 2018; Meltzer et al, 2019; Poe et al, 2019; Port et al, 2014). Third, in our personal experience, as well as literature reports (Mehravar et al, 2019; Port & Boutros, 2022), indicate that the resulting phenotype can produce mosaics in the tissue.

Although the combination of Gateway-based reporters with TRiP-RNAi lines may seem like a fringe case, there are popular reporters that could be screening targets. Potentially the most well-known is the live cell cycle indicator fly-FUCCI system (Zielke et al, 2014), which allows the analysis of the cell cycle in real-time thanks to the expression of two fluorescently tagged degrons. As FUCCI transgenes were constructed with Gateway recombination, they represent targets of the pVALIUM10 TRiP lines. We now include the fly-FUCCI system as an example in addition to 3xHA-tagged FlyORF collection in the Discussion.

REFERENCES

Boisvert FM, Ahmad Y, Gierlinski M, Charriere F, Lamont D, Scott M, Barton G, Lamond AI (2012) A quantitative spatial proteomics analysis of proteome turnover in human cells. Mol Cell Proteomics 11: M111 011429

Gallant P (2007) Control of transcription by Pontin and Reptin. Trends Cell Biol 17: 187-192

Huynh N, Zeng J, Liu W, King-Jones K (2018) A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues. G3 (Bethesda) 8: 3593-3605

Mathieson T, Franken H, Kosinski J, Kurzawa N, Zinn N, Sweetman G, Poeckel D, Ratnu VS, Schramm M, Becher I et al (2018) Systematic analysis of protein turnover in primary cells. Nature Communications 9: 689

Mehravar M, Shirazi A, Nazari M, Banan M (2019) Mosaicism in CRISPR/Cas9-mediated genome editing. Developmental Biology 445: 156-162

Meltzer H, Marom E, Alyagor I, Mayseless O, Berkun V, Segal-Gilboa N, Unger T, Luginbuhl D, Schuldiner O (2019) Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila. Nature Communications 10: 2113

Poe AR, Wang B, Sapar ML, Ji H, Li K, Onabajo T, Fazliyeva R, Gibbs M, Qiu Y, Hu Y et al (2019) Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance in Drosophila. Genetics 211: 459-472

Port F, Boutros M (2022) Tissue-Specific CRISPR-Cas9 Screening in Drosophila. In: Drosophila: Methods and Protocols, Dahmann C. (ed.) pp. 157-176. Springer US: New York, NY

Port F, Chen HM, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111: E2967-2976

Rylee J, Mahato S, Aldrich J, Bergh E, Sizemore B, Feder LE, Grega S, Helms K, Maar M, Britt SG et al (2022) A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation. G3 Genes|Genomes|Genetics: jkac257

Zielke N, Korzelius J, van Straaten M, Bender K, Schuhknecht GFP, Dutta D, Xiang J, Edgar BA (2014) Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues. Cell Rep 7: 588-598

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary:

In their manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes", Stankovic et al. describe off-target silencing of transgenes expressed from Gateway systems when expressed in transgenic RNAi drosophila lines from the VALIUM10 collection. Using fluorescence microscopy and immunostaining, the authors show that this unintended silencing is specific to VALIUM20 lines and is not observed with VALIUM20, KK or GD lines that also allow gene-specific RNAi silencing. This pleiotropic silencing effect was observed in 10 different VALIUM20 lines and affected Gateway-based transgene expressed from an ubiquitous promoter (poly-ubiquitin, ubi) or from Gal4/UAS systems. Finally, the authors identify the molecular basis of VALIUM20 pleiotropic silencing on Gateway transgenes as being due to the presence of short sequences used for PhiC31-based recombination in the Gateway and the VALIUM systems, and could lead to the production of siRNAs against PhiC31 recombination sites in VALIUM10 lines. Using Gateway transgenes lacking the recombination sites (attB1 and attB2), the authors could abrogate silencing of the transgene in VALIUM10 lines, confirming the recombination as shared targets between the Gateway and the VALIUM systems.

Major comments:

• The study is well designed and the key conclusions are convincing.
• However, the authors provide only fluorescence microscopy data to show decreased transgene expression. To confirm pleiotropic RNAi effect on Gateway transgenes in VALIUM10, the authors should assess silencing with another technique. For instance, expression levels of proteins from Gateway transgenes could be measured by Western blot (e.g.: by assessing protein levels of GFP or other tags present in the Gateway transgenes). In addition, the claim on line 141,"These results strongly indicate that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs" , should be modified. The authors only present fluorescence microscopy data to show decreased transgene expression and do not actually provide data on siRNA expression in the pVALUM20 lines. Therefore, with the current data, the authors should only say that their results suggest that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs. In order to substantiate their claim about pleiotropic RNAi effects from VALIUM lines on Gateway transgenes due to the production of attB1- and attB2 -siRNAs, the authors should perform an experiment to show attB1- and attB2 -siRNAs production in VALIUM10 lines and not in VALIUM20, KK or GD lines. Deep-sequencing analysis of siRNA (i.e.: miRNA-seq) from tissue expressing the corresponding RNAi transgenes would be an excellent approach to assess siRNA production in multiple samples at once. Alternatively, the authors could search published miRNA-seq datasets from VALIUM10 and other RNAi lines to assess the presence of attB1- and attB2 -siRNAs only in VALIUM10 lines. This would be free and require only a few days of data mining and analysis, if such datasets exist already. Another cheaper and faster approach (if lacking easy access to sequencing platform or bioinformatics capability) would be to perform small RNA northern blots analysis from fly tissues expressing VALIUM10 vs VALIUM20 (or KK or GD lines) and should take only a few days to do as described in doi: 10.1038/nprot.2008.67.<br /> If such experiments or analyses cannot be performed, then the authors can only conclude that their data suggest that the unintended silencing of Gateway transgenes in VALIUM10 is likely due to the production attB1- and attB2 -siRNAs production.
• The current data and methods are adequately detailed and presented, and the statistical analysis adequate.

Minor comments:

• The current manuscript does not have specific experimental issues.
• Prior studies are referenced appropriately
• Overall the text and figures are clear and accurate except for the following issues with Figure 3 and its legends On lines 396, 397, 399 and 403, the authors refer to "wild-type" ubi-mCherry. This transgene directs the ubiquitous expression of an heterologous reporter gene and thus can not as "wild type". It could instead be referred to as the "original" or "unmodified" transgene. Fig.3 L: the x-axis labels are wrong. Decrease in the mCherry intensity ratio is observed with the ubi-mCherry construct and not in the ubi∆attB-mCherry, where the attB sequences thought to be targeted by the pVALIUM10 have been deleted.
• More space should be added between the first row of images (B-G), the second (H-L) and also the third (M-P) to avoid confusion between the labeling of the figures. Finally, to help contextualize their findings and gauging the extent of the risk of using VALIUM10 lines in RNAi screen where a Gateway transgene is involved, the authors could provide information on the overlap between the VALIUM10 collection and VALIUM20, GD and KK collections. Knowing how many genes are uniquely targeted by VALIUM10, could be helpful.

Significance

• The manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes" by Stankovic et al. is a technical study that sheds light on potential limitations of using common RNAi drosophila lines, namely the VALIUM10 collection.
• The study provides information about very specific genetic screens conditions in Drosophila, that are likely to be rare. A rapid Pubmed search with the following terms: "drosophila TRiP screen" returns only 11 citations, while a similar search with "drosophila CRISPR screen" returns 99 citations. This suggests that in vivo RNAi screen in Drosophila using TRiP RNAi collections might not be as common or powerful as CRISPR-based screens.
• The reported findings might be of interest mostly to a small group of scientists working with Drosophila melanogaster that specifically rely on VALIUM10 lines to perform in vivo RNAi screen in combination with Gateway transgene expression. This very specific combination of parameters is rare, since other RNAi fly stock collections exist (e.g.: VALIUM20, 21, KK, GD...). Furthermore, the advent of CRISPR tools that allows tissue-specific gene knock-out has led to the rapid expansion of CRISPR fly stock collections (https://doi.org/10.7554/eLife.53865). Regardless of the limited scope of the study, this kind information is still valuable, albeit to a very limited audience.
• My relevant fields of expertise for this study are : insect RNAi, RNAi of RNAi screens and drosophila genetics.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Stankovic, Csordas, and Uhlirova show that a specific subset of the TRiP RNAi lines available, namely the pVALIUM10 subset, can cause a knockdown of certain co-expressed transgenes that contain attB1 and attB2 sites. The authors demonstrate that while pVALIUM20 or Vienna KK lines for BuGZ or myc RNAi do not affect RNase H1:GFP expression, pVALIUM10 RNAi lines against BuGZ or myc significantly decrease expression of the RNAseH1:GFP transgene. The authors propose that, due to how these RNAi lines were constructed, the siRNA products could be targeting to attB1 and attB2 sites in transgenes that were made using similar methodology. To support this idea, they ubiquitously express mCherry transgenes encoding mRNAs either containing or lacking attB sites. They find that the knockdown of mCherry seen with several different pVALIUM10 RNAi lines is observed with the reporter mRNA containing attB sites, but is suppressed when the attB sites are removed from mCherry mRNA. They also find that the pVALIUM10 RNAi lines reduce the expression of the FlyORF transgene SmD3:HA.

The paper is very clearly written and the data presented is convincing.

Minor suggestions:

1. Figure 3 L+M The labels for the ubi-mcherry and ubiΔattb-mcherry are switched in these graphs (i.e. ubiΔattb-mcherry should be the one with a higher intensity in the pouch compared to the notum).
2. Figure 3M the labels don't match the RNAi lines used in H-K.
3. Figure 2 and 3. For the images of the transgenes, it seems as if the BuGZ RNAi line has a more drastic effect on RNaseH1 than mCherry, and vice versa for the myc RNAi lines. Did the authors notice a pattern with the decreased expression. Do some of the RNAi lines have a more consistent/severe impact, or might different transgenes be impacted to different extents?
4. Line 150 unnecessary comma after Both Line 131 knockdown should be knocked down Line 133 should be "using an additional" Figure legend 1 wing disc should be at least written out when the abbreviation (WD) is first used.

Significance

Overall, this manuscript is an informative reminder that RNAi lines can have weaknesses that have not yet been considered, and we appreciate the authors work to inform the fly community about this specific issue. These insights are crucial for fly labs to consider when planning experiments that will use the pVALIUM10 RNAi lines in combination with other transgenesis modalities. The manuscript also provides a cautionary note for the usage of similar resources in other model organisms.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

This manuscript by Uhlirova and colleagues identified an unwanted off-target effect in the pVALIUM10 TRiP RNAi lines that are commonly used in the fly community. The pVALIUM10 lines use long double-stranded hairpins and are useful vectors for somatic gene knock-down, hence they are widely used.

Here the authors find that any pVALIUM10 TRiP RNAi line can create the silencing of any transgenes that were cloned with the commonly used Gateway system. this is caused by targeting attB1 and attB2 sequences, which are also present in other Drosophila stocks including the transgenic flyORF collection. Hence, this is an important and useful information for the fly community that should be published quickly. All experiments are well documented and well controlled. I only have a few minor comments.

1. I recommend to mention the number of 1800 pVALIUM10 lines in Bloomington in the abstract rather than 11% to make clear that this is an important number of lines. (1800 of 13,698 lines in Bloomiongton are 13 and not 11 per cent?)
2. The authors may consider to call the 'unspecific' silencing effect an 'off-target' effect compared to intended 'on-target'. Such a nomenclature would be more consensus.
3. Ideally, all the imaging results in Figure 2 and 3 would be quantified. The simple 'V10' label in the Figure 3L and 3M is not the most intuitive, at least it took me a while to figure out what the authors compare.
4. Does the silencing also affect attR sequences? These are present after cassette exchange in many transgenes, most of the time not in the mRNA though, so it might not be so relevant.

Significance

This is an important and useful information for the fly community that should be published quickly.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Response to reviewers

---

Reviewer #1 (Evidence, reproducibility and clarity (Required)): The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.

My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?

We thank the reviewer for this comment. However, we would like to reiterate (as stated in the manuscript) that knockout of Pfs16 in P. falciparum is transmission lethal, i.e. you do not get progression of male gametogenesis. Thus, whilst re-generation of a Pfs16 KO would be interesting in terms of comparing phenotypically with the drug treated parasites, we are not convinced it would add any further evidence of support for or against our conclusion in terms of the ability of the N-4HCS scaffold to target this protein. E.g. we could drug treat a Pfs16 KO but this would not be expected to show gametogenesis irrespective of treatment. Therefore, whilst of academic interest, we believe it is satisfactory to judge our phenotypic work based on published accounts of the Pfs16 KO without having to engage in the costly experiments to regenerate the parasite and work on it side-by-side, especially given the limited resolution it would give towards the overall goal of the work in terms of defining the effect and likely target of this drug class on parasites.

Addressing the second comment, we are happy to alter areas of the paper that may have over-stated the conclusions of the work including the abstract/introduction and discussion.

CROSS-CONSULTATION COMMENTS I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper

Reviewer #1 (Significance (Required)):

The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.

The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.

We thank the reviewer for this positive assessment of our work. Mirroring comments above, our challenge with Pfs16 knockout or mutation is that if we ablate Pfs16 function we cannot assess the effect of drug action. Definition of a mutant that would demonstrate precisely the drug mode of action would require structural resolution of drug bound to target (i.e. to identify which residues to target) – this is a major goal for our research group moving forwards, but likely many years’ work. In general, our core approach here has been one of chemo-proteomic based methods and phenotypic investigation of the novel antimalarial. Further evidence might be forthcoming from molecular genetics/structural biology, but we believe these are beyond the scope of the current work (and our available resources at present). We state future directions in the discussion and can add more to this in any revised manuscript.

This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.

The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.

Major comments: 1. The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway.

Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).

We don’t disagree with reviewer 2’s comments that ETRAMP10.3 could be an additional target. Although not traditionally related there is some similarity between these proteins and it may be that at the macroscopic level there is a structural homology between them. As stated elsewhere we are happy to tone down the assertion that Pfs16 is the only drug target candidate, leaving open the possibility of future follow up work that may yet reveal additional targets. This cannot be explored much further without extensive experimentation, which is beyond our current capacity. Given the strong phenotypic effect on gametocytes, whilst ETRAMP may be upregulated, this paper naturally focused its core attention on Pfs16 as a candidate target. We certainly subscribe to the view that absence of evidence is not evidence of absence.

Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.

We don’t necessarily agree that the evidence is not strong (three methods pointing to the same target is by many accounts solid evidence). Additionally, whilst it is true that the N-4HCS photoaffinity probes likely interact with PVM proteins in first instance, it is also worth noting that this doesn’t necessarily deduct from their likelihood to be true targets, but instead fits with the N-4HCS phenotype. We observe the compounds to inhibit microgametogenesis without any prior incubation and to retain this activity even beyond activation of microgametogenesis, specifically during the window in which the PVM remains associated with the parasite. Our phenotypic observations therefore fit with the notion that the molecules target proteins that lie within the PVM and interact with the molecules at first instance. Whilst we understand the concern that PVM proteins may be likely to be enriched given their abundance and localisation, we believe this to support our phenotypic findings.

The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.

As stated, we are happy to tone down conclusions in this direction. We also note comments above about Pfs16 disruption.

Minor comments: 4. From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.

The modification space of the compounds is explored extensively in previous work from our group, which we feel more than adequately addresses this question. See Rueda-Zubiaurre et al (2020) J Med Chem.

Line 67: references are superscript.

We can change this

Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.

We can change this

Line 116: consider removing 'interdisciplinary'.

We can change this

Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target

We can change this along with the general “tone” of the manuscript.

Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.

We can add this to the final version. The actual IC50 for this compound was 1.7uM. For the SAR study we grouped compounds with IC50 >1uM into discrete groups based on rough IC50 (>1uM, >10uM etc.) hence this fell in the intermediate group.

Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2

This can be addressed in a revised manuscript.

Line 194: was the data deposited in a database?

The proteomics data has not been deposited in a database but is accessible in the extended SI.

Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.

We can embellish the description here.

Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.

We can amend this and label each hit within the scatter plot.

Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).

We can discuss this to a limited extent for future exploration of Etramp10.3.

Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).

We can change this

Line 233 and elsewhere: define 'reversibility' (binding? activity?).

We can change this

Line 240: clarify what is in the cited paper (see major points).

We can clarify this

Line 297: We utilised in-lysate...... clunky sentence, please rephrase.

We can change this

Line 325: reference is missing the year.

We can change this

Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?

We believe that Pfs16 is functionally different at different stages of gametocyte development, this is either in terms of its presentation (e.g. perhaps due to complex formation, though this remains elusive) or the functionality of different domains, as per the effect of different truncation mutants. We can address some of these concerns in a revised manuscript.

Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.

We can change this

Line 405: remove the 'to'

We can change this

Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.

We can state this

Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.

We can make mention of this

Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?

The reviewer is astute to point this out. Limitations with access to the EM facility meant that whilst IFAs were completed for pre-activation treated samples, the post-activation EM became impossible as the EM facility closed during the COVID lockdown. Thus, we do not have a complete set here. However, we do not feel this takes away from the EM observations presented. We can clarify this incompleteness in the revised manuscript.

Line 450: is this really CytB, or was it CytD?

We did indeed used Cytochalasin B here, which whilst less potent than D does still target microfilament formation.

Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.

We can change this

Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.

We can attempt to clarify this in a revised discussion.

Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).

We can clarify this

Line 534: consider replacing 'highly plausible' with something more cautious.

We can change this

Line 550: Given this discussion how stable are N- 4HCS compounds?

We can clarify this.

Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.

We can change this

Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.

We can change this

Figure 3d: Unclear as to why a difference temperature range is displayed here.

We can clarify this

Figure 3e: Unclear % Inhibition compared to what.

We can clarify this

Figure 5G: What is the white arrow pointing to?

We can clarify this

Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.

We are not sure what the comment relates to here but we can endeavour to clarify this

Figure 6: Erythrocyte membrane colour not stated in legend.

We can change this

Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.

We can endeavour to clarify this

Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.

We can attempt to clarify this

Reviewer #2 (Significance (Required)):

Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.

Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.

Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.

Your expertise: malaria, P. falciparum, biology of apicomplexans

Reviewer #3 (Evidence, reproducibility and clarity (Required)): The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.

Major Concerns: The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.

As discussed, we are happy to tone down the conclusions about Pfs16 being an exclusive target for the N-4HCS drug class, however, we feel the reviewer is being unnecessarily negative. There are myriad papers in the literature based on singular proteomics experiments (given their cost, complexity and time -consuming nature) that then facilitate downstream experiments that support findings. We have endeavoured to be as thorough as we could in the work and believe, like others, three replicates of a massive experimental pipeline should be sufficient to make a defined conclusion – whether the additional downstream evidence we have then leaned on is supportive of this (as we judge it to be) is another matter. We agree, proteomics often suffers with low protein abundance. The complexity of growing large quantities of gametocytes is familiar to anyone who has struggled to grow these finicky parasites at a larger scale than 10-25mL dishes. Given the scales we have reached, we believe these might in fact be some of the most comprehensive proteomics studies to date!

Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.

Whilst we understand the concerns with insignificant enrichment in the competition labelling, we believe the enrichment in the presence of photoaffinity probe 2 over background (i.e. DMSO vs. probe experiments) to be of more value given the design of the experiment. The competition experiments were performed by co-treating gametocytes with photoaffinity probe 2 and parent molecule 1 prior to UV irradiation, to enable irreversible conjugation to protein target(s). However, given that both compounds, probe and parent, theoretically bind to Pfs16 at the PVM in a reversible manner (i.e. losing interaction with even gentle washing), UV irradiation is likely to favour probe-binding irrespective of competition with a marginally more potent parent molecule (in this case, parent molecule 1). This is especially true as treated parasites were very thoroughly washed after irradiation, so should the parent molecule have bound the target protein(s), these drug-target interactions were likely lost during stringent washing. The drug-target interactions with parent molecule 1 wouldn’t have been aided by UV irradiation, as the molecule lacks the functional group required for bioconjugation. So, even if parent molecule-target interactions were more abundant than probe-target interactions, interactions between parent molecule 1 were most likely lost and proteins bound by probe were enriched.

This would have been true with more potent N-4HCS derivatives such and DDD01028076 and (-)-DDD01028076 (where potency is tested in the DGFA, independent of bioconjugation), and here we opted for a structurally similar compound of similar potency to not skew competition solely based on potency.

We can embellish on this in the revised manuscript to make our conclusions from this part clear.

A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions: - Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed.

It is certainly the case that Pfs16 is abundant in gametocytes, a reason behind its early discovery. Thus it is challenging to remove it from background. We still believe the enrichment to be specific, highlighting the comparative work with Pfg377 in Figure 2. Further repetitions with more stringent washing might resolve the background, however, this is beyond our current resources to repeat.

-- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.

We don’t disagree with this though this would involve an entire re-running of the experimental workflow which is not possible.

• Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.

Again, the presence of Pfs16 in the flow-through is unsurprising, given its abundance in stage V gametocytes. The relative abundance in the eluate is not an indication that the binding and subsequent enrichment is not specific, rather this shows the compound does not necessarily bind each and every protein – which is not unexpected. The crucial conclusion to be drawn here is the concentration-dependent enrichment of Pfs16 in the eluate in the presence of probe.

• Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.

As we have noted above, it is not unsurprising that modification of the N-4HCS scaffold to yield this probe may introduce a level of irradiation-independent binding, which explains the presence of signal in the UV-independent sample.

• Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.

These experiments are extremely challenging (something that is perhaps beyond the expertise of the reviewer) and what is presented is the result of substantial optimisation. Loss of AzTB fluorescence in the gel which is subsequently analysed by western blot explains this.

• Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.

We do not concur with the reviewer here and their dismissal of what was extremely thorough and well-executed experimtns. These are not like traditional western blots and require substantial optimisation. We refer them to our previous point in reference to the UV controls. With regards to the Pfg377 variability, the experiment itself is inherently variable with such large volumes of parasites. In many cases, for example, the male:female ratio within a mature gametocyte culture can vary and this can contribute to the variability in 377 abundance between replicates.

The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies. - The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?

The individual western blot replicates can be provided in a revised manuscript if judged important.

• The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.

Given all proteins within a lysate will aggregate with thermal treatment, antibody loading controls are not feasible with these experiments. Each sample is normalised prior to thermal stabilisation (ensuring the same protein quantity is treated in both DMSO and drug, at each temperature) and any protein that is not aggregated is loaded – the nature of CETSA itself is to compare the stabilisation between DMSO and drug.

• The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?

We agree that this temperature of stabilisation is unusually high and may require further biochemical validation. Without further investigation we cannot say definitively why the melting temperature of Pfs16 is so high, but suspect its size and membrane localisation may play a role.

• The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).

Please we have already addressed this in the text – refer to line 312 and beyond.

• The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.

Whilst further replicates with different conditions might be preferable, as discussed extensively here, this would be beyond the scope of what we are able to achieve for a revision.

• The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.

We would have to agree to disagree on this point.

• Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.

We don’t agree with this statement, since the drug is binding the protein in native lysate – this may be a multi-meric complex (homo or hetero) which only exists at certain stages. As such we disagree with the reviewer that this argues against Pfs16 being the target.

In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.

We have discussed this in the manuscript and strongly feel the reviewer is being unnecessarily dismissive of a body of work that is coherent. We are happy to tone down the narrative of the paper with Pfs16 being the exclusive target. Structural homology of P. berghei Pfs16 orthologues has never been done but it would not be unprecedented if another target was functionally homologous (an idea we are currently pursuing). Stage specificity is also possible given the nature of Pfs16 (e.g. if it is in a complex). The reviewer appears fixated on a singular entity and unable to imagine a complex scenario where structure or protein-protein interactions might affect drug binding (as it does with other proteins present in complexes, e.g. proteasomal targeting drugs).

Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies.

Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).

We strongly disagree with this reviewer’s entire dismissal of an extensive body of work. In line with other reviewers comments we accept a need to tone down our conclusions, but do not consent to dropping the majority of the paper in favour of a phenotypic descriptive work.

MINOR COMMENTS The manuscript is very well-written and presented.

Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).

We can address the overstatement of conclusions in a revised manuscript.

I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).

This is simple to address – to minimise confusion for readers, we can simply state where photoaffinity labelling and bioconjugation were performed (and not refer to the latter as crosslinking).

The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?

We can simply provide the provide IC50s for compounds of greater potency. We are also happy to provide the curves but with such a large body of work already, this might be unnecessary.

Reviewer #3 (Significance (Required)):

The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.

If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.

Major Concerns:

The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.

Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.

A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions:

• Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed. -- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.
• Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.
• Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.
• Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.
• Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.

The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies.

• The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?
• The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.
• The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?
• The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).
• The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.
• The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.
• Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.

In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.

Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies. Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).

Minor comments

The manuscript is very well-written and presented.

Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).

I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).

The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?

Significance

The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.

If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.

This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.

The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.

Major comments:

1. The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway. Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).
2. Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.
3. The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.

Minor comments:

1. From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.
2. Line 67: references are superscript.
3. Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.
4. Line 116: consider removing 'interdisciplinary'.
5. Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target
6. Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.
7. Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2
8. Line 194: was the data deposited in a database?
9. Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.
10. Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.
11. Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).
12. Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).
13. Line 233 and elsewhere: define 'reversibility' (binding? activity?).
14. Line 240: clarify what is in the cited paper (see major points).
15. Line 297: We utilised in-lysate...... clunky sentence, please rephrase.
16. Line 325: reference is missing the year.
17. Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?
18. Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.
19. Line 405: remove the 'to'
20. Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.
21. Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.
22. Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?
23. Line 450: is this really CytB, or was it CytD?
24. Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.
25. Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.
26. Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).
27. Line 534: consider replacing 'highly plausible' with something more cautious.
28. Line 550: Given this discussion how stable are N- 4HCS compounds?
29. Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.
30. Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.
31. Figure 3d: Unclear as to why a difference temperature range is displayed here.
32. Figure 3e: Unclear % Inhibition compared to what.
33. Figure 5G: What is the white arrow pointing to?
34. Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.
35. Figure 6: Erythrocyte membrane colour not stated in legend.
36. Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.
37. Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.

Significance

Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.

Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.

Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.

Your expertise: malaria, P. falciparum, biology of apicomplexans

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.

My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?

Referees cross-commenting

I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper

Significance

The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.

The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: * Saha et al. characterize Drosophila egg chambers that are mutant for cup and identify an increase in the number of a specialized type of follicle cells, the border cells. They demonstrate that this increase correlates with an expanded domain of STAT activity and reduced Notch signaling in anterior follicle cells. Determining that cup is required in the germline cells, the authors postulate and provide some evidence that cup mutants prevent germline Delta from properly signaling to follicle cells. In line with this, they also show that blocking endocytosis phenocopies some aspects of cup mutants, particularly border cell numbers and Delta levels, which they monitor cytoplasmically and at the cell surface. Lastly, they demonstrate that activation of Rab11 can rescue Delta levels and border cell number in cup mutants. They conclude that a key function of Cup in the germline is to traffic Delta to signal to follicle cells, and that the endocytic processing of Delta is required for its function.*

Major comments:

• The findings of this study are interesting and novel. The authors have completed a lot of experiments and analyzed the results carefully and in great detail. Experimental design is described adequately and statistical analysis is sufficient. While the main results are largely convincing and support the conclusions, there are some weaknesses that need to be addressed.*

Response: We thank the reviewer for appreciating our work and we have tried to address concerns of the reviewers to the maximal possible extent with the hope to strengthen our claims further.

One major concern is that the vast majority of the experiments were conducted with a single homozygous allele for cup. The authors claim this was necessary because other alleles arrest oogenesis, which is understandable, but it leaves the potential problem that the allele, a P-element insertion, may affect other genes, or there may be other unidentified mutations on the mutant chromosome. The authors are able to partially rescue the border cell phenotype with overexpression of Cup and can also mimic the outcome with RNAi in the germline, which helps alleviate some of this concern, but this was only done for one set of experiments (those in figure 1). Similar experiments need to be included to demonstrate the same outcomes when cut is disrupted by other alleles/methods for at least some of the Notch/Delta analyses since this is key to the paper's conclusions.

Response____: We acknowledge the concern raised by reviewer and to address it, we evaluated different allelic combination of Cup to rule out issues with background mutation. We evaluated the Delta count, NICD and border cell numbers in a different allelic background of cup8/ cup01355. Satisfyingly we observed similar results like that observed for cup01355/ cup01355 homozygotes. This result is included as (Fig S1E-G)

In addition, we have specifically downregulated Cup function in the germline employing the RNAi approach and validated the non-cell autonomous effect of Cup function in border cell fate specification. This result is included in (Fig 1M-O)

A second concern is that some evidence is circumstantial or indirect. Specifically, the authors argue that the effect of Cut is due to trafficking of Delta, but do not consider the possibility that Delta could be more directly regulated or that other factors may be relevant. Border cell specification is rescued by increasing recycling in cup mutants, but this could be due to recycling of more factors besides Delta. To address this more directly, the authors should overexpress Delta in the germline of cut mutants. It is possible the disruption of Delta in cut mutants is due to changes in Delta protein stability/levels, so the experiment may also clarify this issue. If this is the case, it may be that hypomorphic Delta mutants would have a defect on border cell number, which could be examined separately. If Delta levels are low, endocytosis and recycling increases may also rescue cut mutants indirectly, but the conclusion about what Cut regulates may differ.

Response: As per the suggestion of the reviewer, we did attempt to over express Delta in the germline of cup mutants egg chambers. Unfortunately, we couldn’t record any Delta overexpression as the available vector (UASt- Delta) can drive stable expression only in the somatic cells but not in the germline cells. However, to check out the possibility if Delta was being directly regulated by Cup, we compared the levels of proteins between wild type and Cup mutant egg chambers (Figure 4E-G). Unlike our expectation we didn’t observe any significant differences in the levels of Delta in Cup compared to the control. This kind of supports our belief that Cup may not be directly regulating the levels of Delta in the germline.

Another concern is that Cup's main role is a confusing since it regulates many things, including cytoskeleton and cytoskeleton is necessary for general health and vesicle trafficking in the egg chamber - how do the authors think Rab11 upregulation is overcoming these defects?

Response: We appreciate the reviewer for raising this concern as it kind of intrigued us to examine if the overexpression Rab11CA was rescuing the cytoskeleton too. Interestingly, we observed that Rab11CA overexpression restored the actin filament in Cup mutant germline(figure S6H-K). This result is in line with report that Rab11 effector Nuf can modulate actin polymerization (Jian Cao et al.,2008).

Rab11CA rescues Delta levels almost completely in cut mutants but only partially rescues Notch activation, suggesting there are other problems in these egg chambers that could contribute to the defects. While exploring possible other factors is beyond the scope of this work, the authors may want to acknowledge this issue.

Response: We do agree with the reviewer that we only observe partial rescue of the NRE GFP with Rab11CA, it suggests that Cup can affect different aspect of egg chamber development independent of Rab11 function.

Minor comments:

It would help the presentation of the paper to introduce Notch/Delta signaling during oogenesis in the introduction. More introduction and clarity about the number of polar cells at early stages and their role in the border cell cluster may also be useful to the reader.

Response: We have modified the introduction to highlight the role of Notch/ Delta signaling in early oogenesis.

It is notable that the primary phenotype of a change in border cell numbers is quite subtle, often only affecting 1-2 cells, and the variation in different genotypes and experiments is sometimes also that large. The authors do a good job of being careful to count the cells at a specific developmental time and do appropriate statistical tests within an experiments. Still, it difficult to be sure that the effects are due to the gene being manipulated specifically or the genetic background. Related to this, a few issues should be addressed. Notably, at earlier stages, Notch signaling impacts cell division, so some of the phenotypes might be explained by there being more total cells in the domain instead of more signaling. The authors show Cut is in the same domain and pH3 is similar, but they didn’t seem assess overall numbers.

Response: As per the suggestion of the reviewer, we assessed the total number of follicle cell nuclei in stage 8 egg chambers. This analysis was done each confocal z slide of the egg chamber taking care that each nuclei (DAPI) was counted only once. Satisfyingly we didn’t observe any significant difference in the number of follicle cell nuclei between wild type and cup mutant egg chambers supporting our earlier claims with pH3 and Cut antibody that cell proliferation is not responsible for the excessive border cell fate in Cup mutants. This result in included in (Fig S2O-Q)

Secondly, for the stat suppression of cut (figure 2L), the authors need to show the stat-/+ control for comparison to make a conclusion about suppression versus additive effects.

Response: As per the suggestion of the reviewer, we have included the data for statp1681/+ control in figure 2L.

In addition, prior work (Wang et al 2007) expressed DN Kuz in border cells and did not see a change in specification, unlike what is claimed here. In the experiment in question, the control has lower than normal numbers of border cells and the DN Kuz has a number more typical of the controls in other experiments- so this is a concern that there is something else in the genetic background influencing the numbers. Other controls could help make this case, but ultimately this result is probably not necessary for the main argument. Thus the authors might consider leaving it out the Kuz analysis or perhaps can comment on the discrepancy with prior published results.

Response: We have removed the data on Kuzbanian and have added data that suggests that Notch activation in the follicle cells downstream of Cup facilitates specification of appropriate number of migratory border cells (Fig 3K-N).

Can the authors comment on why the volume of the border cell cluster increases more dramatically (>2x) than the number of cells (30% more)? * Does the increase in border cell number change the migratory capacity? That is, do the clusters in cut mutant egg chambers migrate normally while the egg chamber looks okay?*

Response: We believe that dramatic increase in the volume of the border cell cluster I (>2x) than the number of cells (30% more) is due the loose arrangement of the cells in the border cell cluster. Interestingly, the cup mutant border cell clusters do exhibit migration defect that we are examine as part of separate study.

Several of the figure legend titles state conclusions that are over interpretations of the data shown:

- Figure 3 legend is overstated- these experiments do not assay STAT activity, only border cell number, so the title can be simplified to say that.

Response: We have modified the Figure legend in line with the data presented.

- For figure 4, both cytoskeleton and Delta are shown to be disrupted in cup mutants, but they are not directly linked, eg, the experiments do not show a change in Delta in cytoskeletal mutants alone. While it is interesting that cup mutants have disrupted cytoskeleton, ultimately this result is not well connected to the main issue of Notch/Delta signaling; in fact, it becomes confusing how anything can be trafficked to the cell surface if there is poor cytoskeletal organization. Since the authors favor the hypothesis that the cytoskeleton is not the key to the border cell specification difference, they may want to move this result out of figure 4.

__Response: __We have included the data that suggests that cytoskeleton organization is critical for Delta trafficking. Specifically we demonstrate that treatment of egg chambers with Cytochalasin D exhibits accumulation of Delta in the nurse cell cytoplasm (Fig S5D-F).

- The Figure 5 legend is also overstated- these experiments show that Delta is higher in cup mutants and endocytosis mutants AND that endocytosis (of something) is required in the germline for border cell number- but these results are not linked in this figure. More evidence for this connection does come later in figure 6. * Some figure legends are quite brief and could benefit from a little more detail on what is being shown*.

__Response: __We have modified the title of the Figure legends with respect to data presented.

Figure layout could be improved by keeping images consistent sizes and making sure graph text is large enough to read easily. Figures in general could be streamlined by having negative results and less pertinent results in supplemental data.

Response: We have reorganized the figures and worked on the graph text for easy read.

Not all papers cited in the text are in the reference list.

Responses: We have modified the title of the figure legends and cross checked our reference list with the papers mentioned in the main text.

CROSS-CONSULTATION COMMENTS

I generally agree with the other reviewers that there are concerns with the precise function of cup in this context, and that some revision is needed, including editing of the writing. In response to reviewer 2, prior published studies only detected Cup in germline, but it is possible that it is expressed in follicle cells at a low level. The mutant clonal experiment in follicle cells that the authors did had no effect on border cells, so that provides some evidence the role is non-autonomous. I agree with reviewer 2's concern that the authors overstate the connection between cup and Delta and border cells based on their data and need a few more experiments to tie things together. I understand reviewer 3's concerns that the experimental effects on border cell numbers are very small and variable- I listed this as a minor concern, though, since this number is mainly being used as a read-out for STAT signaling levels and the data were extensively quantified and statistically tested.

Reviewer #1 (Significance (Required)):

My expertise is in cell migration, developmental biology, and Drosophila genetics. This paper will be of broad interest in these fields as it incorporates aspects of each in its characterization of a new regulatory mechanism to induce a motile cell population non-cell-autonomously, which is an exciting finding. Specifically, the work increases our understanding of the intersection between Notch and Jak/STAT signaling, which many researchers study - these were both known to be involved in border cell specification. The study provides more detailed characterization of the signaling and specification process in general, and makes significant advances in understanding how Delta signals are produced and presented from germline cells to receiving cells in the soma. Cut has not been previously implicated in these signaling pathways, so that is also novel, although its precise mechanistic role here is still somewhat unclear.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this manuscript, Saha et al. made a detailed description of the role of the mRNA binding protein Cup in specifying the number of Border Cells (BC) during Drosophila melanogaster oogenesis. First of all, they show that females homozygote for a hypomorph allele of cup have higher number of BCs compared to Wild Type (WT) females. They present a series of experiments that points towards the phenotype being due to a specific role of cup in the nurse cells that non-cell autonomously regulates BC specification. Also, they show that this phenotype is the result of an increase in the levels of JAK/STAT signalling in the BC, a major determinant of BC

fate. In addition, they show that cup mutant egg chambers exhibit a downregulation of

the Notch (N) pathway function in the BCs and that over-activating Notch results in the rescue of the number of BCs. Moreover, the authors present data on the effect of cup in Delta (Dl) trafficking in the nurse cells: They found that cup mutant egg chambers show increased number of Dl puncta within the cytoplasm of the nurse cells, but reduced numbers in the nurse cell-Anterior Follicle Cell (AFC) boundary as a result of defective Dl endocytosis. Finally, they were able to rescue the Dl trafficking phenotype, as well as the number of BC by overexpressing an active form of Rab11.

Mayor points:

In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:

o The authors show that cup01355 mutant egg chambers have a phenotype in Dl trafficking. Although they analysed in detail the effects on Dl in the nurse cells, their images show that there might be a defect in Dl levels/trafficking in the Follicle Cells (FCs) as well (Fig5A-B). It has been shown that Dl mut FCs have reduced levels of Notch activity due to reduced lateral inhibition (Poulton et al., 2011), so there is a possibility that the reduced levels of Notch activity in the cup01355 egg chambers might be due, partially, to defects in Dl trafficking/levels in the FCs, rather than in the nurse cells. o The authors tested the role of the Notch pathway in the cup mutant phenotypes by measuring the number of NICD puncta in the signal receiving cells as proxy for Notch activity (Fig4). Although I understand the rationale, I am not convinced that they can completely rule out that the changes in NICD puncta number in FCs is not due to some effect of cup LOF on Notch trafficking in these cells.

o In figure 6, the authors show that expression of a constitutively active form of Rab11 specifically in the nurse cells restores the BC number to that of the WT. However, the levels of Dl particles and, especially the levels of NRE-GFP expression, remains slightly lower than in the WT conditions.

Response: We do agree with the reviewer that we only observe partial rescue of the NRE GFP with Rab11CA, it suggests that Cup can affect different aspect of egg chamber development independent of Rab11 function. This has been acknowledged in the main text and it now reads as “We did note that irrespective of partial rescue in the levels of NRE-GFP and Delta puncta count, a complete reversion to wild type border cell numbers was observed when Rab11CA was overexpressed in the cup mutant germline. This may suggest either that border cell fate specification is quite robust beyond a certain base level of signaling or Cup may affect other aspects of egg chamber development independent of Rab11 function.”

One of the main conclusions of this study is that cup regulates BC specification through a non-cell autonomous mechanism that involves communication between nurse cells and AFCs. For that reason, I think in order to conclusively say that, the authors need to try to remove the function of cup specifically in the nurse cells. They mentioned they have tried different ways of doing this unsuccessfully, but do not specify how they have tried. I suggest using the cup-RNAi line combined with a nurse cell specific Gal4 and a ubiquitous gal80ts line (tub-Gal80ts), if they have not try this. I do not expect the authors to repeat all the experiments with this condition, but at least they should test the main findings i.e. number of BCs, JAK/STAT overactivation and Notch attenuation.

Response: To further support the non-autonomous role of Cup in border cell fate specification, we down regulated Cup function in germline nurse cells employing Mat-alpha GAL4 and Cup RNAi. Since Mat-alpha GAL4 driver has weak expression in the nurse cells of early stage chambers, it enabled us to evaluate Cup function during mid oogenesis. Consistent with our expectation, we observed higher number number of border cells in the migratory cluster compared to the control supporting our conclusion that germline Cup modulates the number of adjacent anterior follicle cells that acquire migratory border cell fate. The above results are included in (Fig 1M-O). In addition over expression if Cup cDNA in the anterior follicle cells failed to the rescue the excessive border cells observed in the Cup mutant egg chambers supporting the germline role of Cup further. This result in included in (Fig S1L-O).

• The authors have shown in Figure 3 that there is a decrease in Notch signalling in the AFCs in cup01355 egg chambers. In order to test that the BC number phenotype observe in this condition is due to that effect on Notch signalling they have done a rescue experiment using the antimorphic Notch allele Nax-16. Since in this condition all cells (nurse cells and FCs) have increased levels of Notch, they cannot conclusively say that the increase in Notch function in the FCs rescues the cup

phenotype. If they want to show that the function of Notch is specifically needed in the FCs, they should over-activate Notch exclusively in the AFCs. For instance, they could express a constitutively active form of Notch, such as UAS-NICD (Go et al., 1998) or UAS-NDECD (Fortini et al., 1993), specifically in the AFCs. Otherwise, they should re-write the text since they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype.

Response: Following the suggestion of the reviewer, we attempted over expression of NICD in the follicle using driver slbo-GAL4 in the cup mutant background. Gratifyingly, we observed rescue in the border cell fate of Cup mutant egg chambers. However, we didn’t observe any rescue in the morphology of nurse cell nuclei of Cup mutants. This supports our conclusion that increase in Notch function in the FCs rescues the cup phenotype with respect to the border cell fate only. (Fig 3K-N).

• The authors had made a great effort to prove that proper Delta endocytosis in the nurse cells is essential for adequate Notch signalling in the AFCs and right number of BCs recruitment. Specifically:

o They checked the consequences on Dl trafficking of down-regulation of rab5 or auxilin, but they did not test the effect in BC numbers * o They show that downregulating the function of shi affects the number of BCs, but did not show the effect of this condition in Dl trafficking. * Consequently, they cannot conclusively say that effects on trafficking of Dl affect number of BCs, since they haven't really tested both effects on the same background. I think that for simplification, they should test both, effects on Dl trafficking and number of BCs in one of those genetic backgrounds and leave the other two for supplementary material. Alternatively, they should re-write their conclusion for this section.

Response: As Rab11GTPase over expression rescued the excessive border cell fate in the cup mutants, to test the specificity we downregulated Rab11 function in the germline itself to check Delta trafficking and border cell fate specification. We employed a late expressing GAL4 driver in the germline and observed that down regulation of Rab11 function resulted in more number of follicle cell acquiring border cell fate and decrease in the number of Delta puncta at the interface of Anterior follicle cells and nurse cells. This phenotype is reminiscent of the Cup mutants suggesting that perturbing the recycling component of endocytosis perse affects border cell fate and Delta trafficking. This result in included in (Fig 6D-I)

• Their results clearly show that Dl accumulates in puncta, suggesting that there might be a defect in Dl trafficking, and although their rescue experiments point towards an scenario where Rab11-dependent Dl recycling is being affected, I think there are some weak points on their arguments. The fact that Rab11-KD does not generally affect Notch signalling in the FCs, as shown in (Windler & Bilder, 2010) argues against their conclusion that the effect of cup in nurse cells on Rab11 function is responsible for the defects in Dl trafficking and, subsequently, on Notch activity in AFCs. An alternative explanation is that Rab11 overactivation in the Cup mutant background compensates for a different defect on Dl trafficking, for example, Rab4-dependent recycling pathway. Another possibility is that AFCs could be specially sensitive to changes in Rab11-dependent Dl trafficking defects in the nurse cells. To distinguish between these two possibilities, they should perform some of the following experiments:
• o First of all, there are a number of endosome markers that can be used to check in which step of the endocytic route Dl is being accumulated, including (but not limited to) anti-Rab11 antibody, anti-Rab5, anti-Rab7, tub-Rab4-mcherry. They should do co-localization experiments with Dl and endosomal markers.*
• o Also, they could check what happens to the number of BCs and Dl trafficking when Rab11 function is blocked in the nurse cells, in a similar way to what they did with Auxillin, Rab5 and Shi. They could use some of the tools described in (Satoh et al., 2005)*

Response: We have perturbed Rab11 function during mid oogenesis which is quite distant from early stage egg chambers examined by Windler & Bilder. We observed that down regulation of Rab11 activity in germline affects both border cell fate in the AFCs and Delta trafficking in the germline itself. Protein Trap analysis of Rab11 in wild type and Cup mutant background suggests Rab11 is enriched in the trans-golgi network where the activity of Rab11 is modulated through nucleotide exchange. Over all our results suggest that Rab11 activity is diminished in the cup01355 egg chambers and thus stimulating the recycling endocytosis restores Notch signalling in the AFCs, limiting JAK-STAT activation and restricting BC cell fate specification.

• The authors final model is one in which cup in the nurse cells regulates Rab11 function to ultimately control JAK/STAT signalling in the AFCs. However, they have not looked at the status of JAK/STAT signalling in their Rab11-CA rescue experiments. I think this experiment will really round-up their work.* Response: The border cell fate is linked to activation of JAK-STAT signaling in the anterior follicle cells. As we have already exhausted the STAT antibody, it will difficult to access the levels of STAT perse.

Minor points:

• The authors tested if the extra BC phenotype observed in the cup mutant egg chambers is due to defects in FCs endoreplication. I have two questions related to this section.*

• o First of all, I do not understand the rationale behind this idea that defects in FCs endoreplication would result in extra BCs. Please explain and add any relevant references.*

• o Secondly, they say that they used Cut and Phospho-Histone3 as endoreplication markers. I believe that what they mean is that the absent of these two markers indicates that FCs have exit the cell cycle and enter the endocycle (Sun & Deng, 2005), however they are not markers of endoreplication. Please, re-write to make this clear.*

Response: The follicle cell exhibits a switch from mitotic to endocycle phase at a particular stage of oogenesis (Sun & Deng’ 2005). Our premise is that incase this switch is delayed, will the extra proliferation can account for the excessive border cell fate? In this context we have modified the text to render clarity to this section.

• The authors tested whether the levels of Notch activity were altered in the cup mutant egg chambers. For that, they used an NRE-GFP construct that shows a clear reduction in the levels of Notch activity in the AFCs. They also used the number of NICD and NECD puncta in signal receiving and sending cells respectively, as proxy of Notch activity. Although I understand the rationale, there are other explanations for this phenotype as discussed above. Thus, if they want to have an alternative way of showing the dampening of Notch signalling, they could use the levels of expression of well characterised targets of Notch in the FCs, such us hnt and E(spl)mb-CD2 or E(spl)m7. Response: We believe that our new set of data with NICD over expression (in the AFCs) rescuing border cell fate in Cup mutants coupled with NRE-GFP, NICD, NECD data now lends stronger support to our claim that Notch signaling in the follicle cells is indeed downstream of Cup function in developing egg chambers.

• In M&M the authors explain that NRE-GFP levels were expressed in Fold change. However, in figure 3C the units of the graph are Fluorescence Intensity in a.u. Please,*

check this small inconsistency

Response: We have modified this as per reviewer’s suggestion.

• In figure 4, they show the quantification of tubulin fibres within the nurse cells, however they are missing a similar analysis of Phalloidin (Pha) fibres/levels. I think this experiment and figure will be more complete if the authors added such a quantification of the effects of cup LOF in Pha distribution. Also, the authors do not show the single Pha channel in Fig4C, which would greatly helped to appreciate the differences between the WT and Cup LOF nurse cells. I suggest modifying the figure to better show the changes in Pha distribution. Response: We have modified the figure and included quantitation of actin fibre length in Supplementary figure 6H- K.

• In figure 4F-G the authors are showing the general effect of cup LOF in Delta distribution. They indicate with yellow arrowheads the cytoplasmic Dl puncta accumulation in the nurse cells, however it is almost impossible to see such puncta with that level of magnification/resolution. I suggest removing the arrowheads, since the figure 4H-I shows the same puncta more clearly. Response: We have modified the figure to render clarity

• In the Dl trafficking experiments (Fig4 H-I,K,L and Fig5A-C), the authors measured the number of puncta in the anterior nurse cell-follicle cell junction. In order to do those types of quantifications they need to be able to tell the cell boundaries that separate FCs from the nurse cells. Please, clarify the criteria for determining if the puncta are within the FCs or the underlying nurse cells. Response: Delta, NICD, NECD proteins marks the apical surface of the follicle cells. We used this as a reference to segregate nurse cell puncta with respect to follicle cells. This has been elaborated in the Material & Method section.

• In figure 6C-D the authors show example images of egg chambers expressing Rab11-CA-YFP using the germline specific nos-Gal4. However, in the images it looks like the YFP signal is coming from the surrounding stretched FCs. Please check that these are the right images or explain the inconsistency.

Response: We have crosschecked the images and the YFP signaling is from nurse cell periphery which gives the wrong impression that it is from stretched follicle cells.

• In figures 1R, 2L, 3Q, 6I, 6M, the authors should show the results of the statistical analysis between all the conditions tested. I think that this is crucial to be able to tell whether some of the rescues are complete or only partial. *Responses: To avoid cramming the Figures, we have including some of the p values in the Figure legends. *

• Line 174: should say "mutant egg chambers".*
• Line 281: There is a reference that is missing from reference list: Liu et al., 2010;*
• Line 292: The reference for the NRE-GFP construct is not the correct one, since that references to a review article. Please, add the correct reference.*
• In line 462 of the manuscript you have a reference that is missing from your reference list.*
• In line 394 the authors say: "protein, it's enrichment in the cytoplasmic fraction of the cup mutant egg chambers", but I think that they meant mutant nurse cells.*

Response: We have modified the text as per the all the suggestions above Reviewer #2 (Significance (Required)):

The BC migration is an excellent model to study collective cell migration and how epithelial cells can acquire migratory behaviours. After years of study, there is good understanding of the signals and genetic circuits that regulate BCs specification and migration (Montell et al., 2012), but there are not many studies, to my knowledge, that describe a role of nurse cells in specifying or guiding the migration of these cells. Thus, this study by Saha and colleagues is one of the first studies that show a role for nurse cells in specifying the number of BCs.

My field of expertise is in cell-cell communication through different pathways, including Notch and Integrin signalling. I have studied the role of endocytosis in regulating Notch signalling in various contexts, including follicular epithelium in Drosophila ovaries.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This manuscript describes an investigation into the signaling that induces the differentiation of follicle cells into border cells in the Drosophila ovary. Previous studies have established the border cells as an informative model for studying how epithelial cells delaminate and undergo collective cell migration, and have identified the JAK-STAT and Notch pathways as important regulators of the process. Here, the authors performed a forward genetic screen and identified cup as another gene that is involved in the regulation of border cell differentiation. Their findings are consistent with a model in which cup is required in germ cells for the endocytosis of the Notch ligand, Delta. In cup mutants, impaired trafficking of Delta leads to decreased Notch signaling in follicle cells, which allows for increased JAK-STAT expression in follicle cells and an increase in the number of follicle cells that differentiate into border cells. Overall, the approach is thorough and the phenotypes are clear and well-described. The quantification of phenotype penetrance and of aspects of the images, such as pixel intensities and the number of particles in a region is a strength of the paper. The use of multiple independent methods to test key points is another strength. However, there are several concerns that should be addressed before the paper is considered for publication:

• 1. The central phenotype that this paper is based on is a difference in the number of border cells per cluster in wildtype and mutant genotypes. However, this phenotype is fairly subtle in some cases (e.g. in Fig. 2L, it varies by only about 10% between control and mutant) and it is somewhat variable. For example, the number of cells in border cell clusters of the controls range from 4.49 in Fig. 3M to 6.41 in Fig. 1F. Considering that the mutant values fall within this range in some cases (e.g. 5.98 in Fig 3M) and the difference between the means from control and mutant genotypes is often less than two, the significance of this phenotype is unclear. How does this compare to other mutants that have been described to affect border cell specification? Are there any consequences for the differentiation of the follicle or the function of the egg caused by this defect?*

Response: We are using the border cell number as readout for the output of JAK-STAT signaling. Though the difference in numbers may appear to be subtle, we believe our data clearly demonstrates that Cup non cell autonomously regulates border cell fate by modulating Notch signaling in the follicle cells*. *

• Wang, et al. (PMID 17010965) have described previously that Notch signaling, and*

Kuzbanian specifically, is required for border cell migration. The authors should cite this paper and discuss their findings in light of this study. For example, if Notch signaling is impaired in cup mutants, is border cell migration also impaired? Likewise, the citation of the Assa-Kunik, 2007 study as evidence that Notch and JAK-STAT signaling act antagonistically (Line 286) is a bit of an oversimplification. While that study does show that Notch and JAK-STAT act antagonistically at earlier stages of follicle development, Fig. 6 of that paper shows that a Notch reporter and a JAK-STAT reporter are both expressed concomitantly in border cells of a Stage 10 follicle and in the anterior follicle cells of what looks like a Stage ~8 follicle. The authors should discuss the apparent contradiction between their findings and this study.

Response: We provide genetic evidence to support our claims that Cup in the germline modulates Notch activation in the anterior follicle cells thus limiting border cell fate specification to a few. The overlap in the expression of Notch reporter m7-lacz and STAT in the follicle cells and border cells is interesting and will need further investigation in real time to decipher any comparison between the two studies.

• Lastly, the manuscript contains many grammatical errors, incomplete sentences, improper punctuation and spacing, and informal writing, such as the use of contractions. It should be thoroughly edited for content and clarity.*

Response: We have tried to edit the manuscript with the aim to improve on the language, grammar and punctuations.

Reviewer #3 (Significance (Required)):

Although the identification of cup as a contributor to the regulation of border cell differentiation is novel, the other main regulators investigated in this study, including Notch and JAK-STAT signaling, have been identified previously. The role of cup in this context seems to be to fine tune Notch signaling and it seems to play a relatively minor role in the process of border cell specification. In addition, the conclusions of this paper are not well-integrated into the existing literature on Notch and JAK-STAT signaling in border cells, and the discussion about the broader implications of this study for the understanding of Notch signaling was not well-developed. However, the careful documentation and quantification of the phenotypes reported in this study adds rigor and allows for firm conclusions. For these reasons, this study may have a lasting but perhaps somewhat incremental impact on the study of border cell migration in the Drosophila ovary.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

This manuscript describes an investigation into the signaling that induces the differentiation of follicle cells into border cells in the Drosophila ovary. Previous studies have established the border cells as an informative model for studying how epithelial cells delaminate and undergo collective cell migration, and have identified the JAK-STAT and Notch pathways as important regulators of the process. Here, the authors performed a forward genetic screen and identified cup as another gene that is involved in the regulation of border cell differentiation. Their findings are consistent with a model in which cup is required in germ cells for the endocytosis of the Notch ligand, Delta. In cup mutants, impaired trafficking of Delta leads to decreased Notch signaling in follicle cells, which allows for increased JAK-STAT expression in follicle cells and an increase in the number of follicle cells that differentiate into border cells. Overall, the approach is thorough and the phenotypes are clear and well-described. The quantification of phenotype penetrance and of aspects of the images, such as pixel intensities and the number of particles in a region is a strength of the paper. The use of multiple independent methods to test key points is another strength. However, there are several concerns that should be addressed before the paper is considered for publication:

1. The central phenotype that this paper is based on is a difference in the number of border cells per cluster in wildtype and mutant genotypes. However, this phenotype is fairly subtle in some cases (e.g. in Fig. 2L, it varies by only about 10% between control and mutant) and it is somewhat variable. For example, the number of cells in border cell clusters of the controls range from 4.49 in Fig. 3M to 6.41 in Fig. 1F. Considering that the mutant values fall within this range in some cases (e.g. 5.98 in Fig 3M) and the difference between the means from control and mutant genotypes is often less than two, the significance of this phenotype is unclear. How does this compare to other mutants that have been described to affect border cell specification? Are there any consequences for the differentiation of the follicle or the function of the egg caused by this defect?
2. Wang, et al. (PMID 17010965) have described previously that Notch signaling, and Kuzbanian specifically, is required for border cell migration. The authors should cite this paper and discuss their findings in light of this study. For example, if Notch signaling is impaired in cup mutants, is border cell migration also impaired? Likewise, the citation of the Assa-Kunik, 2007 study as evidence that Notch and JAK-STAT signaling act antagonistically (Line 286) is a bit of an oversimplification. While that study does show that Notch and JAK-STAT act antagonistically at earlier stages of follicle development, Fig. 6 of that paper shows that a Notch reporter and a JAK-STAT reporter are both expressed concomitantly in border cells of a Stage 10 follicle and in the anterior follicle cells of what looks like a Stage ~8 follicle. The authors should discuss the apparent contradiction between their findings and this study.
3. Lastly, the manuscript contains many grammatical errors, incomplete sentences, improper punctuation and spacing, and informal writing, such as the use of contractions. It should be thoroughly edited for content and clarity.

Significance

Although the identification of cup as a contributor to the regulation of border cell differentiation is novel, the other main regulators investigated in this study, including Notch and JAK-STAT signaling, have been identified previously. The role of cup in this context seems to be to fine tune Notch signaling and it seems to play a relatively minor role in the process of border cell specification. In addition, the conclusions of this paper are not well-integrated into the existing literature on Notch and JAK-STAT signaling in border cells, and the discussion about the broader implications of this study for the understanding of Notch signaling was not well-developed. However, the careful documentation and quantification of the phenotypes reported in this study adds rigor and allows for firm conclusions. For these reasons, this study may have a lasting but perhaps somewhat incremental impact on the study of border cell migration in the Drosophila ovary.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In this manuscript, Saha et al. made a detailed description of the role of the mRNA binding protein Cup in specifying the number of Border Cells (BC) during Drosophila melanogaster oogenesis. First of all, they show that females homozygote for a hypomorph allele of cup have higher number of BCs compared to Wild Type (WT) females. They present a series of experiments that points towards the phenotype being due to a specific role of cup in the nurse cells that non-cell autonomously regulates BC specification. Also, they show that this phenotype is the result of an increase in the levels of JAK/STAT signalling in the BC, a major determinant of BC fate. In addition, they show that cup mutant egg chambers exhibit a downregulation of the Notch (N) pathway function in the BCs and that over-activating Notch results in the rescue of the number of BCs. Moreover, the authors present data on the effect of cup in Delta (Dl) trafficking in the nurse cells: They found that cup mutant egg chambers show increased number of Dl puncta within the cytoplasm of the nurse cells, but reduced numbers in the nurse cell-Anterior Follicle Cell (AFC) boundary as a result of defective Dl endocytosis. Finally, they were able to rescue the Dl trafficking phenotype, as well as the number of BC by overexpressing an active form of Rab11.

Major points:

• In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:
  • The authors show that cup01355 mutant egg chambers have a phenotype in Dl trafficking. Although they analysed in detail the effects on Dl in the nurse cells, their images show that there might be a defect in Dl levels/trafficking in the Follicle Cells (FCs) as well (Fig5A-B). It has been shown that Dlmut FCs have reduced levels of Notch activity due to reduced lateral inhibition (Poulton et al., 2011), so there is a possibility that the reduced levels of Notch activity in the cup01355 egg chambers might be due, partially, to defects in Dl trafficking/levels in the FCs, rather than in the nurse cells.
  • The authors tested the role of the Notch pathway in the cup mutant phenotypes by measuring the number of NICD puncta in the signal receiving cells as proxy for Notch activity (Fig4). Although I understand the rationale, I am not convinced that they can completely rule out that the changes in NICD puncta number in FCs is not due to some effect of cup LOF on Notch trafficking in these cells.
  • In figure 6, the authors show that expression of a constitutively active form of Rab11 specifically in the nurse cells restores the BC number to that of the WT. However, the levels of Dl particles and, especially the levels of NRE-GFP expression, remains slightly lower than in the WT conditions.

One of the main conclusions of this study is that cup regulates BC specification through a non-cell autonomous mechanism that involves communication between nurse cells and AFCs. For that reason, I think in order to conclusively say that, the authors need to try to remove the function of cup specifically in the nurse cells. They mentioned they have tried different ways of doing this unsuccessfully, but do not specify how they have tried. I suggest using the cup-RNAi line combined with a nurse cell specific Gal4 and a ubiquitous gal80ts line (tub-Gal80ts), if they have not try this. I do not expect the authors to repeat all the experiments with this condition, but at least they should test the main findings i.e. number of BCs, JAK/STAT overactivation and Notch attenuation. - The authors have shown in Figure 3 that there is a decrease in Notch signalling in the AFCs in cup01355 egg chambers. In order to test that the BC number phenotype observe in this condition is due to that effect on Notch signalling they have done a rescue experiment using the antimorphic Notch allele Nax-16. Since in this condition all cells (nurse cells and FCs) have increased levels of Notch, they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype. If they want to show that the function of Notch is specifically needed in the FCs, they should over-activate Notch exclusively in the AFCs. For instance, they could express a constitutively active form of Notch, such as UAS-NICD (Go et al., 1998) or UAS-NECD (Fortini et al., 1993), specifically in the AFCs. Otherwise, they should re-write the text since they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype. - The authors had made a great effort to prove that proper Delta endocytosis in the nurse cells is essential for adequate Notch signalling in the AFCs and right number of BCs recruitment. Specifically: - They checked the consequences on Dl trafficking of down-regulation of rab5 or auxilin, but they did not test the effect in BC numbers - They show that downregulating the function of shi affects the number of BCs, but did not show the effect of this condition in Dl trafficking. Consequently, they cannot conclusively say that effects on trafficking of Dl affect number of BCs, since they haven't really tested both effects on the same background. I think that for simplification, they should test both, effects on Dl trafficking and number of BCs in one of those genetic backgrounds and leave the other two for supplementary material. Alternatively, they should re-write their conclusion for this section. - Their results clearly show that Dl accumulates in puncta, suggesting that there might be a defect in Dl trafficking, and although their rescue experiments point towards an scenario where Rab11-dependent Dl recycling is being affected, I think there are some weak points on their arguments. The fact that Rab11-KD does not generally affect Notch signalling in the FCs, as shown in (Windler & Bilder, 2010) argues against their conclusion that the effect of cup in nurse cells on Rab11 function is responsible for the defects in Dl trafficking and, subsequently, on Notch activity in AFCs. An alternative explanation is that Rab11 overactivation in the Cup mutant background compensates for a different defect on Dl trafficking, for example, Rab4-dependent recycling pathway. Another possibility is that AFCs could be specially sensitive to changes in Rab11-dependent Dl trafficking defects in the nurse cells. To distinguish between these two possibilities, they should perform some of the following experiments: - First of all, there are a number of endosome markers that can be used to check in which step of the endocytic route Dl is being accumulated, including (but not limited to) anti-Rab11 antibody, anti-Rab5, anti-Rab7, tub-Rab4-mcherry. They should do co-localization experiments with Dl and endosomal markers.<br /> - Also, they could check what happens to the number of BCs and Dl trafficking when Rab11 function is blocked in the nurse cells, in a similar way to what they did with Auxillin, Rab5 and Shi. They could use some of the tools described in (Satoh et al., 2005) - The authors final model is one in which cup in the nurse cells regulates Rab11 function to ultimately control JAK/STAT signalling in the AFCs. However, they have not looked at the status of JAK/STAT signalling in their Rab11-CA rescue experiments. I think this experiment will really round-up their work.

Minor points:

• The authors tested if the extra BC phenotype observed in the cup mutant egg chambers is due to defects in FCs endoreplication. I have two questions related to this section.
  • First of all, I do not understand the rationale behind this idea that defects in FCs endoreplication would result in extra BCs. Please explain and add any relevant references.
  • Secondly, they say that they used Cut and Phospho-Histone3 as endoreplication markers. I believe that what they mean is that the absent of these two markers indicates that FCs have exit the cell cycle and enter the endocycle (Sun & Deng, 2005), however they are not markers of endoreplication. Please, re-write to make this clear.
• The authors tested whether the levels of Notch activity were altered in the cup mutant egg chambers. For that, they used an NRE-GFP construct that shows a clear reduction in the levels of Notch activity in the AFCs. They also used the number of NICD and NECD puncta in signal receiving and sending cells respectively, as proxy of Notch activity. Although I understand the rationale, there are other explanations for this phenotype as discussed above. Thus, if they want to have an alternative way of showing the dampening of Notch signalling, they could use the levels of expression of well characterised targets of Notch in the FCs, such us hnt and E(spl)m-CD2 or E(spl)m7.
• In M&M the authors explain that NRE-GFP levels were expressed in Fold change. However, in figure 3C the units of the graph are Fluorescence Intensity in a.u. Please, check this small inconsistency
• In figure 4, they show the quantification of tubulin fibres within the nurse cells, however they are missing a similar analysis of Phalloidin (Pha) fibres/levels. I think this experiment and figure will be more complete if the authors added such a quantification of the effects of cup LOF in Pha distribution. Also, the authors do not show the single Pha channel in Fig4C, which would greatly helped to appreciate the differences between the WT and Cup LOF nurse cells. I suggest modifying the figure to better show the changes in Pha distribution.
• In figure 4F-G the authors are showing the general effect of cup LOF in Delta distribution. They indicate with yellow arrowheads the cytoplasmic Dl puncta accumulation in the nurse cells, however it is almost impossible to see such puncta with that level of magnification/resolution. I suggest removing the arrowheads, since the figure 4H-I shows the same puncta more clearly.
• In the Dl trafficking experiments (Fig4 H-I,K,L and Fig5A-C), the authors measured the number of puncta in the anterior nurse cell-follicle cell junction. In order to do those types of quantifications they need to be able to tell the cell boundaries that separate FCs from the nurse cells. Please, clarify the criteria for determining if the puncta are within the FCs or the underlying nurse cells.
• In figure 6C-D the authors show example images of egg chambers expressing Rab11-CA-YFP using the germline specific nos-Gal4. However, in the images it looks like the YFP signal is coming from the surrounding stretched FCs. Please check that these are the right images or explain the inconsistency.
• In figures 1R, 2L, 3Q, 6I, 6M, the authors should show the results of the statistical analysis between all the conditions tested. I think that this is crucial to be able to tell whether some of the rescues are complete or only partial.
• Line 174: should say "mutant egg chambers".
• Line 281: There is a reference that is missing from reference list: Liu et al., 2010;
• Line 292: The reference for the NRE-GFP construct is not the correct one, since that references to a review article. Please, add the correct reference.
• In line 462 of the manuscript you have a reference that is missing from your reference list.
• In line 394 the authors say: "protein, it's enrichment in the cytoplasmic fraction of the cup mutant egg chambers", but I think that they meant mutant nurse cells.

Significance

The BC migration is an excellent model to study collective cell migration and how epithelial cells can acquire migratory behaviours. After years of study, there is good understanding of the signals and genetic circuits that regulate BCs specification and migration (Montell et al., 2012), but there are not many studies, to my knowledge, that describe a role of nurse cells in specifying or guiding the migration of these cells. Thus, this study by Saha and colleagues is one of the first studies that show a role for nurse cells in specifying the number of BCs.

My field of expertise is in cell-cell communication through different pathways, including Notch and Integrin signalling. I have studied the role of endocytosis in regulating Notch signalling in various contexts, including follicular epithelium in Drosophila ovaries.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

Saha et al. characterize Drosophila egg chambers that are mutant for cup and identify an increase in the number of a specialized type of follicle cells, the border cells. They demonstrate that this increase correlates with an expanded domain of STAT activity and reduced Notch signaling in anterior follicle cells. Determining that cup is required in the germline cells, the authors postulate and provide some evidence that cup mutants prevent germline Delta from properly signaling to follicle cells. In line with this, they also show that blocking endocytosis phenocopies some aspects of cup mutants, particularly border cell numbers and Delta levels, which they monitor cytoplasmically and at the cell surface. Lastly, they demonstrate that activation of Rab11 can rescue Delta levels and border cell number in cup mutants. They conclude that a key function of Cup in the germline is to traffic Delta to signal to follicle cells, and that the endocytic processing of Delta is required for its function.

Major comments:

The findings of this study are interesting and novel. The authors have completed a lot of experiments and analyzed the results carefully and in great detail. Experimental design is described adequately and statistical analysis is sufficient. While the main results are largely convincing and support the conclusions, there are some weaknesses that need to be addressed. One major concern is that the vast majority of the experiments were conducted with a single homozygous allele for cup. The authors claim this was necessary because other alleles arrest oogenesis, which is understandable, but it leaves the potential problem that the allele, a P-element insertion, may affect other genes, or there may be other unidentified mutations on the mutant chromosome. The authors are able to partially rescue the border cell phenotype with overexpression of Cup and can also mimic the outcome with RNAi in the germline, which helps alleviate some of this concern, but this was only done for one set of experiments (those in figure 1). Similar experiments need to be included to demonstrate the same outcomes when cut is disrupted by other alleles/methods for at least some of the Notch/Delta analyses since this is key to the paper's conclusions.

A second concern is that some evidence is circumstantial or indirect. Specifically, the authors argue that the effect of Cut is due to trafficking of Delta, but do not consider the possibility that Delta could be more directly regulated or that other factors may be relevant. Border cell specification is rescued by increasing recycling in cup mutants, but this could be due to recycling of more factors besides Delta. To address this more directly, the authors should overexpress Delta in the germline of cut mutants. It is possible the disruption of Delta in cut mutants is due to changes in Delta protein stability/levels, so the experiment may also clarify this issue. If this is the case, it may be that hypomorphic Delta mutants would have a defect on border cell number, which could be examined separately. If Delta levels are low, endocytosis and recycling increases may also rescue cut mutants indirectly, but the conclusion about what Cut regulates may differ.

Another concern is that Cup's main role is a confusing since it regulates many things, including cytoskeleton and cytoskeleton is necessary for general health and vesicle trafficking in the egg chamber - how do the authors think Rab11 upregulation is overcoming these defects? Rab11CA rescues Delta levels almost completely in cut mutants but only partially rescues Notch activation, suggesting there are other problems in these egg chambers that could contribute to the defects. While exploring possible other factors is beyond the scope of this work, the authors may want to acknowledge this issue.

Minor comments:

It would help the presentation of the paper to introduce Notch/Delta signaling during oogenesis in the introduction. More introduction and clarity about the number of polar cells at early stages and their role in the border cell cluster may also be useful to the reader.

It is notable that the primary phenotype of a change in border cell numbers is quite subtle, often only affecting 1-2 cells, and the variation in different genotypes and experiments is sometimes also that large. The authors do a good job of being careful to count the cells at a specific developmental time and do appropriate statistical tests within an experiments. Still, it difficult to be sure that the effects are due to the gene being manipulated specifically or the genetic background. Related to this, a few issues should be addressed. Notably, at earlier stages, Notch signaling impacts cell division, so some of the phenotypes might be explained by there being more total cells in the domain instead of more signaling. The authors show Cut is in the same domain and pH3 is similar, but they didn't seem assess overall numbers. Secondly, for the stat suppression of cut (figure 2L), the authors need to show the stat-/+ control for comparison to make a conclusion about suppression versus additive effects. In addition, prior work (Wang et al 2007) expressed DN Kuz in border cells and did not see a change in specification, unlike what is claimed here. In the experiment in question, the control has lower than normal numbers of border cells and the DN Kuz has a number more typical of the controls in other experiments- so this is a concern that there is something else in the genetic background influencing the numbers. Other controls could help make this case, but ultimately this result is probably not necessary for the main argument. Thus the authors might consider leaving it out the Kuz analysis or perhaps can comment on the discrepancy with prior published results.

Can the authors comment on why the volume of the border cell cluster increases more dramatically (>2x) than the number of cells (30% more)?

Does the increase in border cell number change the migratory capacity? That is, do the clusters in cut mutant egg chambers migrate normally while the egg chamber looks okay?

Several of the figure legend titles state conclusions that are over interpretations of the data shown:

• Figure 3 legend is overstated- these experiments do not assay STAT activity, only border cell number, so the title can be simplified to say that.
• For figure 4, both cytoskeleton and Delta are shown to be disrupted in cup mutants, but they are not directly linked, eg, the experiments do not show a change in Delta in cytoskeletal mutants alone. While it is interesting that cup mutants have disrupted cytoskeleton, ultimately this result is not well connected to the main issue of Notch/Delta signaling; in fact, it becomes confusing how anything can be trafficked to the cell surface if there is poor cytoskeletal organization. Since the authors favor the hypothesis that the cytoskeleton is not the key to the border cell specification difference, they may want to move this result out of figure 4.
• The Figure 5 legend is also overstated- these experiments show that Delta is higher in cup mutants and endocytosis mutants AND that endocytosis (of something) is required in the germline for border cell number- but these results are not linked in this figure. More evidence for this connection does come later in figure 6. Some figure legends are quite brief and could benefit from a little more detail on what is being shown.

Figure layout could be improved by keeping images consistent sizes and making sure graph text is large enough to read easily. Figures in general could be streamlined by having negative results and less pertinent results in supplemental data.

Not all papers cited in the text are in the reference list.

Referees cross-commenting

I generally agree with the other reviewers that there are concerns with the precise function of cup in this context, and that some revision is needed, including editing of the writing. In response to reviewer 2, prior published studies only detected Cup in germline, but it is possible that it is expressed in follicle cells at a low level. The mutant clonal experiment in follicle cells that the authors did had no effect on border cells, so that provides some evidence the role is non-autonomous. I agree with reviewer 2's concern that the authors overstate the connection between cup and Delta and border cells based on their data and need a few more experiments to tie things together. I understand reviewer 3's concerns that the experimental effects on border cell numbers are very small and variable- I listed this as a minor concern, though, since this number is mainly being used as a read-out for STAT signaling levels and the data were extensively quantified and statistically tested.

Significance

My expertise is in cell migration, developmental biology, and Drosophila genetics. This paper will be of broad interest in these fields as it incorporates aspects of each in its characterization of a new regulatory mechanism to induce a motile cell population non-cell-autonomously, which is an exciting finding. Specifically, the work increases our understanding of the intersection between Notch and Jak/STAT signaling, which many researchers study - these were both known to be involved in border cell specification. The study provides more detailed characterization of the signaling and specification process in general, and makes significant advances in understanding how Delta signals are produced and presented from germline cells to receiving cells in the soma. Cut has not been previously implicated in these signaling pathways, so that is also novel, although its precise mechanistic role here is still somewhat unclear.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary:

In this manuscript, authors establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. They used B. subvibroides and C. crescentus specific mutants in pseI and legI genes involved in the Pse and Leg biosynthesis, respectively, and cross-complementation assays with orthologous genes from different bacterial species, analysing motility and flagellin glycosylation. These assays show that Pse and Leg biosynthetic pathways are genetically different and recognize the LegX enzyme as a critical element in the Leg-specific enzymatic biosynthesis. Since that legX orthologous were only identified in the genome of bacteria with Leg biosynthetic pathways, it becomes a good marker to distinguish Leg from Pse biosynthesis pathways and a novel bioinformatic criterion for the assignment and discrimination of these two pathways. Reconstitution of Leg biosynthetic pathway of B. subvibroides in the C. crescentus mutant that lack flagellins, PseI and FlmG, complemented with both flagellin and FlmG of B. subvibroides, identified a new class of FlmG protein glycosyltransferases that modify flagellin with legionaminic acid. Furthermore, the construction of a chimeric FlmG through domain substitutions, allowed to reprogram a Pse-dependent FlmG into a Leg-dependent enzyme and reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

Major comments:

The conclusions obtained are convincing and well-supported. However, I think some points should be specify or clarify.

1.- In the mutants (pseI, legI, flmG,...) the non-glycosylated flagellin are exported and assembled in a flagellum filament shorter than the WT strain. However, motility in plates is absent or very reduced. This might be produced by instability of the flagellum filament when rotating in a semi-solid surface. MET was performed from plates or liquid cultures? Do the author analyses motility in liquid media? If they did, changes in motility were observed?

Response: The Caulobacter ΔpseI mutant accumulates low levels of flagellin in the supernatant. TEM analysis reveals that the flagellar filament is not assembled and only the hook structure is visible (PMID: 33108275). Brevundimonas subvibrioides ΔlegI or ΔflmG cells feature a shorter filament compared to WT by TEM. In all these analyses, TEM was performed on cells grown in broth to exponential growth phase as detailed in the Experimental procedures section. These mutant cells do not swim when analyzed by phase contrast microscopy. While is not known if swimming on semi-solid medium would further destabilize the flagellar structures seen in liquid cultures by TEM, there is more residual motility in B. subvibrioides mutants that make a short filament compared to C. crescentus mutants that lack the flagellar filament. Thus, our analyses point to a positive correlation between the residual motility and residual filament length when comparing the B. subvibrioides and C. crescentus mutants.

2.- In page 5, lines 158-163, the analysis, by HPLC, of derivatized nonulosonic acid from B. subvibroides flagella, shows a major peak at 9.8 minutes retention and a minor peak at 15.3 minutes. Since that Pse-standard have retentions peaks at 9.7 and 13 minutes, and Leg-standard at 12.3 minutes, the authors cannot infer, only with these data, the flagella sugar is a legionaminic acid derivative. In my opinion, should be included that inference comes from the data obtained by HPLC analysis and genetic approaches. Thanks. Corrected. 3.- In page 5, line 173-175. Authors indicate, "While no difference in the abundance of flagellin was observed in extracts from mutant versus WT cells, flagellin was barely detectable in the supernatants of mutant cultures, suggesting flagellar filament formation is defective in these mutants". MET images show that the flagellum filament length is shorter in the mutants than in the WT strain. Therefore, if the same number of mutants and WT cells has been used in the immunodetection assays, there should be more flagellin monomers in the WT samples than in the mutants ones and flagellin bands should be less intense in mutant samples corresponding to the anchored flagellum. Why bands corresponding to flagellin in mutants and WT show similar intensity in the immunodetection assays (Figure 3C and D)? Furthermore, in lane 177-178, authors suggest that LegI and FlmG govern flagellin glycosylation and export (or stability after export). However, if filament stability is affected, the amount of flagellin monomers in the supernatant of mutants should be higher than in the WT. However, immunodetection assays show less abundance of flagelin monomers in the supernatant of mutants. Please, can you clarify this? In relation to this point, I suggest that authors include, in the experimental procedures, how they obtained the supernatants to flagellin immunodetection, as well as why they used anti- FljKCc anti-serum to detect the B. subvibroides flagellin.

We thank the reviewer for raising this point. We have now clarified this question in the updated Experimental procedures section. Our immunoblots harbor the same number of cells harvested in exponential phase (OD=0.4). One mL of cells was harvested from cultures by centrifugation at full speed. The supernatant that was used for the immunodetection corresponds to the supernatant after the centrifugation. The supernatant fraction contains flagella that have been shed during the cell cycle at the swarmer cell to stalked cell (G1-S) transition of C. crescentus and B. subvibrioides.

Thus, it is clear that the majority of flagellins detected by immunoblotting are in fact cell associated and specifically the intracellular flagellins. The evidence for this is that the levels are comparable between WT and ΔflmG mutant cells, even though the latter has shorter or no flagellar filaments. Moreover, while C. crescentus cells are not constantly flagellated during the cell cycle, flagellins are detectable on cell-associated samples by immunoblotting even when cells do not yet or no longer have a flagellar filament. Based on these two points, we conclude that the total flagellin levels associated with cells do not reflect the levels of flagellin assembled into a flagellar filament, but rather the flagellin bulk present in the cytoplasm.

Consistent with this view, we previously reported that C. crescentus ΔpseI cells have the same amount of flagellins in cell lysates compared to the WT strain (PMID: 33108275), even though the mutant cells lack a flagellar filament. Thus, the results obtained here are consistent with previous observations and indicate that B. subvibrioides flagellin glycosylation mutants also still produce comparable amounts of flagellins intracellularly like the WT strain, despite the absence of flagellin glycosylation and inefficient assembly into a flagellar filament.

Concerning the potential role of LegI and FlmG in flagellin stability after export, we were referring to protein stability (half-life), not filament stability. Glycosylation may impact the half-life of extracellular flagellins since glycosylation can protect from proteolytic degradation of proteins, possibly in this case by different proteases that may accumulate in the supernatant. Thus, non-glycosylated flagellins could be more easily degraded by extracellular proteases once they are exported, ultimately resulting in a lower amount in the supernatant.

Addressing the final question about the specificity of the anti-FljKCc antiserum: we used this anti-serum because it detects the B. subvibrioides flagellins owing to the high sequence similarity between B. subvibrioides flagellins and C. crescentus flagellins. We previously showed that the anti-FljKCc anti-serum detects all six flagellins from C. crescentus, as determined by individually expressing each flagellin in a strain deleted for all six flagellin genes (Δfljx6) (PMID: 33108275). FljKCc (against which the antibody was raised) is 65% similar to the most distant C. crescentus flagellin, FljJ. As the similarity of FljKCc to the three B. subvibrioides flagellins ranges from 74% -67% sequence similarity, they should be even better recognized by the anti- FljKCc antibody than C. crescentus FljJ. However, on immunoblots we cannot attribute the signal to any individual B. subvibrioides flagellin as they could all co-migrate on SDS-PAGE and therefore all flagellins might reside in the same immunoblot band. However, we can clearly demonstrate that the immunoblot band corresponds to flagellins: a B. subvibrioides ΔflaF mutant (see below) that we constructed revealed that the flagellin signal is lost, as is the case for a C. crescentus ΔflaF mutant (PMID: 33113346). In the case of C. crescentus, the FlaF secretion chaperone is required for flagellin translation (synthesis) and we suspect that this also the case for B. subvibrioides FlaF. This experiment provides additional evidence that the B. subvibrioides flagellins are recognized by the anti-FljK (C. crescentus) anti-serum.

4.- Authors demonstrate the specificity of the GT-B domain of FlmG, using a chimeric FlmGCc-Bs in a mutant of C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides. However, since that TPR comes from C. crescentus, this chimeric protein, could be transfer the legionaminic acid to the flagellin of B. subvibroides? Furthermore, the complementation of this mutant with the FlmGBs did not support efficient flagellin modification and this might be related to the TPRCc domain. Therefore, in my opinion, the chimeric protein should be introduced in the B. subvibroides∆flmG background. The answer to the first question is “No” or “very inefficiently” as determined from immunoblot analyses of B. subvibrioides ΔflmG cells expressing the chimeric FlmG_Cc-Bs protein that we now show in Fig S2B.

Expression of the different FlmG (FlmG_Cc, FlmG_Bs, FlmG_Cc-Bs) in C. crescentus cells producing Pse or Leg revealed that FlmG_Bs does not support efficient flagellin modification with Pse in C. crescentus, likely because FlmG_Bs interacts poorly with the C. crescentus flagellins. By using the FlmG_Cc-Bs chimera we hoped to overcome this interaction problem with the C. crescentus flagellins (because the FlmG chimera harbors the C. crescentus TPR to bind the C. crescentus flagellins), however glycosyltransfer still does not occur efficiently because the GT domain from FlmG_Bs does not function with Pse. However, FlmG_Cc-Bs can modify the C. crescentus flagellins once C. crescentus is genetically modified to produce CMP-Leg (instead of CMP-Pse). This confirms that the FlmG TPR from C. crescentus is important for flagellin modification through the FlmG/flagellin interaction and that GT_B type glycosyltransferase only transfers Leg. In addition, we have now added as Fig S2B an immunoblot and as Fig S2C a motility test of B. subvibrioides ΔflmG cells expressing the FlmG_Cc-Bs chimeric protein in which we only observed little modification of B. subvibrioides flagellins and a poor motility, respectively. We extended our discussion of these results.

5.- Page 8, line 299-301. Authors point out that C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides and the chimeric FlmGCc-Bs, although it has a glycosylated flagellin, whose mobility in SDS-PAGE is like the WT strain, is non-motile. They suggest that additional factors exist in the flagellation pathway that exhibit specificity towards the glycosyl group that is joined to flagellins. However, would be interesting to see if the flagellum filament has similar length to the WT strain or at least, it has increased in relation to the flagella length of the mutant. If flagella length has not increased, it could suggest that changes in the glycan type might affects the flagellin assembly or the stability of the flagellum filament. Therefore, would be also important to analyse its motility in liquid media.

To investigate why the C. crescentus cells that produce Leg and express the chimeric FlmGCc-Bs glycosyltransferase are non-motile (Figure S5B) despite flagellin modification (by immunoblotting, Figure 7C), we employed two strategies. First, we performed immunoblot analyses on the supernatant fraction from these cells to determine if flagellins accumulate extracellularly. As now showed in Figure S5A, only low amounts of C. crescentus flagellins modified by Leg are present in the SN fraction. Second, we conducted TEM analyses of cells grown to exponential growth phase in broth. As shown in Figure S5C, the C. crescentus cells producing Leg and expressing FlmG_Cc-Bs glycosyltransferase harbor a shorter flagellum compared to those expressing the FlmG_Cc in which C. crescentus flagellins are modified by Pse. Altogether these results explain why these cells are non-motile both on soft agar plate and in liquid.

Minor comments: 1.- Pag 3 line102. Please change ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes only PseI...." to ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes a PseI...." 2.- Figure 2 A. Plasmid nomenclature (Plac-neuB) is confusing because C.c. ΔpseI cells express predicted LegI or PseI synthases. Please change to Plac, as in Figure 2B and 4. Figure 2A and 2B do not contain any complementation with Bacillus subtilis (Basu), however two complementation are labelled as Bs in Figure 2A and 2B. Furthermore, no Bs are present in the Figure 2 legend. 3.- Legend of figure 3 should include B. subvibrioides abreviation Bs. Line 774: Please change ".......glycosylation and secretion in B. subvibrioides." to ".......glycosylation and secretion in B. subvibrioides (Bs)." 4.- Figure 3. In order to keep a similar nomenclature in all plasmids, plasmid Plac-legI syn and Plac-flmG should be labelled as Plac-legIBs syn and Plac-flmGBs.

5.- Legend of figure 4 should include B. subvibrioides abreviation Bs. Line 791: Please change "....... complementation of the B.subvibrioides ΔlegI mutant with ...." to "....... complementation of the B.subvibrioides (Bs)ΔlegI mutant with ...." Furthermore, Legend of figure 4 indicate in line 795, that immunoblots reveal the intracellular levels of flagellin, however figure 2 and 3 show immunoblot of cell extracts. Please, correct this sentence. 6.- Legend of figure 5, 6 and 7 should include B. subvibrioides abreviation Bs. Line 808: Please change "Predicted Leg biosynthetic pathway in B. subvibrioides " to"Predicted Leg biosynthetic pathway in B. subvibrioides (Bs)" Line 834: Please change "....affects motility, flagellin glycosylation and secretion in B. subvibrioides."to "....affects motility, flagellin glycosylation and secretion in B. subvibrioides (Bs).Line 852: Please change "...acetyltransferase in flagellar motility of B. subvibrioides cells." to ""...acetyltransferase in flagellar motility of B. subvibrioides (Bs) cells." Furthermore, figure 5 should include C. crescentus abbreviation. Line 815: Please change "....whole cell lysates from C. crescentus mutant cultures......." to "....whole cell lysates from C. crescentus (Cc) mutant cultures......." 7.- In my opinion it would be useful to include a scheme of the gene organization involved in Leg biosynthesis in B. subvibrioides.

8.- Legend of figure S1 should include B. subvibrioides (Bs) and C. crescentus (Cc) abbreviations. Line 888-867: Please change "...C. crescentus ΔpseI cells and B. subvibrioides ΔlegI cells with plasmids expressing..." to "...C. crescentus (Cc) ΔpseI cells and B. subvibrioides (Bs) ΔlegI cells with plasmids expressing..." Furthermore, the name and abbreviations (Mm, So, Ku, Pi, Dv) of the species used should be included in the legend. Why the authors used a plasmid with a Pvan promoter in these assays? Why the authors changed the code color of pseI and legI orthologous genes? It would be more useful and understandable follow the code color used in figure 2 and 4.

Page 6 line 200, Please change ".....complementing synthases exhibit greater overall sequence similarity to LegI than Pse of C. jejuni. 22268,....." to ".....complementing synthases exhibit greater overall sequence similarity to LegI than PseI of C. jejuni. 22268,....." 10.- Page 7 line 231, Please change ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and P. sp. Irchel 3E13..." to ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and Pseudomonas sp. Irchel 3E13..." 11.- Introduce a line break between line 503 and 504. 12.- Page 14 line 543, please change "XbaI" to "XbaI" Thanks for the careful editing. We changed the text as suggested by the reviewer. We also added a scheme showing the genetic organization of the genes involved in Leg production and present as Figure 1B. When this study was initiated, the pMT335 plasmid with a Pvan promoter was used before we switched to using the pSRK plasmid with Plac promoter for better induction. Note that the results with Pvan or Plac are comparable regarding the PseI synthases interchangeability. Color code is now homogenous through the manuscript.

Reviewer #1 (Significance (Required)):

This is an interesting manuscript that contributes to the knowledge of the legionaminic biosynthetic pathway and establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. The analysis of Leg patway allowed to identify a gene (legX) that can be used to distinguish Leg from Pse biosynthesis pathways, becoming a bioinformatic tool for the assignment and discrimination of these two pathways. Furthermore, a new class of FlmG protein glycosyltransferases, able to transfer Leg to the flagellin, has been identified and its analysis reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: Viollier and co-workers present a study in which they preform an elegant and rigorous genetic profiling of the the legionaminic and pseudaminic acid biosynthesis and flagellar glycosylation pathways in C. crescentus (native Pse) and B. subvibrioides (native Leg). They use motility as a representative readout for functional flagellar glycosylation with these microbial sialic acids. They discover orthologous Pse synthase genes can replace the function of the native synthase in C. crescentus and orthologous legionaminic acid synthase genes can achieve the same in B. subvibrioides. However, not vice versa indicating a strong preference for each microbial sialic acid stereoisomer in these species. For the Leg biosynthesis pathway, which requires GDP-GlcNAc, the authors also identify LegX as an essential component to synthesize this sugar nucleotide and thus a marker for Leg biosynthesis pathways. Upstream in theses pathways, they also identify a new class of FlmG flagellar protein glycosyltransferases. Importantly, through heterologous reconstitution experiments to uncovered that these glycosyltransferases possess two distinct domains, a transferase domain the determines specificity for either CMP-Leg or CMP-Pse, and a flagellin-binding domain to achieve selectivity for the substrate. Interestingly, by creating chimeric FlmG for these two domains between C. crescentus and B. subvibrioides they show that these two modular parts can be interchanged to adapt flagellin glycosyltransferase specificity in these species. Major comments: The key conclusions of the manuscript by Viollier and co-workers are convincing and well supported by their experiments and used methods, with respect to the insulation of the Leg and Pse biosynthetic pathways, they key role of LegX in launching the Leg pathway and the successful reconstitution of Leg glycosylation in a previously Pse-producing C. crescentus strain. Finally, they convincingly show that a chimeric version of the involved glycosyltransferases is functional, which besides intriguing future glycoengineering possibilities also emphasizes the two discrete domains in these transferases that dictate their sugar nucleotide and acceptor specificity. There is one additional experiment I would suggest with relation to the detection and confirmation of Pse and Leg on flagella of respectively, C. crescentus and B. subvibrioides. In the case of C. crescentus the detected DMB derivatized monosaccharide co-elutes with a validated standard of tri-acetylated Pse, which is convincing evidence of its identity. However, for B. subvibrioides. Their DMB derivatized monosaccharides from its flagella, results in a peak the does not co-elute with the only Leg standard (Leg5Ac7Ac) they have, it does elute at the same time as their Pse standard. Although it cannot of course be Pse as B. subvibrioides. Does not possess a Pse biosynthesis pathway, it also does not provide enough evidence to conclude that it is a Leg derivative. An MS(-MS) measurement of the eluted signal would not be a big investment in time and resources and would provide additional evidence to at least assign this peak to microbial sialic acid related to the present Leg biosynthesis pathway. It the identified mass would lead to identification of the derivative, it would also add to the proper characterization of the flagella glycosylation in the bacterium.

We have now added the glycopeptide analyses as requested. They are described in the last experimental section and confirm our results.

The data and the methods presented in this study are presented with sufficient detail so that they can be reproduced? However, I would suggest as is common nowadays in most journals that the authors include images of the raw unprocessed blot in de supporting info.

*The motility pictures are representative of three independent experiments and the immunoblots are representative of at least two independent experiments. This has now been mentioned in the Experimental procedures. The raw unprocessed blots have now been added as supporting info. *

Minor comments: There are a few textual errors that the authors should fix: -page 2, line 70: change "used" to "use" -page 11, line 407: add the word "are" after Pse On page 2, line 36, the authors state that "most eubacteria and the archaea typically decorate their cell surface structures with (5-, 7-)diacetamido derivatives, either pseudaminic acid (Pse) and/or its stereoisomer legionaminic acid (Leg,". This should be nuanced as to my knowledge it is not most eubacteria, but more a subset as identified by Varki in his seminal PNAS paper. The authors clearly present their data and conclusions in the figures of this manuscript. However, I would recommend the take a critical look at the drawing of their monosaccharide chair conformations and the positioning of the axial and equatorial groups on these chairs in Figure 1 and 5, as these are in most cases drawn a bit crooked, which can easily be corrected. We corrected the text as the reviewer suggested. We changed the sentence in the introduction to be more nuanced. The drawing of the monosaccharide has been improved.

Reviewer #2 (Significance (Required)):

The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in specific microbes. Especially symbiotic and pathogenic microbes associated with the humans express a wide array of unique microbial sialic acids for which their functional roles are not well understood and the associated glycosylhydrolase and glycosyltransferase have in most cases not been identified yet. The authors present an impressive insight into flagellar glycosylation with Pseudaminic and Legionaminic acid in two bacterial species, using genomic analysis, rewiring, immunoblots and motility assays as their main tools. They provide compelling evidence on the insulation of the Pse of Leg pathway in these species, the flexibility in exchanging between biosynthetic enzymes from the same pathway between various species. Crucially, most glycosyltransferases that add the Pse or Leg glycoform onto various acceptor sites in bacteria, have up to this point remained elusive in most cases. It is therefore very valuable information that the authors here provide on the involved glycosyltransferases. Especially, on the two domains that govern their sugar nucleotide and acceptor specificity, and that these can be reengineered as chimeric glycosyltransferases. To me as a chemical glycobiologist this provides compelling possibilities for glycoengineering possibilities in future studies in the field to elucidate the functional roles of Pse and Leg glycosylation.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary of the findings and key conclusions (including methodology and model system(s) where appropriate): Kint et al describe a neat study of bacterial flagellin glycosylation by a recently identified class of protein glycosyltransferases called FlmG. The experiments are well designed, the data presented is convincing and the conclusions drawn are mostly in line with the experimental evidence presented. These are the key findings. Kint et al show that genetic tools and motility can be used as a readout to probe the sugar biosynthesis pathway in bacteria. Using the recently characterized system of Caulobacter crescentus, they have performed a survey of different PseI/LegI/NeuB genes from various bacteria, checking whether they could rescue the motility defect in C. crescentus ΔpseI cells. They found that those genes that did confer motility also had higher sequence similarity to C. jejuni PseI than to C. jejuni LegI or C. jejuni NeuB. They also found that these genes also restored flagellin glycosylation as checked by mobility shift on gel electrophoresis with immunoblotting to anti-FljK antibody. This survey brought up an interesting finding that the PseI/LegI/NeuB orthologs of the closely related Brevundimonas species were unable to confer motility to C. crescentus ΔpseI cells, and were more similar to C. jejuni LegI than to C. jejuni PseI or C. jejuni NeuB. They also performed similar glycoprofiling experiments using B. subvibrioides ΔlegIBs cells and various PseI/LegI/NeuB orthologs from different bacteria, which indicated the restoration of motility by putative LegI synthases. Kint et al demonstrate flagellin glycosylation in B. subvibrioides by performing in-frame deletions of FlmG, and LegI genes in B. subvibrioides and checking for motility, presence of flagella, and flagellin glycosylation by motility shift on gel electrophoresis. Further, they confirm the critical nature of GDP-GlcNAc for Leg biosynthesis by assessing flagellin glycosylation and motility in B. subvibrioides with an in-frame deletion in PtmE/LegX and by performing heterologous complementation with an M. humiferra PtmE ortholog. They also reconstitute the legionaminic acid biosynthesis pathway in C. crescentus cells that lack flagellins, PseI and FlmG, and show that the heterologously expressed B. subvibrioides flagellin is glycosylated by heterologously expressed B. subvibrioides FlmG. Finally, they also show that whereas the CcFlmG cannot substitute for BsFlmG and vice versa, a chimeric FlmG bearing the TPR domain from C. crescentus FlmG (that recognizes C. crescentus FljK) and the GT domain from B. subvibrioides FlmG (that transfers CMP-Leg) modifies CcFljK in C. crescentus cells that lack CcFlmG but express both Pse (endogenously) and Leg (from the reconstituted pathway). This demonstrates the modularity of the FlmG glycosyltransferases. Kint et al provide the chemical nature of C. crescentus flagellin glycosylation. Kint et al have analyzed the glycans released from the flagellin by acid hydrolysis and clearly shown the nature of the glycan in C. crescentus flagellin to be Pse4Ac5Ac7Ac by use of Pse standards. The glycan from B. subvibrioides was distinct from the Leg standard used, and could be a Leg derivative distinct from Leg5Ac7Ac.

Major comments: 1. Table 1 and Text in Results, lines 116-119, "In support of the notion that derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg, our glyco profiling analysis revealed that putative PseI proteins (identified by sequence comparisons to C. jejuni 11168, Table S1) conferred motility to C. crescentus ΔpseI cells, whereas putative LegI synthases did not." Not clear how putative PseI and LegI synthases were identified. Table 1 only lists overall percent sequence identity and similarity to Cj PseI, LegI and NeuB, and percent identities and similarities of the various nonulosonic synthases to these proteins are in the similar range, as expected. In the absence of sequence alignments indicating the presence of conserved residues, particularly related to the substrate binding region, that are distinct in these paralogs, calling out the type of synthase based on the highest percent identity (to Cj PseI, LegI or NeuB) is speculative. Also, Shewanella oneidensis does not follow the pattern of highest similarity to NeuB3. Second, in the absence of data showing that the Leg and Pse found in these different organisms actually are different derivatives, this does not support that "derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg". Putative PseI and LegI were proposed based on BlastP analyses in which the protein sequences of interest were aligned to the three experimentally validated synthases from C. jejuni 11168: PseI, LegI, NeuB as well as PseI from C. crescentus, as indicated in Table S1. While, the assignment of the donor sugar is based only on the sequence identity and similarity to LegI or PseI, this assignment corresponds well according to the restoration of the motility of the C. crescentus ΔpseI mutant upon expression of PseI ortholog and B. subvibrioides ΔlegI mutant with heterologous LegI expression.

It is true that for Shewanella oneidensis the assignment as PseI or LegI is ambiguous, exhibiting nearly identical similarity, but it is quite distinct from NeuB. This actually makes the S. oneidensis synthase a very interesting case to explore the enzymology of its Pse/LegI ortholog, knowing that it has been previously shown that this bacterium glycosylates its flagellins with Pse derivatives (PMID: 24039942). The results from our genetic complementation analysis are however very clear (PseI ortholog) and very consistent with the functional analysis in S. oneidensis.

Concerning the different derivatives of Pse or Leg: McDonald and Boyd (PMID 32950378) recently published a review giving some examples of Bacteria/Archaea experimentally shown to contain Pse/Leg-derivatives: C. jejuni 11168 modifies its flagellin with 5,7-N-acetyl Pse, Sinorhizobium fredii NGR234 (not used in this study but in our previous work PMID 33113346 and showed to restore the motility of C. crescentus ΔpseI cells) modifies its capsule with 5-acetamido-7-3-hydroxybutyramido-Pse), Treponema denticola modifies its flagellin with 7-(2-metoxy-4,5,6-trihydroxy-hexanoyl-Pse, A. baumannii LAC-4 produces 5,7-N-acetyl-8-epi-Leg to decorate the capsule, Halorubrum sp. PV6 modifies the LPS with N-formylated Leg and L. pneumophila produces 5-acetamidino-Leg.

The reviewer is right in that we do not know the exact version of Pse or Leg produced in C. crescentus and B. subvibrioides, HOWEVER, the fact that complementation works with the majority of the orthologs of PseI and LegI including many from bacteria that are known to produce modified Pse derivatives for example in Shewanella oneidensis and Treponema denticola, the most likely explanation is that derivatization occurs after the PseI or LegI step, but we concede that the results are also compatible with a promiscuous enzyme that can accept different Pse derivatives or different Leg derivatives.

1. Related to (1), Text in Results, lines 130-131, "We conclude from our survey that (heterologous) PseI synthase activity generally confers motility to C. crescentus ΔpseI cells, whereas LegI-type (or NeuB-type) synthases are unable to do so." There is no a priori evidence provided indicating that these were PseI or LegI type synthases. So the conclusion really is that assuming only PseI type synthases would be able to rescue the motility defect in C. crescentus ΔpseI cells, this glyco-profiling motility assay now provides the first biochemical evidence telling us which synthases are Pse-type, and which are Neu/Leg-type. And in my view, this is the conclusion of greater significance in the field - to be able to now identify which is a PseI and which a LegI based on these complementation assays. However, if the authors still wish to retain their original conclusion, they could cite or provide evidence (either biochemical evidence in this work or reported literature regarding the sugar synthesized or bioninformatics analysis regarding the presence of distinct genes such as the Ptm genes for legionaminic acid biosynthesis pathway or genes that differ in their enzyme activities and overall fold such as PseB/LegB or PseG/LegG in the gene neighborhood) indicating or suggesting the PseI/LegI/NeuB nature of the different synthases. Also, methods for the bioinformatics analysis (eg. BLASTp settings used, dates of searches, whether regular BLAST or PSI-BLAST was used, etc.) are missing in the manuscript, and need to be included. We agree that for many PseI or LegI tested, there is no provided biochemical evidence. HOWEVER, this is not the case for some of them including the PseI, LegI and NeuB from Campylobacter jejuni (PMID 19282391), some A. baumannii strains (α-epi-legionaminic acid for A. baumannii LAC-4 PMID 24690675), Shewanella oneidensis (Pseudaminic acid with methylation PMID 23543712), Legionella pneumophila (Legionaminic acid PMID 18275154) or Halorubrum sp. PV6 (N-formylated legionaminic acid PMID 30245679). Thus, we maintain the two conclusions: the PseI and LegI synthases are generally interchangeable and the complementation assays can enable to identify and assign PseI and LegI function. BLAST2P was used to compare the protein sequences of the tested NeuB-like synthases with NeuB1, LegI (NeuB2) and PseI (NeuB3) from Campylobacter jejuni but also with PseI from C. crescentus. BLOSUM62 matrix was used as well as a word of size 3 for the comparison. We have now added this procedure in the legend of the Table S1.
2. It is interesting that there is still a signification amount of flagellin secretion/assembly in the B. subvibrioides LegI and FlmG mutants. It will be good to see a discussion about whether this is likely from due to low level of function despite the in-frame deletion of genes; how many flagellin subunits are likely to have managed secretion and assembly in these short flagella; whether there is any redundancy of LegI / FlmG (perhaps with lower levels of expression); considering Parker and Shaw's findings of glycosylation being required for flagellin binding to the chaperone and subsequence secretion in A. caviae whether there is a FlaJ homolog in B. subvibrioides. Also, can the authors rule out the possibility that absence of glycosylation does not affect flagellin assembly but makes the flagellum prone to shear/breaks in B. subvibriodes, resulting in smaller flagella? How many flagellins are there in B. subvibrioides? Is it possible that one is glycosylated but another/others are not, and that is the reason for the small flagellum in these mutants? The number of flagellin subunits that are assembled into a full-length flagellar filament is unknown in C. crescentus and in B. subvibrioides. There are 3 different flagellin genes that are now presented schematically in Figure 1C. No redundancy has been found for LegI or FlmG. It is possible that the B. subvibrioides is better in exporting non-glycosylated flagellin or that the capping proteins can function better with sugar modification or that the filament of B. subvibrioides mutants is less fragile when it is non-glycosylated or that its flagellins “stick” better. It is also possible that short filaments are not actually containing flagellins mounted on the hook but another protein that polymerizes aberrantly in the absence of Leg or FlmG. This remains to be investigated and compared to the situation of Pse and FlmG mutants of C. crescentus.

B. subvibrioides possesses an ortholog of the C. crescentus flagellin secretion chaperon FlaF (PMID 33113346). As observed in C. crescentus, FlaF likely has a role in flagellin translation as its inactivation totally prevents flagellins production (see answer to reviewer #1). For C. crescentus, bacterial two hybrid experiments revealed that FlaF can interact with non-glycosylated flagellins in E. coli. Thus, it is strongly possible that FlaF/flagellins interaction is not dependent on the flagellins glycosylation state. In addition, the short flagellum filament observed in B. subvibrioides ΔlegI or ΔflmG mutants argues that at least some flagellins are secreted while not glycosylated.TEM pictures have been performed in liquid medium from exponential growth phase. In this condition, no fragment of flagella was observed in the culture medium by TEM but only small flagella with a hook structure attached. Also, flagella breaks might result in more random length of flagellum.

Three flagellins are in B. subvibrioides (Bresu_2403 is 59% identical with FljLCc, Bresu_2638 is 57% identical with FljKCc and Bresu_2636 is 62% identical with FljJCc). We now show this genetic organization of the flagellins in Fig. 1C. The three flagellins are all detected by the anti-FljKCc anti serum (see answer and figure to reviewer #1). We cannot attribute the immunoblot signal to any individual B. subvibrioides flagellin as they could all co-migrate on SDS-PAGE. However, the signal often looks like a doublet (as shown in Figure 4B for example) suggesting that at least two flagellins are detected and this doublet is always found to migrate faster in absence of glycosylation that could indicate that all B. subvibrioides flagellins (or at least 2) are modified.

Text in Results, lines 170-171, "We then probed the resulting ΔlegIBs and ΔflmGBs single mutants for motility defects in soft agar and analyzed flagellin glycosylation by immunoblotting using antibodies to FljKCc". Was the antibody to FljKCc determined to also specifically bind to FljKBs? Also, how many flagellins are there in B. subvibrioides? Are all detected with this antibody? Antibodies raised to FljKCc were raised against His6-FljK produced in E. coli (previously published in Ardissone et al, 2020). This serum recognizes the 6 flagellins from C. crescentus (PMID: 33108275). It recognized the three flagellins from B.s. (see answer to reviewer #1).

It is interesting that C. cresentus cells expressing Pse (endogenously) and Leg (reconstituted pathway), and BsFlmG and BsFljK (corresponding to Figure 5C) are not motile. Was the motility assay done for the experiment of figure 5B as well? Are the C. crescentus cells lacking Pse and FlmG but with heterologous expression of Leg and BsFljK and BsFlmG also non-motile? Also, it will be good to see the TEM images for these cells.

C. crescentus cells that produce Pse (endogenously) or Leg (reconstituted pathway) and BsFlmG and BsFljK (formerly Figure 5C and now as Figure 7C) are indeed not motile as shown by the motility tests presented in Figure S5B. Motility assays with cells used in the former Figure 5B (now Figure 7B) have also been done and are now presented Figure S4B. These cells are non-motile because BsFljK is not efficiently secreted (or unstable after secretion) as shown on the immunoblot of the supernatant fraction in Figure S4A lower panel. As a result, flagellar filament is not properly assembled as only a short flagellum was observed by TEM in such cells compared to the WT C. crescentus (Figure S4C and S4D).

Immunoblotting of the supernatants should be shown (in addition to the cell extracts) for Figures 5B and 5C so that the reader can appreciate whether glycosylation has taken place but secretion/assembly has not. Further, HPLC of the acid extracts from flagellin could be done to unambiguously show whether the CcFlmG has transferred Pse and the BsFlmG and Cc-BsFlmG have transferred Leg on to the CcFljK in Figure 5c, and the identity of the sugar, if any, transferred by CcFlmG in the absence of Pse, and BsLeg genes or BsLegX gene in figure 5B.

*__ Immunoblots of the supernatants for Figure 5B (now Figure 7B) have been done and been added (Figure S4A lower panel). BsFljK is barely detected in the supernatant whatever its glycosylation state (with or without Leg). Note that in the supporting info where the raw unprocessed blot used for this panel is shown, a positive control of blotting (C. crescentus Δfljx6 mutant expressing CcFljK from pMT463) has been used. Immunoblots of the supernatant from Figure 5C (now 7C) have been done and been added in figure S5A. The CcFljK modified with Leg is poorly secreted (or unstable after secretion). As a result, these cells only harbor a short flagellum compared to those that are able to modify CcFljK with Pse (Figure S5C).

HPLC of the acid extracts from flagellins have been performed on purified flagella obtained by ultracentrifugation. As C. crescentus cells expressing BsFlmG and Cc-BsFlmG harbor no or short flagellar filament, the purification by ultracentrifugation is limited. Thus, to further confirm that CcFlmG has transferred Pse and Cc-BsFlmG (and BsFlmG) has transferred Leg on CcFljK (former Figure 5C and now Figure 7C), we performed immunoblots on the cell extracts of C. crescentus ΔflmG ΔpseI cells that cannot produce Pse but able to produce Leg (reconstituted pathway). These experiments, now presented in Figure 7C (lower panel) confirmed that no modification of CcFljK was observed in C. crescentus cells expressing CcFlmG whereas CcFljK is modified in C. crescentus expressing Cc-BsFlmG, confirming that Cc-BsFlmG has transferred Leg (the only NulO produced in this condition).__*

Text in discussion, lines 334-338, "By extension, having recognized the LegX/PtmE enzyme as a critical element in the Leg-specific enzymatic biosynthesis step (Figure 6) likewise offers another functional, but also a novel bioinformatic, criterion for the correct assignment and discrimination of predicted stereoisomer biosynthesis routes residing in ever-expanding genome databases" It will be nice to see a discussion on the prevalence of PtmE versus GlmU (or equivalent gene), PtmF, PtmA, PgmL in the Leg synthesizing organisms. Is the PtmE but not the other genes found in all cases, which makes it better as a molecular determinant for bioinformatics predictions of the type of pathway? Also, on whether PtmE has any homology to genes in other pathways (not associated with flagellin glycosylation) and how reliable a marker it is to differentiate Leg biosynthesis from Neu5Ac biosynthesis pathways.

GlmU is a potential bifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase that can be part of both Pse and Leg pathway (PMID 19282391). Accordingly, a GlmU ortholog is found in C. crescentus and B. subvibrioides that we showed are producing Pse and Leg, respectively. Thus, GlmU cannot be attributed to a Leg pathway signature. On the other hand, PtmE is barely found in the organisms from which PseI orthologs restore the motility of C. crescentus ΔpseI cells.

PtmF, PtmA, PgmL and GlmS are proposed to act upstream of the production of GlcN-1-P that is a precursor of both UDP-GlcNAc and GDP-GlcNAc, the precursors of Pse and Leg respectively. In addition, orthologs of these genes are not prevalent in the Leg synthetizing organisms present in Table S2 using BlastP analyses with C. jejuni proteins as templates.PtmE ortholog is found in most of the Leg synthetizing organisms as shown in Table S2 and often genetically linked with other genes coding for proteins involved in Leg production (shown with the asterisk * in table S2). Of note, PtmE is found not only in organisms that modify flagellin(s) with Leg but also in organisms that add Leg on capsule such as A. baumannii LAC-4.

It is not clear from the methods or the figure legends how many times the immunoblotting, motility experiments were done; how many experiments/trials are the images representative of? The motility pictures are representative of three independent experiments. The immunoblots are representative of at least two independent experiments. This information is now added in the Experimental procedures section.

Minor comments:

1. The gene for GlcN-1-P guanylyltransferase in the Leg-specific enzymatic biosynthesis step is already known as PtmE from the work of Schoenhofen's group. For the sake of consistency, it would be better to retain the nomenclature as PtmE throughout the manuscript instead of introducing the name LegX, which makes it sound like it is a previously unknown gene.

2. Text in abstract, lines 15-17: "Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages" The glycosyl donor is the nucleotide sugar and not the nonulosonic acid or sialic acid... rephrasing required for accuracy. Done

3. Text in abstract, lines 18: "a new class of FlmG protein glycosyltransferases that modify flagellin" The authors are presumably referring to FlmG as the new class of protein glycosyltransferases... rephrasing required for accuracy Corrected
4. Text in introduction, lines 41-42 "Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments" It should be "derivatives of Pse and Leg precursors" and not "Pse and Leg derivatives" corrected

5. Text in introduction, line 46 "Pse- or Leg-decorated flagella may also be immunogenic." This sentence is not referenced and it is not clear why it is written here.

6. Text in introduction, lines 63-66 "The synthesis of CMP-Pse or CMP-Leg proceeds enzymatically by series of steps [20-22], ultimately ending with the condensation of an activated 6-carbon monosaccharide (typically N-acetyl glucosamine, GlcNAc) with 3-carbon pyruvate (such as phosphoenolpyruvate, PEP) by Pse or Leg synthase paralogs, PseI or LegI, respectively" The synthesis begins with activated GlcNAc. The substrate for condensation is not activated GlcNAc. It is 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose in case of LegI and 2,4-diacetamido-2,4,6-trideoxy-b-L-altropyranose in case of PseI. Indeed, we modified the sentence.

7. Text in introduction, line 70 "for used as glycosyl donors" Typographical error, "for use as glycosyl donors" Corrected
8. Text in Results, line 102, "C. crescentus only encodes only PseI" Do the authors mean "only one PseI"? Corrected
9. Text in Results, lines 108 and 109, "Such modifications could occur before the PseI synthase acts or afterwards. In the latter case, most (if not all) synthases would be predicted to produce the same Pse molecule," Do the authors know of any reports of modifications occurring before the PseI synthase? Please cite references, if known. Why "most (if not all)"? If the former case is true, the PseI synthase might not be able to accept the substrate. Correct. Because we cannot test all enzymes we must keep the statement non-committing.

“Most (if not all)” refers to the latter case i.e. the modification occurs after PseI synthase. In this context, PseI should do the same reaction, however, there might be some exceptions.

There is, to our knowledge, no reports showing that modifications occur before the PseI synthase. The glyco-profiling experiments all suggest that modification occurs after Pse production based on our motility readout. It is possible that PseI enzymes that condense a modified precursor would not be functional in our motility assay.

Text in Results, lines 141-143, "our bioinformatic searches using C. jejuni 11168 as reference genome identified all six putative enzymes in the B. subvibrioides ATCC15264 genome (CP002102.1) predicted to execute the synthesis of Leg from GDP-GlcNAc" Not clear how this was done. Do the authors mean that they used the genes from C. jejuni 11168 as the query genes to identify homologs in B. subvibrioides ATCC15264 genome (CP002102.1)? Or did they use putative genes from B. subvibrioides ATCC15264 genome (CP002102.1) and pull out homologs from C. jejuni 11168 by using C. jejuni 11168 as the reference genome? We now have modified the sentence to make it clearer.

At first reading, the flow of the manuscript is difficult to follow due to the figures not appearing in full in order of their occurrence. For instance, Figures 5B and 5C are discussed only in the end of the manuscript after the results of Figures 6 and 7. Other instances also exist. The authors may consider re-ordering the figure parts if possible so that all parts of each figure appear in order of occurrence in the manuscript text. Thanks for raising this issue. We have now tried to address this concern by re-organizing the order of occurrence of the figures. Notably we have now exchanged Figure 5 (on Leg pathway reconstitution and FlmG rewiring) with Figure 7 (on LegB and LegH). We modified the text accordingly. We hope that it makes the manuscript and corresponding figures easier to follow.

Reviewer #3 (Significance (Required)):

The nonulosonic acids, Pseudaminic acid and Legionaminic acid, are abundant in bacterial systems in the capsular and lipopolysaccharides as well as in glycoprotein glycans. The Ser/Thr-O-nonulosonic acid glycosylation of flagellins has been studied with respect to the system of Maf glycosyltransferases in Campylobacter jejuni, C. coli, Helicobacter pylori, Aeromonas caviae, Magnetospirillum magneticum, Clostridium botulinum and Geobacillus kaustophilus, and recently with respect to the system of FlmG glycosyltransferases by Viollier's group in Caulobacter crescentus. However, the determinants that govern the glycosyltransferase function are not still well known. Kint et al have performed excellent work using bacterial genetics tools to (1) highlight the "functional insulation" of the Leg and Pse biosynthesis pathways, (2) demonstrate the modularity of the FlmG glycosyltransferase proteins with respect to the flagellin binding and glycosyltransferase domains. This work makes a significant advance in the field with respect to (1) understanding flagellin glycosylation by FlmG, (2) making designer protein Ser/Thr-O-glycosyltransferases, and (3) bioinformatics analysis of genomes with respect to the Pse/Leg/Neu nonulosonic acid biosynthetic potential encoded. The findings will be of great interest to scientific audiences working in the areas of glycobiology and bacteriology. My area of expertise: Maf flagellin glycosyltransferases

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary of the findings and key conclusions (including methodology and model system(s) where appropriate): Kint et al describe a neat study of bacterial flagellin glycosylation by a recently identified class of protein glycosyltransferases called FlmG. The experiments are well designed, the data presented is convincing and the conclusions drawn are mostly in line with the experimental evidence presented.<br /> These are the key findings. Kint et al show that genetic tools and motility can be used as a readout to probe the sugar biosynthesis pathway in bacteria. Using the recently characterized system of Caulobacter crescentus, they have performed a survey of different PseI/LegI/NeuB genes from various bacteria, checking whether they could rescue the motility defect in C. crescentus ΔpseI cells. They found that those genes that did confer motility also had higher sequence similarity to C. jejuni PseI than to C. jejuni LegI or C. jejuni NeuB. They also found that these genes also restored flagellin glycosylation as checked by mobility shift on gel electrophoresis with immunoblotting to anti-FljK antibody. This survey brought up an interesting finding that the PseI/LegI/NeuB orthologs of the closely related Brevundimonas species were unable to confer motility to C. crescentus ΔpseI cells, and were more similar to C. jejuni LegI than to C. jejuni PseI or C. jejuni NeuB. They also performed similar glycoprofiling experiments using B. subvibrioides ΔlegIBs cells and various PseI/LegI/NeuB orthologs from different bacteria, which indicated the restoration of motility by putative LegI synthases. Kint et al demonstrate flagellin glycosylation in B. subvibrioides by performing in-frame deletions of FlmG, and LegI genes in B. subvibrioides and checking for motility, presence of flagella, and flagellin glycosylation by motility shift on gel electrophoresis. Further, they confirm the critical nature of GDP-GlcNAc for Leg biosynthesis by assessing flagellin glycosylation and motility in B. subvibrioides with an in-frame deletion in PtmE/LegX and by performing heterologous complementation with an M. humiferra PtmE ortholog. They also reconstitute the legionaminic acid biosynthesis pathway in C. crescentus cells that lack flagellins, PseI and FlmG, and show that the heterologously expressed B. subvibrioides flagellin is glycosylated by heterologously expressed B. subvibrioides FlmG. Finally, they also show that whereas the CcFlmG cannot substitute for BsFlmG and vice versa, a chimeric FlmG bearing the TPR domain from C. crescentus FlmG (that recognizes C. crescentus FljK) and the GT domain from B. subvibrioides FlmG (that transfers CMP-Leg) modifies CcFljK in C. crescentus cells that lack CcFlmG but express both Pse (endogenously) and Leg (from the reconstituted pathway). This demonstrates the modularity of the FlmG glycosyltransferases. Kint et al provide the chemical nature of C. crescentus flagellin glycosylation. Kint et al have analyzed the glycans released from the flagellin by acid hydrolysis and clearly shown the nature of the glycan in C. crescentus flagellin to be Pse4Ac5Ac7Ac by use of Pse standards. The glycan from B. subvibrioides was distinct from the Leg standard used, and could be a Leg derivative distinct from Leg5Ac7Ac.

Major comments:

1. Table 1 and Text in Results, lines 116-119, "In support of the notion that derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg, our glyco profiling analysis revealed that putative PseI proteins (identified by sequence comparisons to C. jejuni 11168, Table S1) conferred motility to C. crescentus ΔpseI cells, whereas putative LegI synthases did not." Not clear how putative PseI and LegI synthases were identified. Table 1 only lists overall percent sequence identity and similarity to Cj PseI, LegI and NeuB, and percent identities and similarities of the various nonulosonic synthases to these proteins are in the similar range, as expected. In the absence of sequence alignments indicating the presence of conserved residues, particularly related to the substrate binding region, that are distinct in these paralogs, calling out the type of synthase based on the highest percent identity (to Cj PseI, LegI or NeuB) is speculative. Also, Shewanella oneidensis does not follow the pattern of highest similarity to NeuB3. Second, in the absence of data showing that the Leg and Pse found in these different organisms actually are different derivatives, this does not support that "derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg".
2. Related to (1), Text in Results, lines 130-131, "We conclude from our survey that (heterologous) PseI synthase activity generally confers motility to C. crescentus ΔpseI cells, whereas LegI-type (or NeuB-type) synthases are unable to do so." There is no a priori evidence provided indicating that these were PseI or LegI type synthases. So the conclusion really is that assuming only PseI type synthases would be able to rescue the motility defect in C. crescentus ΔpseI cells, this glyco-profiling motility assay now provides the first biochemical evidence telling us which synthases are Pse-type, and which are Neu/Leg-type. And in my view, this is the conclusion of greater significance in the field - to be able to now identify which is a PseI and which a LegI based on these complementation assays. However, if the authors still wish to retain their original conclusion, they could cite or provide evidence (either biochemical evidence in this work or reported literature regarding the sugar synthesized or bioninformatics analysis regarding the presence of distinct genes such as the Ptm genes for legionaminic acid biosynthesis pathway or genes that differ in their enzyme activities and overall fold such as PseB/LegB or PseG/LegG in the gene neighborhood) indicating or suggesting the PseI/LegI/NeuB nature of the different synthases. Also, methods for the bioinformatics analysis (eg. BLASTp settings used, dates of searches, whether regular BLAST or PSI-BLAST was used, etc.) are missing in the manuscript, and need to be included.
3. It is interesting that there is still a signification amount of flagellin secretion/assembly in the B. subvibrioides LegI and FlmG mutants. It will be good to see a discussion about whether this is likely from due to low level of function despite the in-frame deletion of genes; how many flagellin subunits are likely to have managed secretion and assembly in these short flagella; whether there is any redundancy of LegI / FlmG (perhaps with lower levels of expression); considering Parker and Shaw's findings of glycosylation being required for flagellin binding to the chaperone and subsequence secretion in A. caviae whether there is a FlaJ homolog in B. subvibrioides. Also, can the authors rule out the possibility that absence of glycosylation does not affect flagellin assembly but makes the flagellum prone to shear/breaks in B. subvibriodes, resulting in smaller flagella? How many flagellins are there in B. subvibrioides? Is it possible that one is glycosylated but another/others are not, and that is the reason for the small flagellum in these mutants?
4. Text in Results, lines 170-171, "We then probed the resulting ΔlegIBs and ΔflmGBs single mutants for motility defects in soft agar and analyzed flagellin glycosylation by immunoblotting using antibodies to FljKCc". Was the antibody to FljKCc determined to also specifically bind to FljKBs? Also, how many flagellins are there in B. subvibrioides? Are all detected with this antibody?
5. It is interesting that C. cresentus cells expressing Pse (endogenously) and Leg (reconstituted pathway), and BsFlmG and BsFljK (corresponding to Figure 5C) are not motile. Was the motility assay done for the experiment of figure 5B as well? Are the C. crescentus cells lacking Pse and FlmG but with heterologous expression of Leg and BsFljK and BsFlmG also non-motile? Also, it will be good to see the TEM images for these cells.
6. Immunoblotting of the supernatants should be shown (in addition to the cell extracts) for Figures 5B and 5C so that the reader can appreciate whether glycosylation has taken place but secretion/assembly has not. Further, HPLC of the acid extracts from flagellin could be done to unambiguously show whether the CcFlmG has transferred Pse and the BsFlmG and Cc-BsFlmG have transferred Leg on to the CcFljK in Figure 5c, and the identity of the sugar, if any, transferred by CcFlmG in the absence of Pse, and BsLeg genes or BsLegX gene in figure 5B.
7. Text in discussion, lines 334-338, "By extension, having recognized the LegX/PtmE enzyme as a critical element in the Leg-specific enzymatic biosynthesis step (Figure 6) likewise offers another functional, but also a novel bioinformatic, criterion for the correct assignment and discrimination of predicted stereoisomer biosynthesis routes residing in ever-expanding genome databases" It will be nice to see a discussion on the prevalence of PtmE versus GlmU (or equivalent gene), PtmF, PtmA, PgmL in the Leg synthesizing organisms. Is the PtmE but not the other genes found in all cases, which makes it better as a molecular determinant for bioinformatics predictions of the type of pathway? Also, on whether PtmE has any homology to genes in other pathways (not associated with flagellin glycosylation) and how reliable a marker it is to differentiate Leg biosynthesis from Neu5Ac biosynthesis pathways.
8. It is not clear from the methods or the figure legends how many times the immunoblotting, motility experiments were done; how many experiments/trials are the images representative of?

Minor comments:

1. The gene for GlcN-1-P guanylyltransferase in the Leg-specific enzymatic biosynthesis step is already known as PtmE from the work of Schoenhofen's group. For the sake of consistency, it would be better to retain the nomenclature as PtmE throughout the manuscript instead of introducing the name LegX, which makes it sound like it is a previously unknown gene.
2. Text in abstract, lines 15-17: "Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages" The glycosyl donor is the nucleotide sugar and not the nonulosonic acid or sialic acid... rephrasing required for accuracy.
3. Text in abstract, lines 18: "a new class of FlmG protein glycosyltransferases that modify flagellin" The authors are presumably referring to FlmG as the new class of protein glycosyltransferases... rephrasing required for accuracy
4. Text in introduction, lines 41-42 "Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments" It should be "derivatives of Pse and Leg precursors" and not "Pse and Leg derivatives"
5. Text in introduction, line 46 "Pse- or Leg-decorated flagella may also be immunogenic." This sentence is not referenced and it is not clear why it is written here.
6. Text in introduction, lines 63-66 "The synthesis of CMP-Pse or CMP-Leg proceeds enzymatically by series of steps [20-22], ultimately ending with the condensation of an activated 6-carbon monosaccharide (typically N-acetyl glucosamine, GlcNAc) with 3-carbon pyruvate (such as phosphoenolpyruvate, PEP) by Pse or Leg synthase paralogs, PseI or LegI, respectively" The synthesis begins with activated GlcNAc. The substrate for condensation is not activated GlcNAc. It is 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose in case of LegI and 2,4-diacetamido-2,4,6-trideoxy-b-L-altropyranose in case of PseI.
7. Text in introduction, line 70 "for used as glycosyl donors" Typographical error, "for use as glycosyl donors"
8. Text in Results, line 102, "C. crescentus only encodes only PseI" Do the authors mean "only one PseI"?
9. Text in Results, lines 108 and 109, "Such modifications could occur before the PseI synthase acts or afterwards. In the latter case, most (if not all) synthases would be predicted to produce the same Pse molecule," Do the authors know of any reports of modifications occurring before the PseI synthase? Please cite references, if known. Why "most (if not all)"? If the former case is true, the PseI synthase might not be able to accept the substrate.
10. Text in Results, lines 141-143, "our bioinformatic searches using C. jejuni 11168 as reference genome identified all six putative enzymes in the B. subvibrioides ATCC15264 genome (CP002102.1) predicted to execute the synthesis of Leg from GDP-GlcNAc" Not clear how this was done. Do the authors mean that they used the genes from C. jejuni 11168 as the query genes to identify homologs in B. subvibrioides ATCC15264 genome (CP002102.1)? Or did they use putative genes from B. subvibrioides ATCC15264 genome (CP002102.1) and pull out homologs from C. jejuni 11168 by using C. jejuni 11168 as the reference genome?
11. At first reading, the flow of the manuscript is difficult to follow due to the figures not appearing in full in order of their occurrence. For instance, Figures 5B and 5C are discussed only in the end of the manuscript after the results of Figures 6 and 7. Other instances also exist. The authors may consider re-ordering the figure parts if possible so that all parts of each figure appear in order of occurrence in the manuscript text.

Significance

The nonulosonic acids, Pseudaminic acid and Legionaminic acid, are abundant in bacterial systems in the capsular and lipopolysaccharides as well as in glycoprotein glycans. The Ser/Thr-O-nonulosonic acid glycosylation of flagellins has been studied with respect to the system of Maf glycosyltransferases in Campylobacter jejuni, C. coli, Helicobacter pylori, Aeromonas caviae, Magnetospirillum magneticum, Clostridium botulinum and Geobacillus kaustophilus, and recently with respect to the system of FlmG glycosyltransferases by Viollier's group in Caulobacter crescentus. However, the determinants that govern the glycosyltransferase function are not still well known. Kint et al have performed excellent work using bacterial genetics tools to (1) highlight the "functional insulation" of the Leg and Pse biosynthesis pathways, (2) demonstrate the modularity of the FlmG glycosyltransferase proteins with respect to the flagellin binding and glycosyltransferase domains. This work makes a significant advance in the field with respect to (1) understanding flagellin glycosylation by FlmG, (2) making designer protein Ser/Thr-O-glycosyltransferases, and (3) bioinformatics analysis of genomes with respect to the Pse/Leg/Neu nonulosonic acid biosynthetic potential encoded. The findings will be of great interest to scientific audiences working in the areas of glycobiology and bacteriology. My area of expertise: Maf flagellin glycosyltransferases

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary:

Viollier and co-workers present a study in which they preform an elegant and rigorous genetic profiling of the the legionaminic and pseudaminic acid biosynthesis and flagellar glycosylation pathways in C. crescentus (native Pse) and B. subvibrioides (native Leg). They use motility as a representative readout for functional flagellar glycosylation with these microbial sialic acids. They discover orthologous Pse synthase genes can replace the function of the native synthase in C. crescentus and orthologous legionaminic acid synthase genes can achieve the same in B. subvibrioides. However, not vice versa indicating a strong preference for each microbial sialic acid stereoisomer in these species. For the Leg biosynthesis pathway, which requires GDP-GlcNAc, the authors also identify LegX as an essential component to synthesize this sugar nucleotide and thus a marker for Leg biosynthesis pathways. Upstream in theses pathways, they also identify a new class of FlmG flagellar protein glycosyltransferases. Importantly, through heterologous reconstitution experiments to uncovered that these glycosyltransferases possess two distinct domains, a transferase domain the determines specificity for either CMP-Leg or CMP-Pse, and a flagellin-binding domain to achieve selectivity for the substrate. Interestingly, by creating chimeric FlmG for these two domains between C. crescentus and B. subvibrioides they show that these two modular parts can be interchanged to adapt flagellin glycosyltransferase specificity in these species.

Major comments:

The key conclusions of the manuscript by Viollier and co-workers are convincing and well supported by their experiments and used methods, with respect to the insulation of the Leg and Pse biosynthetic pathways, they key role of LegX in launching the Leg pathway and the successful reconstitution of Leg glycosylation in a previously Pse-producing C. crescentus strain. Finally, they convincingly show that a chimeric version of the involved glycosyltransferases is functional, which besides intriguing future glycoengineering possibilities also emphasizes the two discrete domains in these transferases that dictate their sugar nucleotide and acceptor specificity. There is one additional experiment I would suggest with relation to the detection and confirmation of Pse and Leg on flagella of respectively, C. crescentus and B. subvibrioides. In the case of C. crescentus the detected DMB derivatized monosaccharide co-elutes with a validated standard of tri-acetylated Pse, which is convincing evidence of its identity. However, for B. subvibrioides. Their DMB derivatized monosaccharides from its flagella, results in a peak the does not co-elute with the only Leg standard (Leg5Ac7Ac) they have, it does elute at the same time as their Pse standard. Although it cannot of course be Pse as B. subvibrioides. Does not possess a Pse biosynthesis pathway, it also does not provide enough evidence to conclude that it is a Leg derivative. An MS(-MS) measurement of the eluted signal would not be a big investment in time and resources and would provide additional evidence to at least assign this peak to microbial sialic acid related to the present Leg biosynthesis pathway. It the identified mass would lead to identification of the derivative, it would also add to the proper characterization of the flagella glycosylation in the bacterium. The data and the methods presented in this study are presented with sufficient detail so that they can be reproduced? However, I would suggest as is common nowadays in most journals that the authors include images of the raw unprocessed blot in de supporting info.

Minor comments:

There are a few textual errors that the authors should fix:

• page 2, line 70: change "used" to "use"
• page 11, line 407: add the word "are" after Pse
• On page 2, line 36, the authors state that "most eubacteria and the archaea typically decorate their cell surface structures with (5-, 7-)diacetamido derivatives, either pseudaminic acid (Pse) and/or its stereoisomer legionaminic acid (Leg,". This should be nuanced as to my knowledge it is not most eubacteria, but more a subset as identified by Varki in his seminal PNAS paper. The authors clearly present their data and conclusions in the figures of this manuscript. However, I would recommend the take a critical look at the drawing of their monosaccharide chair conformations and the positioning of the axial and equatorial groups on these chairs in Figure 1 and 5, as these are in most cases drawn a bit crooked, which can easily be corrected.

Significance

The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in specific microbes. Especially symbiotic and pathogenic microbes associated with the humans express a wide array of unique microbial sialic acids for which their functional roles are not well understood and the associated glycosylhydrolase and glycosyltransferase have in most cases not been identified yet. The authors present an impressive insight into flagellar glycosylation with Pseudaminic and Legionaminic acid in two bacterial species, using genomic analysis, rewiring, immunoblots and motility assays as their main tools. They provide compelling evidence on the insulation of the Pse of Leg pathway in these species, the flexibility in exchanging between biosynthetic enzymes from the same pathway between various species. Crucially, most glycosyltransferases that add the Pse or Leg glycoform onto various acceptor sites in bacteria, have up to this point remained elusive in most cases. It is therefore very valuable information that the authors here provide on the involved glycosyltransferases. Especially, on the two domains that govern their sugar nucleotide and acceptor specificity, and that these can be reengineered as chimeric glycosyltransferases. To me as a chemical glycobiologist this provides compelling possibilities for glycoengineering possibilities in future studies in the field to elucidate the functional roles of Pse and Leg glycosylation.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

In this manuscript, authors establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. They used B. subvibroides and C. crescentus specific mutants in pseI and legI genes involved in the Pse and Leg biosynthesis, respectively, and cross-complementation assays with orthologous genes from different bacterial species, analysing motility and flagellin glycosylation. These assays show that Pse and Leg biosynthetic pathways are genetically different and recognize the LegX enzyme as a critical element in the Leg-specific enzymatic biosynthesis. Since that legX orthologous were only identified in the genome of bacteria with Leg biosynthetic pathways, it becomes a good marker to distinguish Leg from Pse biosynthesis pathways and a novel bioinformatic criterion for the assignment and discrimination of these two pathways. Reconstitution of Leg biosynthetic pathway of B. subvibroides in the C. crescentus mutant that lack flagellins, PseI and FlmG, complemented with both flagellin and FlmG of B. subvibroides, identified a new class of FlmG protein glycosyltransferases that modify flagellin with legionaminic acid. Furthermore, the construction of a chimeric FlmG through domain substitutions, allowed to reprogram a Pse-dependent FlmG into a Leg-dependent enzyme and reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

Major comments:

The conclusions obtained are convincing and well-supported. However, I think some points should be specify or clarify.

1. • In the mutants (pseI, legI, flmG,...) the non-glycosylated flagellin are exported and assembled in a flagellum filament shorter than the WT strain. However, motility in plates is absent or very reduced. This might be produced by instability of the flagellum filament when rotating in a semi-solid surface. MET was performed from plates or liquid cultures? Do the author analyses motility in liquid media? If they did, changes in motility were observed?
2. • In page 5, lines 158-163, the analysis, by HPLC, of derivatized nonulosonic acid from B. subvibroides flagella, shows a major peak at 9.8 minutes retention and a minor peak at 15.3 minutes. Since that Pse-standard have retentions peaks at 9.7 and 13 minutes, and Leg-standard at 12.3 minutes, the authors cannot infer, only with these data, the flagella sugar is a legionaminic acid derivative. In my opinion, should be included that inference comes from the data obtained by HPLC analysis and genetic approaches.
3. • In page 5, line 173-175. Authors indicate, "While no difference in the abundance of flagellin was observed in extracts from mutant versus WT cells, flagellin was barely detectable in the supernatants of mutant cultures, suggesting flagellar filament formation is defective in these mutants". MET images show that the flagellum filament length is shorter in the mutants than in the WT strain. Therefore, if the same number of mutants and WT cells has been used in the immunodetection assays, there should be more flagellin monomers in the WT samples than in the mutants ones and flagellin bands should be less intense in mutant samples corresponding to the anchored flagellum. Why bands corresponding to flagellin in mutants and WT show similar intensity in the immunodetection assays (Figure 3C and D)? Furthermore, in lane 177-178, authors suggest that LegI and FlmG govern flagellin glycosylation and export (or stability after export). However, if filament stability is affected, the amount of flagellin monomers in the supernatant of mutants should be higher than in the WT. However, immunodetection assays show less abundance of flagelin monomers in the supernatant of mutants. Please, can you clarify this? In relation to this point, I suggest that authors include, in the experimental procedures, how they obtained the supernatants to flagellin immunodetection, as well as why they used anti- FljKCc anti-serum to detect the B. subvibroides flagellin.
4. • Authors demonstrate the specificity of the GT-B domain of FlmG, using a chimeric FlmGCc-Bs in a mutant of C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides. However, since that TPR comes from C. crescentus, this chimeric protein, could be transfer the legionaminic acid to the flagellin of B. subvibroides? Furthermore, the complementation of this mutant with the FlmGBs did not support efficient flagellin modification and this might be related to the TPRCc domain. Therefore, in my opinion, the chimeric protein should be introduced in the B. subvibroides∆flmG background.
5. • Page 8, line 299-301. Authors point out that C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides and the chimeric FlmGCc-Bs, although it has a glycosylated flagellin, whose mobility in SDS-PAGE is like the WT strain, is non-motile. They suggest that additional factors exist in the flagellation pathway that exhibit specificity towards the glycosyl group that is joined to flagellins. However, would be interesting to see if the flagellum filament has similar length to the WT strain or at least, it has increased in relation to the flagella length of the mutant. If flagella length has not increased, it could suggest that changes in the glycan type might affects the flagellin assembly or the stability of the flagellum filament. Therefore, would be also important to analyse its motility in liquid media.

Minor comments:

1. • Pag 3 line102. Please change ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes only PseI...." to ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes a PseI...."
2. • Figure 2 A. Plasmid nomenclature (Plac-neuB) is confusing because C.c. ΔpseI cells express predicted LegI or PseI synthases. Please change to Plac, as in Figure 2B and 4. Figure 2A and 2B do not contain any complementation with Bacillus subtilis (Basu), however two complementation are labelled as Bs in Figure 2A and 2B. Furthermore, no Bs are present in the Figure 2 legend.
3. • Legend of figure 3 should include B. subvibrioides abreviation Bs. Line 774: Please change ".......glycosylation and secretion in B. subvibrioides." to ".......glycosylation and secretion in B. subvibrioides (Bs)."
4. • Figure 3. In order to keep a similar nomenclature in all plasmids, plasmid Plac-legI syn and Plac-flmG should be labelled as Plac-legIBs syn and Plac-flmGBs.
5. • Legend of figure 4 should include B. subvibrioides abreviation Bs. Line 791: Please change "....... complementation of the B.subvibrioides ΔlegI mutant with ...." to "....... complementation of the B.subvibrioides (Bs)ΔlegI mutant with ...." Furthermore, Legend of figure 4 indicate in line 795, that immunoblots reveal the intracellular levels of flagellin, however figure 2 and 3 show immunoblot of cell extracts. Please, correct this sentence.
6. • Legend of figure 5, 6 and 7 should include B. subvibrioides abreviation Bs. Line 808: Please change "Predicted Leg biosynthetic pathway in B. subvibrioides " to"Predicted Leg biosynthetic pathway in B. subvibrioides (Bs)" Line 834: Please change "....affects motility, flagellin glycosylation and secretion in B. subvibrioides."to "....affects motility, flagellin glycosylation and secretion in B. subvibrioides (Bs).Line 852: Please change "...acetyltransferase in flagellar motility of B. subvibrioides cells." to ""...acetyltransferase in flagellar motility of B. subvibrioides (Bs) cells." Furthermore, figure 5 should include C. crescentus abbreviation. Line 815: Please change "....whole cell lysates from C. crescentus mutant cultures......." to "....whole cell lysates from C. crescentus (Cc) mutant cultures......."
7. • In my opinion it would be useful to include a scheme of the gene organization involved in Leg biosynthesis in B. subvibrioides.
8. • Legend of figure S1 should include B. subvibrioides (Bs) and C. crescentus (Cc) abbreviations. Line 888-867: Please change "...C. crescentus ΔpseI cells and B. subvibrioides ΔlegI cells with plasmids expressing..." to "...C. crescentus (Cc) ΔpseI cells and B. subvibrioides (Bs) ΔlegI cells with plasmids expressing..." Furthermore, the name and abbreviations (Mm, So, Ku, Pi, Dv) of the species used should be included in the legend. Why the authors used a plasmid with a Pvan promoter in these assays? Why the authors changed the code color of pseI and legI orthologous genes? It would be more useful and understandable follow the code color used in figure 2 and 4.
9. Page 6 line 200, Please change ".....complementing synthases exhibit greater overall sequence similarity to LegI than Pse of C. jejuni. 22268,....." to ".....complementing synthases exhibit greater overall sequence similarity to LegI than PseI of C. jejuni. 22268,....."
10. • Page 7 line 231, Please change ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and P. sp. Irchel 3E13..." to ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and Pseudomonas sp. Irchel 3E13..."
11. • Introduce a line break between line 503 and 504.
12. • Page 14 line 543, please change "XbaI" to "XbaI"

Significance

This is an interesting manuscript that contributes to the knowledge of the legionaminic biosynthetic pathway and establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. The analysis of Leg patway allowed to identify a gene (legX) that can be used to distinguish Leg from Pse biosynthesis pathways, becoming a bioinformatic tool for the assignment and discrimination of these two pathways. Furthermore, a new class of FlmG protein glycosyltransferases, able to transfer Leg to the flagellin, has been identified and its analysis reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

the authors do not wish to provide a response at this time.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

The manuscript by Wong et al., provides the first report of a homozygous loss-of-function mutation of the RAF1 gene in humans. The mutation (T543M) was found in two siblings of a consanguineous family in association with perinatal death and multiple developmental abnormalities. These abnormalities show strong similarities with a rare congenital malformation syndrome with unknown aetiology named Acro-cardio-facial syndrome (ACFS, MIM600460). Conversely, reported abnormalities are different from those observed in RASopathies, congenital diseases caused by gain-of-function mutations in either the RAF gene or genes implicated in the same signaling pathway (MAPK) and that induce its ectopic/over-activation. By performing functional experiments in cellular systems (cell line where the mutated RAF was either overexpressed or knocked-in) and in Xenopus Laevis embryos, the authors demonstrate that the reason for the phenotypic differences compared to RASopathies is that the RAF1T543M variant impairs the signaling activity of RAF and blunts MAPK pathway activation. In particular, the RAF1T543M variant: 1) is not actively phosphorylated at key activating residues, 2) is unable to transduce MAPK signaling towards MEK/ERK substrates, 3) is inherently unstable and prone to proteasome-mediated degradation and 4) is unable to block stress-induced apoptosis. On the basis of increased apoptosis detected in cellular systems and some morphological defects observed in the probands, the author classify this novel syndrome as a segmental progeroid syndrome.

Major comments:

The authors perform a thorough analysis of the structural and functional defects of the RAF1T543M protein, using in silico analyses and in vitro systems. The data are corroborated by an elegant and clear-cut experiment in Xenopus embryos that demonstrates that the mutated RAF is not able to transduce MAPK signaling when overexpressed in an in vivo model. The lack of patients' material prevents a validation in human cells, but I think the evidence collected in the manuscript is supportive of the loss-of-function mutation in RAF as the causative mutation of the observed phenotype.

The concept illustrated in the last sentence of the discussion, i.e. that different mutations in the same genes can either cause cancer (when overactivating) or premature aging (when blunting the activity of the enzyme) is fascinating. I think discussing this concept in the context of this manuscript is appropriate. However, the authors classify the syndrome caused by the RAF1T543M variant as a "novel segmental progeroid syndrome" in the title, abstract and first sentence of the discussion. I don't think that presented data are convincing for this classification. Indeed, two affected siblings die at very early post-natal stages (7 and 50 days, respectively) likely because of malformations that are not compatible with life and that are due to altered in uthero development. Progeroid syndromes are a heterogeneous group of syndromes, a minority of which is characterized by malformations at birth. The more general concept is that physical abnormalities are progressively acquired during post-natal life, in specific tissues that show typical features associated with aging, including senescence, decline of the stem cell compartment, increased inflammation. In the case of individuals carrying the RAF1T543M variant, none of these tissue abnormalities have been reported (due to the lack of material from patients) and the classification as a progeroid syndrome is based on external inspection of organs. Reported phenotypes are not specific and a failure of in uthero development of heart, limbs and other organs seems like the absolutely predominant trait. This in uthero phenotype is perfectly consistent with the physiological role of the MAPK pathway downstream multiple RTKs that transduce morphogenetic, in addition to mitogenic, signals. As for the authors words: "endogenous FGF/FGFR signaling is required for proper mesoderm and neural induction" and supports the author claim that the analysis of individuals carrying the RAF1T543M variant "underscores the importance of RTK signaling during human development". I think that the "progeroid" phenotypes are less clear. It is still important to present the phenotypes reminiscent of progeroid syndromes and discuss them and perhaps more clearly explain the logical connection with the increased apoptosis observed in in vitro experiments.

In regard to the apoptosis experiment in figure 4, it supports the claim that RAF1 activity is necessary for protection from stress-induced apoptosis. However, the cartoon presented in figure 4C also shows that this process is mediated by increased ASK1/MST2 signaling. This part of the cartoon is based on the literature and has not been formally demonstrated. The authors can try to rescue the apoptotic status by silencing ASK1 in the RAF1T543M cells, similar to what has been done in Yamaguchi, O., 2004, Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937-943. Literature also suggests that RAF1-mediated inhibition of apoptosis is kinase-independent. This part is particularly interesting since the variant produces a protein that is both kinase-inactive and unstable. It would be a nice addition to the matuscript if the authors could clarify, at least in their cellular model, whether the increased apoptosis is due to the loss of either the protein or the kinase activity.

Minor comments:

A minor comment to the Xenopus ISH: the number of embryos that have been analyzed per condition is not reported anywhere. Figure legend explains that presented images are "representative pictures", but knowing the number of tested embryos and the penetrance of the phenotype would help undertsand the relevance of the RAF mutation in the signaling pathway under investigation.

Significance

This work by Wong and colleagues provides conceptual advance and will be of interest for researchers dealing with RTK/MAPK signaling in multiple contexts including oncology, developmental biology and cardiomyopathy.

The gene under investigation is widely studied in cancer, where gain-of-function, oncogenic mutations are common. The role of RAF1 during embryonic development is less known. A couple of studies have investigated the developmental phenotype of mouse models carrying either a knock-out allele (Mikula, M, et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J. 2001. 20:1952-1962) or an allele producing a truncated protein (Wojnowski L et al., (1998) Craf-1 protein kinase is essential for mouse development. Mech Dev, 76, 141-149). A few studies have investigated the conditional inactivation in specific tissues, such as the cardiac muscle (Yamaguchi, O., 2004, Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937-943.). And one study has found heterozygous carriers of a loss-of-function mutation among cohorts of children affected by dilated cardiomyopathy (Dhandapany, P.S., et al. (2014). RAF1 mutations in childhood-onset dilated cardiomyopathy. Nat. Genet. 46, 635-639). The manuscript reports the spectrum of defects acquired during embryonic development by carriers of a pathogenic mutation in the RAF1 gene. The mutation impairs both stability and kinase activity of the protein. The manuscript points out the non-redundant role of RAF1-mediated signaling in specific organs during embryonic development.

The person who is reviewing the manuscript has expertise in cellular signaling, mouse embryonic development and human aging.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary:

The authors describe a homozygous missense variant in RAF1 identified in a child with a lethal malformation syndrome. The T543M variant not only blocks the kinase activity, but also destabilizes the RAF1 protein. In combination, this leads not only to a loss of MAPK signaling, but also to elevated apoptosis upon cell stress, which both together very likely explain the dramatic phenotype, which may correspond to the rarely described acro-cardio-facial syndrome.

Major comments:

The results of the functional analysis presented by the authors are highly convincing and clearly demonstrate a LOF effect of the identified variant. The experiments are described in sufficient detail and the statistical analysis is sound. Depending on the target journal the clinical information might be a bit scarce. There are neither clinical pictures nor molecular data from the first affected sibling. For a more clinically oriented journal, a summary of clinical features in form of a table and a comparison to ACFS will be a prerequisite and facilitates appreciation of this very special phenotype. The affected child seems much older than his chronological age, which seems due to the hypoplastic viscerocranium and the loss of subcutaneous fat tissue. The heart and limb defects have no link to chronological aging. Whether such phenotypes should be really called progeroid can be debated, but it is common practice (and always a good selling point). The authors do not discuss their findings in the light of chronological aging. The hypothesis that the RAF1 LOF liberates ASK1 leading to increased apoptosis is very attractive. It would be very nice to show some experimental data proving this, but such data is not pivotal for the paper.

Minor comments:

The facial and overall progeroid phenotype of the affected child is quite different from most of the described ACFS patients. In contrast, the prominent neurocranium with low hairline and the hypoplastic viscerocranium remind this reviewer of Gorlin-Chaudhry-Moss/Fontaine-Petty syndrome. This differential diagnosis is also interesting since the disorder is caused by GOF variants in a mitochondrial ATP transporter increasing the sensitivity for apoptosis. This underlines the suggested link between RAF1 and the apoptosis inducer ASK1. There is probably no cellular function that has not been linked to the MAPK pathway. One of these connections is to cellular aging. Strongly activating variants in pathway members lead to oncogene-induced cellular senescence. Here, we have a progeroid phenotype, but a variant with LOF effect. This might be worthwhile to discuss. The discussion could also use some sentences on the SHSF mechanism. FGFs produced by the AER are important for proliferation of the limb mesenchyme. What is the connection between MAPK and p63?

Significance

This is a central signaling pathway and a prominent and historically outstanding oncogene. Up to now all variants in the MAPK pathway have been described as GOF and this is the first phenotype related to a LOF. Thus, this is a landmark paper widening the view on the pathway. The paper is interesting for clinical and molecular geneticists, tumor biologists, and cell biologists. This reviewer is a biochemist and clinical geneticist with research focus on mechanisms of skeletal and connective tissue disorders.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In the present manuscript, Wong et al describe for the first time the human phenotype resulting from the bi-allelic germline T543M mutation in the RAF1 proto-oncogene. Although somatic RAF1 mutations are outnumbered by BRAF mutations, they also occur in human cancer, but so far germline RAF1 mutations, usually dominant-acting ones abrogating RAF1 autoinhibition, have been mainly associated with RASopathies, in particular with Noonan syndrome. Here, Wong et al describe for the first time that the two individuals with a homozygous T543M mutation is associated with a neonatal lethal progeroid syndrome presenting with cutaneous, craniofacial, cardiac and limb anomalies. Moreover, the affected residue, despite its conservation across RAF1 orthologues and in the ARAF and BRAF paralogues, has neither been described as a target of somatic and germ-line mutation in cancer and RASopathies, respectively. The authors then use a combination of ectopic expression experiments in HEK293T cells and Xenopus embryos as well as CRISPR/Cas9 genome editing and structural bioinformatics to provide several lines of evidence that RAF1 T543M represents a hypomorph suppressing ERK activation. In summary, this is a very interesting and well-written manuscript with a stimulating discussion of the novel and convincing findings and the mechanisms driving the described pathology. The suggestions below might further strengthen this otherwise already very advanced manuscript.

Major:

1. The data presented in this manuscript imply that RAF1 T543M represents a hypomorph suppressing ERK pathway activity. Nevertheless, it remains unclear whether this mutation reduces/abolishes the intrinsic activity of RAF1 or acts by a dominant-negative mechanism, e.g. by sequestrating critical components of RAF complexes, such as KSR proteins, or MEKs. To distinguish between both possibilities, it would be helpful, if the authors were able to document the intrinsic kinase activity of immunoprecipitated RAF1 T543M (and appropriate controls such as wildtype RAF1, the S257L gain-of-function allele and a truly kinase-dead variant, e.g. by mutating the aspartate of the DFG motif) towards recombinant and commercially available GST-MEK1.
2. RAF kinases engage in complex homo- and heterodimerization events. Does RAF1 T543M form homo- and heterodimers, e.g. with BRAF or ARAF, to a similar or different extent than wildtype RAF1?

Minor:

1. Abstract. To my knowledge, RAS but not RAF was the first human oncogene identified, albeit the discovery of RAF and its viral counterparts also took place very early in the oncogene discovery era.
2. Introduction, p. 2 "...while Braf-/- mice are embryonic lethal due to vascular defects.7" For a more balanced review, I would suggest citing additional and more recent papers describing the complex phenotypes of Braf deficient mice, e.g. PMIDs: 18332218 and PMID: 16432225
3. Figure 1D. Here, CRAF is used instead of RAF1. As the latter is the official gene/protein name RAF1 should be used to avoid confusion to readers outside of the RAF field.

Significance

Very interesting novel findings to researchers being active in the signalling, cancer and developmental biology fiels as well as to human geneticists and pediatricians.

My expertise: MAPK signalling, functional characterization of oncogenic mutations

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

In this paper we exploit the model system of the Y loops in Drosophila spermatocytes to provide a super-resolution view of the topology of active transcription. We are pleased that Reviewer 1 assesses the work as “highly significant”. They also mention the caveat that findings may be “specific to the model system”. Reviewer 2 provides a counter to this caveat by pointing out that our findings are in line with some previous reports in mammalian cells. The interpretation in previous studies has however been hindered by the density of chromatin in the nucleus making it difficult to clearly identify individual fibres and Reviewer 2 points out that our work provides a significant advance as in our system we are able to “visualize the organization of chromatin within an individual, isolated loop”.

*Reviewer #1 *

∙ Have you assessed if you are under-labeling the nucleosomes? or RNA Polymerase?

Yes, we tried a range of antibody concentrations which did not change the observations. We have added a statement in the M&M; “To control for under-labelling of structures, we tested a range of antibody concentrations and found no significant difference in the observed nucleosome or RNAPol distribution.”

*∙ Can you stimulate and inhibit transcription to see how changes in RNA Polymerase and nucleosome organisation occur? *

We are not aware of an approach to further stimulate transcription in this system, however we have some preliminary analyses of the effects of transcription inhibition that we plan to firm up within weeks. We agree with the reviewer that it may potentially be interesting and informative to include such studies but we do not consider this as central to the significance of the paper. So our plan is to attempt to quickly gather data on this for potential inclusion but we would plan to only include this material if we can rapidly achieve a clear understanding of the effects of transcription inhibition on nucleosome cluster organisation.

∙ The 300 msec exposure time is very long for typical STORM experiments - blinking would usually be lost at this time scale?

Apologies, this was a typo. Now corrected to 30ms.

∙ Please quote laser power in absolute values rather than percentage

Done.

∙ Collecting variable number of frames (20K - 50K) can impact the number of detections/clusters. How can you account for this variation in your analysis?

Although data was collected with variable number of frames (20K-50K), in the original paper we only used data from 50K samples for quantitative analysis. In response to reviewer #2, we have now performed the analysis on a larger data set with variable frame number. Analysis of the two data sets controls for any impact of frame number variation and makes very little difference to the results of the analysis e.g the median cluster FWHM remains at 52 nm.

Reviewer #2

∙ Pages 2/3, the authors estimate the number of nucleosomes per cluster identified in the Y loops. They do so "based on a simple volume calculation" but the quantification method and reference values used to reach the estimate of a "maximum of 158 nucleosomes" is not reported in the methods. Also, the authors should discuss how do they account for multiple blinking effect in the estimate of cluster measures. Methods based on normalization curves with nucleosome arrays (Ricci et al Cell 2015) and on DNA origami (Cella Zanacchi et al Nat Meth 2017) among others were previously used to address similar questions. The authors should consider applying a normalization approach to achieve a more accurate estimate or at least discuss the caveats of their strategy.

We have now specified the details of our estimation of the number of nucleosomes per cluster in the Materials and Methods. Techniques to quantify clusters based on localisation number have been used in the past on SMLM data, but the normalisations are difficult to validate as there is uncertainty around numbers of antibodies binding, differences in frame numbers affecting blink number, as well as unknown binding efficiencies of antibodies. Our cluster quantification instead is based on the size of the clusters (cluster width), as this does not vary significantly across frame number once signal saturation has been achieved and avoids the difficulties associated with normalisation of localisation number. As we have mentioned in the Discussion, our “simple volume calculation” does have some caveats arising from the STORM precision, the labelling method and the EM-derived estimate of packing density; nevertheless, as we say in the Discussion we consider it provides a useful “starting point for interpretation of the observed labelling in terms of underlying chromatin structure.”

∙ Methods, STORM imaging: the authors should provide the imaging buffer composition as the papers they refer to make use of an array of buffers. If possible, the authors should include laser powers in standard units (kW/cm^2) to increase clarity and reproducibility.

We have provided the imaging buffer composition and the laser power in standard units.

∙ Methods, Cluster analysis: the authors should specify the values for the parameters used in "First a description of the average cluster was established using spatial statistics, then a clustering algorithm using the average parameters provided by the initial spatial statistics further refined the description"

As detailed in response to the next comment, we have clarified the text in the Materials and Methods to specify the parameters and workflow of the analysis.

∙ Given the availability of broadly used methods for clustering such as DBScan (Ester, et al. A density-based algorithm for discovering clusters in large spatial databases with noise. Kdd, 1996), Bayesian methods (Rubin-Delanchy et al. Bayesian cluster identification in single-molecule localization microscopy data. Nature methods, 2015), (Ricci et al. Cell 2015), Voronoi based tessellation (Levet et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nature methods, 2015), and more recently machine learning approaches (Williamson et al. Machine learning for cluster analysis of localization microscopy data. Nature communications, 2020) the clustering method of choice is peculiar. It makes more difficult the direct comparisons with previous work. The authors could justify the choice or compare results using other more broadly used methods.

We have added text to justify our selected analysis method and to clarify the analysis workflow. We do not consider our approach “peculiar” as very similar approaches have been employed previously (e.g. Sengupta et al Nat. Methods 8, 969–975 (2011), Veatch et al PLoS ONE 7, e31457 (2012) and Cisse Science 341:664-667 (2013)). In selecting this approach we compared various approaches and we found Meanshift to perform best with respect to identifying clusters closely positioned along a fibre. (Example data comparing the performance of Meanshift with DBscan in cluster identification with varying radius input parameter was provided to reviewers). We concluded that MeanShift provides a more robust cluster identification and that the radius value of 2Xsigma derived from the PCF is appropriate.

∙ No statistical tests were performed in the study, although no comparisons between experimental conditions are shown. The authors mention the number of replicates performed only on Fig 3 ("13 regions of interest (ROI) were selected from 3 cells"), which is a low number of cells analyzed. The authors should enlarge the analyzed datasets and mention how many replicates were used throughout the work.

As mentioned above, the quantitative analysis originally presented used the data with the constant, highest frame number (3 cells, 895 clusters). In the revised paper we present the analysis of the wider data set (12 cells, 2473 clusters). In practice this makes very little difference to the results, e.g the median cluster FWHM remains at 52 nm.

∙It is unclear why the authors did not perform STORM on EU-labeled samples immunolabeled with Pol2 antibodies. In principle, they could have applied the same imaging protocol used in previous figures gaining spatial resolution to better characterize Y nascent transcripts and how Pol2 is arranged with respect to nascent RNA.

There are indeed a number of questions that can be approached by super-resolution imaging of nascent transcripts in association with co-labelling for RPol. We are exploring this interesting direction but this is a whole separate project and we propose to report our findings on this in a subsequent publication.

∙ The text is well organized and clear as well as the figures but the authors should revise the text in the section "linking RNA polymerase distribution and Y loop transcription" to include missing references to Figure 10. For example, the statement "This distribution of nascent transcript along the Y loops fits with the distribution of RNA-PSer2," refers to results shown in Fig 10A.

We are pleased the reviewer found the text and figures well organised and clear. We have added/modified figure references in this section to clarify the relationship between text and figures.

∙ At end of page 5 the authors describe the location of RNA Pol2 pSer2 with respect to nucleosomes/RNA. As these results are related to findings reported by Castells-Garcia Nucl Acids Res 2022, cited elsewhere in the manuscript, the authors could cite the work here.

Done.

∙ It is unclear for which experiments goat anti-mouse Ig-Alexa Fluor 405 has been used, remove from antibody list if not relevant.

This antibody has been removed.

∙ The use of the 488 laser as the activatory laser is peculiar, can the authors better explain this choice?

Empirically we found the 488 laser performed better as an activatory laser- as it gave a higher signal to noise than the more commonly used 405 laser. We have added this statement to the M&M.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary

The authors investigate the nucleosome and RNA Pol2 arrangement along the Y loop of Drosophila primary spermatocytes with super-resolution (STORM) and confocal microscopy. Given the peculiarities of the model system (extremely long transcriptional unit, transcriptional expression restricted to spermatogenesis, well-characterized nuclear location), Y loops are an interesting choice to investigate the organization of chromatin loops as they allow to physically isolate one specific DNA filament from the rest of the genome. Ball and colleagues found that, although this loop undergoes transcription and is under a decondensed state, chromatin is arranged as a chain of nucleosome clusters of variable sizes and not as a 10 nm fiber. They also show that RNA Pol 2 pSer2 is interspersed along the loop, preferentially at the periphery of nucleosome clusters and at linker sites and do not form transcription factories. RNA Pol 2 pSer5 instead is less associated with the loop and is preferentially found close to the nucleolus consistent with its role in transcriptional initiation. Given the relative scarcity of RNA Pol 2 with respect to nucleosomes along the Y loop, the authors conclude that nucleosome arrangement in chains of clusters is not determined by the transcriptional activity.

Major comments

• Pages 2/3, the authors estimate the number of nucleosomes per cluster identified in the Y loops. They do so "based on a simple volume calculation" but the quantification method and reference values used to reach the estimate of a "maximum of 158 nucleosomes" is not reported in the methods. Also, the authors should discuss how do they account for multiple blinking effect in the estimate of cluster measures. Methods based on normalization curves with nucleosome arrays (Ricci et al Cell 2015) and on DNA origami (Cella Zanacchi et al Nat Meth 2017) among others were previously used to address similar questions. The authors should consider applying a normalization approach to achieve a more accurate estimate or at least discuss the caveats of their strategy.
• Methods, STORM imaging: the authors should provide the imaging buffer composition as the papers they refer to make use of an array of buffers. If possible, the authors should include laser powers in standard units (kW/cm^2) to increase clarity and reproducibility.
• Methods, Cluster analysis: the authors should specify the values for the parameters used in "First a description of the average cluster was established using spatial statistics, then a clustering algorithm using the average parameters provided by the initial spatial statistics further refined the description"
• Given the availability of broadly used methods for clustering such as DBScan (Ester, et al. A density-based algorithm for discovering clusters in large spatial databases with noise. Kdd, 1996), Bayesian methods (Rubin-Delanchy et al. Bayesian cluster identification in single-molecule localization microscopy data. Nature methods, 2015), (Ricci et al. Cell 2015), Voronoi based tessellation (Levet et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nature methods, 2015), and more recently machine learning approaches (Williamson et al. Machine learning for cluster analysis of localization microscopy data. Nature communications, 2020) the clustering method of choice is peculiar. It makes more difficult the direct comparisons with previous work. The authors could justify the choice or compare results using other more broadly used methods.
• No statistical tests were performed in the study, although no comparisons between experimental conditions are shown. The authors mention the number of replicates performed only on Fig 3 ("13 regions of interest (ROI) were selected from 3 cells"), which is a low number of cells analyzed. The authors should enlarge the analyzed datasets and mention how many replicates were used throughout the work.
• It is unclear why the authors did not perform STORM on EU-labeled samples immunolabeled with Pol2 antibodies. In principle, they could have applied the same imaging protocol used in previous figures gaining spatial resolution to better characterize Y nascent transcripts and how Pol2 is arranged with respect to nascent RNA.

Minor comments

• The text is well organized and clear as well as the figures but the authors should revise the text in the section "linking RNA polymerase distribution and Y loop transcription" to include missing references to Figure 10. For example, the statement "This distribution of nascent transcript along the Y loops fits with the distribution of RNA-PSer2," refers to results shown in Fig 10A.
• At end of page 5 the authors describe the location of RNA Pol2 pSer2 with respect to nucleosomes/RNA. As these results are related to findings reported by Castells-Garcia Nucl Acids Res 2022, cited elsewhere in the manuscript, the authors could cite the work here.
• It is unclear for which experiments goat anti-mouse Ig-Alexa Fluor 405 has been used, remove from antibody list if not relevant.
• The use of the 488 laser as the activatory laser is peculiar, can the authors better explain this choice?

Referees cross-commenting

I believe the comments of the other reviewer are very good and in line with the comments I made. I do not have much to add.

Significance

The conclusions reached by the authors are in line with previous reports (Ricci et al. Cell 2015, Ou et al. Science 2017, Castells-Garcia et al Nucl Acids Res 2022, among others). With different SR techniques, these works show that chromatin in mammalian cells is arranged in heterogeneous groups of nucleosomes and that RNA Pol 2 is not organized in transcription factories but rather binds the chromatin fiber forming small clusters at the periphery of nucleosomes. The use of Y loops as a paradigm of transcriptionally active allows the authors to confirm previous findings on another organism, Drosophila Melanogaster, and to visualize the organization of chromatin within an individual, isolated loop, without the need for FISH-like labeling. The work is potentially interesting to the field of genome and chromatin organization and SR microscopy, it is also relevant to scientists working on transcriptional regulation.

Field of expertise: Chromatin organization - SR microscopy - transcription.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The authors use STORM imaging to investigate transcription/spatial organisation of Drosophila Y loops. They find that the Y loop is organised in to small sections of clustered nucleosomes. Active RNAP is then found on the periphery of these clusters - furthermore, RNAP is distributed along the fibre rather than in to distinct "factory" units. The RNAP foci were less frequent than the nucleosome clusters therefore RNAP is not directing organising of the chromatin at these sites.

Comments:

1. Have you assessed if you are under-labeling the nucleosomes? or RNA Polymerase?
2. Can you stimulate and inhibit transcription to see how changes in RNA Polymerase and nucleosome organisation occur?
3. The 300 msec exposure time is very long for typical STORM experiments - blinking would usually be lost at this time scale?
4. Please quote laser power in absolute values rather than percentage.
5. Collecting variable number of frames (20K - 50K) can impact the number of detections/clusters. How can you account for this variation in your analysis?

Referees cross-commenting

I agree with the other reviewer statements/comments.

Significance

Highly significant work to visualise active transcription. It provides an interesting finding that transcription is not acting to determine chromatin organisation. Nevertheless, this may be specific to the model system.

Field of expertise: Transcription, Super resolution imaging, single molecule imaging, spatial organisation.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

The reviews are on balance an accurate, thoughtful, thorough assessment of the manuscript. We appreciate the careful engagement with the B cell differentiation aspect of our work. We identify 2 major critiques from the reviews:

1. The manuscript should make stronger connections with existing literature on ____in-vitro _and _in-vivo ____B cell differentiation. We agree the manuscript should be revised to interact more holistically and carefully with relevant B cell differentiation research. In this respect, the reviewers both help by pointing to high-quality and relevant literature that will be discussed and cited.

The cytokine mixture we used on the B cells was not defined / described in the manuscript. This fact hinders the interpretation of the data because B cells will respond to diverse stimuli in quite different ways.

We agree this hinders interpretation of the data, and the reviewers bring up astute points about different types of stimuli (TD vs. TI vs. TLR vs. BCR). Unfortunately, the manufacturer of the product, Stem Cell Technologies, will not disclose exactly what is in the product. Given we are in strong agreement with the reviewers on this point, we analyzed the cytokine contents of the cocktail and our cell culture supernatants using a luminex cytokine panel. We present a discussion of our findings on this data in a supplementary note and figure. We acknowledge this analysis is non-exhaustive, because it does not include possible additions of non-cytokine stimulants. However, we maintain it adds much clarity to the interpretation of the data.

We note that the contents of the stimulation cocktail are knowable and well-defined. These attributes are in contrast to almost all B cell stimulation protocols of which are aware. Typical stimulation protocols use various types of feeder cells, cytokines, and FBS (Fetal Bovine Serum). In particular, the feeder cells and FBS, are highly variable between labs, lots, and even experiments. FBS has a myriad of issues which are described here (____https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349753/____). Major variability, from genomic to phenotypic, has been described in laboratory cell lines like the ones used as feeder cells. With respect to B cells specifically, large differences in B cell activation programs are observed between lots of FBS, as described here (____https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854248/#r5____). Additionally, we have observed the presence of bovine viruses and other contaminants in FBS (unpublished data). Thus, the stimulation protocol we used is reproducible and robust in ways generally unseen by us in B cell stimulation literature. In summary, we view this cocktail as useful in a similar way to how FBS is useful to biologists – a major difference being that this cocktail is better defined and controlled. We provide similar thoughts in our supplementary note.

A final general point we will make is about the significance of our work, which appears to be lost on Reviewer #1. Similar technical and conceptual advances by our lab have been cited 1000s of times. Thus, we think the impact of our scientific approach speaks for itself. Many of our results confirm and expand on previous literature about B cells. We deliberately chose to make this novel technical and conceptual advance in the well-studied system of B cell differentiation. This allows us to integrate our findings with prior literature and helps validate the general approach. Reviewer #1 has performed a scholarly service by independently verifying our findings are coherent with existing literature, and we thank them for that.

In response to the reviews, we have edited the manuscript to reference even more of the papers in the field which report similar findings. Thus, our concordance with prior literature should be viewed as a strength of the manuscript. It shows readers of the manuscript the conceptual framework we use here is valid and can generate similar insights in less well-studied systems. For example, the approach developed here could be used in non-B cells, non-human immune systems, or even non-model organisms. In response to the reviewers critique, we modified the discussion of our work in multiple places to emphasize these points.

Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

1) Which B cell activation protocol was used? No information is provided in the main text or supplementary information. Yet, this information is key to fully understand many of the conclusions of this work (e.g., ... memory B cells are intrinsically two-fold more persistent in vitro (2A)), which largely depend on the nature of the stimuli used in the in vitro B cell culture.

We used the B cell activation protocol developed by StemCell Technologies as described in our methods section. We agree the reader and scientific community would benefit from additional information about this cocktail. To this end, we added a discussion of the cocktail to the supplementary information. We also used a cytokine analysis panel to analyze the cocktail, which provided detailed although non-exhaustive information about what is in the cocktail.

2) It would be informative to use more than one B cell activation program, e.g., CD40L with or without a cytokine as well as CD40L vs. CpG-DNA. Authors make broad statements about B cell fates without discussing the impact of a given signal on a given B cell fate. For instance, do memory B cells follow the same differentiation program upon stimulation with CD40L, IL-4 or a combination of CD40L and IL-4? How about differences between a TD signaling program such as that provided by CD40L and IL-10 and TI signaling program such as that provided by CpG-DNA and IL-10?

This is a good point. We agree stimulation using a panel of different agents would be a worthwhile experiment. It stands as a goalpost for future studies. Currently, performing single cell RNA sequencing on so many samples is both beyond the scope of this manuscript and very resource intensive. ____

3) Page 3, first line: After low quality and non-B cells (Fig S1A & B). What does this statement mean? The sentence seems incomplete.

Thank you for catching this typo. It is now clarified in the manuscript that we removed these cells bioinformatically.

4) First, we noted that non-B cells present in the input rapidly became undetectable by day 4, which shows the specificity of the cytokines for B cell expansion. Which cytokines are we talking about? No detail is provided.

We now provide our analysis of the stimulation cocktail, in Supplementary note 1 and Supplementary figure 1A. We still believe it is an interesting observation that this cocktail specifically stimulates B cells because many cytokines are not specifically B cell division signals, there were some impurities in the input population, and many cytokines are produced by the cultured cells themselves.

5) Plasmablasts were not distinguished from plasma cells.

We agree this is an interesting and important distinction to make. We have now distinguished between these classes of cells.

6) Critically, we observed no appreciable evidence of hypermutation in vitro (S2C), consistent with prior literature (Bergthorsdottir et al. 2001). This statement is vague, misleading and likely inaccurate for the following reasons. (a) The B cell culture conditions used by the authors are completely unknown. (b) It was shown that SHM can be achieved under specific in vitro B cell culture conditions that include the presence of activated CD4+ T cells (PMID: 9052835; PMID: 10092799; PMID: 10878357; PMID: 12145648). Did authors try to recapitulate those culture conditions?

We see how this statement could be misunderstood. We only claim not to observe evidence of hypermutation in our specific culture conditions, which is important for the inferences we make. We added language to make this more clear.

We did not try to recapitulate the conditions in the references supplied by the reviewer. We note that these references use cell lines and not B cells. While there is immensely valuable work done on cell lines, they behave very differently from actual cells and these findings may not be relevant to our human B cells.

7) Some of the reported findings are repetitive of previously published results and provide no additional new information. For example:

1. a) "Interestingly, we found mutated B cells were far more likely to express genes involved in T cell interaction (2B), suggesting Memory B cells are intrinsically licensed to enter an inflammatory state which activates T cells". This evidence is already published (PMID: 7535180 among many other published studies).

We will cite this paper, which is a landmark study. We don’t claim we are the first to discover a propensity of mutated B cells to present help to T cells, but note that we were able to observe this fact via lineage tracing in a single experiment, which is a conceptual and technical advance. Additionally, we report an entire transcriptional module of genes which are upregulated in memory B cells vs. Naive B cells exposed to the same stimulus. This adds to the systematic understanding of the Memory B Cell activation program.

1. b) "Instead, Naive B cells were biased toward expressing lectins and CCR7, suggesting Naive B cells are intrinsically primed to home into the lymphatic system and germinal centers (2B)". This evidence is already published (PMID: 9585422 among many other published studies).

While this is an interesting and important paper referenced by the reviewer, we are unable to find anything similar to our claim about naive B cells in the reference provided. The investigators do not discuss intrinsic differences between memory and naive subsets when responding to the same stimulus.

8) We quantified the in vitro dynamics of CSR through the lens of mutation status, which revealed strongly different fate biases between germline and mutated cells (2D). Most strikingly, B cells which switched to IGHE were almost exclusively derived from germline progenitors: the ratio of germline IGHE cells to mutated IGHE cells was (8-fold - inf, 95 % CI). Also this evidence is not novel (PMID: 34050324 among other published studies) and, again, must reflect the presence of specific culture conditions that remain completely undisclosed. This is incredibly confusing.

Thank you for providing this reference, we were not aware of this interesting study. These studies are quite different and complementary. Differences between these studies likely reflect the fact that their B cells are isolated from a niche, rather than generated ____in-vitro_. Most of the tissue-resident cells in their study are quite mutated, and thus are not the Naive B cells we are making a claim abountj. In fact, despite their claim of low mutational load, these cells would fall into the “mutated” or even “heavily mutated” categories we defined in our paper. Cells with mutation levels of 5% are not thought to be Naive in any classification scheme. Our study showed that, _in vitro____, IGHE B cells effectively came exclusively from germline progenitors, their study shows no such result. The novelty of this finding was appreciated by reviewer 2.

9) Authors should mention that non-switched memory B cells include IgDlowIgM+CD27+ and IgD-IgM+CD27+ memory B cells. Some authors define these distinct memory B cell fractions as marginal zone (MZ) or MZ-like B cells (please, notice that splenic MZ B cells recirculate in humans) and IgM-only B cells, respectively (PMID: 28709802; PMID: 9028952; PMID: 10820234; PMID: 11158612; PMID: 26355154; PMID: 15191950; and PMID: 24733829 among many other published studies).

We appreciate these points. We attempted to classify our B cells within this taxonomy and found no such separation clearly exists in single-cell RNA-seq profiles. Instead, we opted to re-classify our data with a state-of-the-art algorithm called celltypist (DOI: 10.1126/science.abl5197____ )____ which harmonizes cell annotations across a growing number of single-cell RNA sequencing studies. While this classification system is not currently mutually exclusive / completely exhaustive, we believe using this system provides standardization and data availability that are key for sharing results. As single-cell RNA-seq and flow/mass cytometry harmonize their classification systems, anyone should be able to transfer their preferred classification scheme to the cells profiled here.

10) Thus, CSR from IGHM cells did not meaningfully contribute to the abundance of IGHA+ cells in the population. Also this conclusion may be misleading and/or inaccurate. Indeed, an efficient class switching to IgA requires the exposure of naïve B cells to the cytokine TFG-beta in addition to a robust TD (CD40L) or TI (CpG DNA or BAFF or APRIL) co-signal. Was TGF-beta present in this culture?

This is a good point about TGF-beta and switching to IgA. Here is a clear example of the novelty and power of our approach, as well as the benefits of using a well-characterized system such as B cell differentiation. Lineage tracing clarifies between two explanations for why there are IgA cells in the output population. One explanation is that non-IgA B cells in the input switch to IgA, driven by TGF-beta. Another explanation is that IgA cells in the input expand modestly and account for IgA cells in the output. Lineage tracing offers clear evidence that the latter explanation is true. Following from this, our approach allows us to make a strong inference that TGF-beta is not present in the incompletely determined cytokine mixture. We are not sure how this conclusion may be misleading or inaccurate, as it is a clear and simple description of our data, not a claim about what factors are necessary for switching.

11) In contrast, we noted that many intraclonal class-switching events appeared to be directly from IGHM to IGHE. Explanations involving unobserved cells with intermediate isotypes notwithstanding, these data illustrate the relative ease with which B cells can switch directly to IGHE. It is very difficult to interpret this statement, as no information regarding the B cell-stimulating conditions used is provided. In addition, relevant literature is not quoted (e.g., PMID: 34050324).

We clarify our discussion here to claim the ease with which peripheral blood IGHM B cells switch to IGHE. Again, lineage tracing has allowed us to distinguish between two very different population-level phenomena. One explanation is that undetected IGHE+ progenitors in the input population expanded rapidly and account for the IGHE+ cells. Another explanation is that cells class-switch to IGHE. Our data are consistent with the latter. We note that this validates the conceptual use of lineage tracing to understand rapid population dynamics in immune responses and cell differentiation protocols. This is a strength of our manuscript. We appreciate the the reviewer has furnished relevant studies, which we will cite.

12) Our data for IGHE cells contrasts with in vivo data which show IgE B cells to be: (1) very rare, (2) apparently derived from sequential switching (e.g. from IgG1 to IgE) (Horns et al., 2016; Looney et al., 2016), and (3) often heavily hypermutated (Croote et al., 2018).

While this reviewer agrees with the first comment (switching to IgE is relatively rare in vivo, at least in healthy individuals), the other statements are quite inaccurate. Indeed, unmutated extrafollicular naïve B cells from tonsils and possibly other mucosal districts directly class switch from IgM to IgE in healthy individuals, thereby generating a low-affinity IgE repertoire. In principle, low-affinity IgE antibodies may protect against allergy by competing with high-affinity IgE specificities. In allergic patients, high-affinity IgE clones emerge from class-switched and hypermutated memory B cells that sequentially switch from IgG1 or IgA1 to IgE as a result of specific environmental conditions, including an altered skin barrier (PMID: 22249450; PMID: 30814336; PMID: 32139586).

Moreover, in contrast to what stated by authors, sequential IgG1/IgA1-to-IgE class switching mostly occurs in allergic patients but is less frequent in healthy individuals, where IgE specificities are less mutated (PMID: 30814336). Along the same lines, IgE is heavily mutated only in allergic individuals with significant molecular evidence of sequential IgG1/IgA1-to-IgE class switching (PMID: 30814336; PMID: 32139586). Overall, the data provided by Swift M. et al. are largely confirmatory of previously published evidence.

We appreciate the clarification of this complex field and will cite the relevant literature. We also agree with the reviewers assessment that our data are validated by other approaches and groups. We see that our discussion of IgE B cells should have included that caveat that we are discussing IgE B cells detected in the peripheral blood. We have restricted claim to the suggestion that if our conditions mimic such niches where B cells switch to IgE, there are clearly efficient mechanisms which limit the amount of circulating IGHE B cells mechanisms in comparison to other isotypes.____

Taken together, these data suggest that while direct switching to IGHE from Naive progenitors is trivial in vitro, niche factors or intrinsic death programs efficiently limit their generation or lifetime in vivo." I cannot understand this conclusion, which seems to contradict earlier statements.

We hope we have clarified via the above comment.

13) I am not sure I learned much regarding the "cell-intrinsic" fate bias and transcriptional memory of B cells after reading this elegantly presented but confusing and superficially discussed manuscript (please, see also comments 15-23).

We understand the reviewer is confused about various aspects of our manuscript and appreciate the opportunities to clarify. We show cells with broad identities (such as germline vs. mutated or naive vs. memory) respond differently to the same stimulus. These are cell intrinsic fate biases. We quantify them and provide statistical bounds on the effect sizes of these differences, which to our knowledge has not been done. We agree with the reviewer that in the case of memory and naive B cells, much is already known about their biases. We recapitulate some of this knowledge, while adding a quantitative and an unbiased transcriptomic lens with which to view the biases. However, our analysis moves beyond cell types broadly defined, and focuses on the concept that each clone is a cell state or identity, where some of the identity may be faithfully propagated over generations and other information may not be. To this end, we tracked the transcriptome of clones during differentiation. We show that B cell clones share highly similar cell fates, implicating cell-intrinsic heterogeneity as a major contribution to diversity in immune responses. We note this reviewer did not critique this aspect of our work. The review also did not critique Figure 3 or 4, in which we present a quantitative analysis of which transcriptional programs are maintained by B cells and contribute to their clonal identity. Finally, via our analysis of human long-lived plasma cells, we report these transcriptional identities are observed in-vivo, over long time scales. This type of cell-intrinsic bias has not been studied or described to our knowledge. These findings were of particular interest to reviewer 2 and other readers of the manuscript.

MINOR COMMENTS

Thank you for reading the manuscript carefully and providing these comments and observations. We have fixed all clerical errors that were pointed out. We also responded to some of these minor comments here, and made changes to the manuscript to clarify.

1) Figures 1B, S1C and S1D are not referred to in the text. x

2) 2B in the Text is 2D in the Figure. x

3) 2D in the Text is 2E in the Figure. x

4) Figure 2D seems to show only 10 genes. Please, clarify.

We clarify in the manuscript that we present the top differentially expressed genes

5) 2E in the text is 2F in the Figure. x

6) Figure S3B is not indicated in the text. x

7) Figure 3E is not indicated in the text. x

8) Figure S4A is not indicated in the text. x

9) In some sections of the text, Figure panels are not sequentially discussed, which makes the text very difficult to follow. x

Reviewer 2:

Major comments:

On p.3 the authors assume that a B cell with an unmutated BCR in the time course arose from a naive B cell progenitor. However, it is also possible that it arose from a IgM memory B cells since they also contain a non-negligible proportion of cells with 0 mutations. This was initially seen already in the Klein, J Exp Med, 1998 paper and later confirmed by e.g. Weller et al, J Exp Med, 2008 and Wu et al, Front Immunol, 2011. And since the authors herein and others have demonstrated that IgM memory B cells have a high proliferative capacity it is possible that IgM memory B cells are overrepresented among those unmutated BCRs seen in the cultures.

The finding that IgM memory B cells are highly proliferative is not novel. It has been demonstrated by other groups before and one good example is Seifert et al, PNAS, 2015 where IgM memory B cells proliferated significantly more to BCR stimulation than naive or IgG memory B cells. However, it is also shown that IgG memory B cells are more responsive to TLR9 stimulation than IgM memory B cells as demonstrated by e.g. Marasco et al, Eur J Immunol, 2017. This is not discussed by the authors and should be added into the discussion for context of their finding by scRNAseq methods.

These are astute points. We incorporated a more nuanced discussion of the prior literature about highly proliferative IgM memory B cells, which have been reported before. We also added a figure which identifies the genes associated with proliferative clones in Figure 3d, which adds to our understanding of the gene regulatory networks which govern IgM memory B cell behavior. We appreciate the reference to the Seifert et al paper, which is relevant and high quality work. We concur that a discuss of Marasco would be helpful, especially because it is unknown if a TLR9 agonist is in the stimulation cocktail, but their data would suggest there is not.

The notion that a memory transcriptional program can be induced without SHM is not novel and this should be brought up in the discussion. One paper showing a memory transcriptional program in unmutated memory B cells is Kibler et al, Front Immunol, 2022.

We were not aware of this literature and have now cited it in our discussion of this finding.

The observation that memory B cells are more likely to enter an inflammatory state and support T cells has been suggested by other groups (Seifert et al, PNAS, 2015; Magri et al, Immunity, 2017, Grimsholm et al, Cell Reports, 2020).

We have now cited and discussed a number of papers which contain similar findings. We note that we add to the holistic understanding of this phenomenon via our single cell transcriptomic approach.

Please provide the age distribution of the peripheral blood samples as well.

We have now provided the age distribution of the peripheral blood samples

Please show flow cytometry analysis of the cultures to assist in assessing subset distribution, viability and plasma cell differentiation for each time point. This can be provided as supplementary information.

We did not use flow cytometry for subset distribution and measurements of differentiation per se, only to exclude non-viable cells and we have now made this clearer in the methods section. We also now include representative plots show our sorting strategy.

The stimulation cocktail used for this study, what does it contain? This needs to be specified in the manuscript and not only referring to the manufacturer. This has major impact on the results since different stimulatory agents will induce different pathways.

This is a valid point that we addressed in our response to reviewer 1. See supplementary note 1 and Figure S1A for our analysis of the stimulation cocktail.

Minor comments:

Please avoid the term plasma B cells, does it refer to plasmablasts and/or plasma cells?

Thank you for the suggestion, we have modified our language to refer to plasmablasts and plasma cells separately.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

I have with great interest read the manuscript "Fate bias and transcriptional memory of human B cells" by Swift et al where they use an in vitro approach and single cell RNA sequencing to elucidate fate decisions of human B cells. The authors demonstrate that genes related to cell fate determination in human B cells are persistent both in their in vitro culture and in ex vivo plasma cells from bone marrow. This is a very important finding in this manuscript and the technical execution of the paper is overall of high quality. The finding of switching to IgE in vitro from unmutated precursor is very interesting and does not necessarily contradict existing data in the field but rather adds new important information. However, some of the findings are not totally novel and need to be put in context of earlier findings (outlined below). The discussion does not acknowledge literature on memory B cell transcriptional programs and mutation status in a balanced manner. Important literature by leading groups in the field are left out.

Major comments:

On p.3 the authors assume that a B cell with an unmutated BCR in the time course arose from a naive B cell progenitor. However, it is also possible that it arose from a IgM memory B cells since they also contain a non-negligible proportion of cells with 0 mutations. This was initially seen already in the Klein, J Exp Med, 1998 paper and later confirmed by e.g. Weller et al, J Exp Med, 2008 and Wu et al, Front Immunol, 2011. And since the authors herein and others have demonstrated that IgM memory B cells have a high proliferative capacity it is possible that IgM memory B cells are overrepresented among those unmutated BCRs seen in the cultures.

The finding that IgM memory B cells are highly proliferative is not novel. It has been demonstrated by other groups before and one good example is Seifert et al, PNAS, 2015 where IgM memory B cells proliferated significantly more to BCR stimulation than naive or IgG memory B cells. However, it is also shown that IgG memory B cells are more responsive to TLR9 stimulation than IgM memory B cells as demonstrated by e.g. Marasco et al, Eur J Immunol, 2017. This is not discussed by the authors and should be added into the discussion for context of their finding by scRNAseq methods. The notion that a memory transcriptional program can be induced without SHM is not novel and this should be brought up in the discussion. One paper showing a memory transcriptional program in unmutated memory B cells is Kibler et al, Front Immunol, 2022.

The observation that memory B cells are more likely to enter an inflammatory state and support T cells has been suggested by other groups (Seifert et al, PNAS, 2015; Magri et al, Immunity, 2017, Grimsholm et al, Cell Reports, 2020). Please provide the age distribution of the peripheral blood samples as well.

Please show flow cytometry analysis of the cultures to assist in assessing subset distribution, viability and plasma cell differentiation for each time point. This can be provided as supplementary information.

The stimulation cocktail used for this study, what does it contain? This need to be specified in the manuscript and not only referring to the manufacturer. This has major impact on the results since different stimulatory agents will induce different pathways.

Minor comments:

Please avoid the term plasma B cells, does it refer to plasmablasts and/or plasma cells?

Referees cross-commenting

Comments on reviewer 1 report: The reviewer raised the same significant objection to the manuscript as it stands today i.e. the content of the stimulation cocktail is not described. Without this information the manuscript is hard to put into the perspective of the existing literature.

Significance

See above comments.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary

Swift M. et al. explored how gene expression responds to extracellular stimuli by dissecting the transcriptional programs that underlie cell-intrinsic clonal fate bias. They also verified how extrinsic signals, intrinsic state, and clonal population structure interacted to determine the dynamics of the B cell immune response. To address these questions, lineage and single-cell RNA sequencing measurements were performed in human B cells stimulated in vitro through an undisclosed protocol. The B cell receptor (BCR) gene was used as a genetic lineage marker and this information was paired with that gained from transcriptomics, which provided an additional readout of cellular identity. Authors concluded that B cells have intrinsic biases towards specific cell fates and identified specific transcriptional memory states.

Major comments

While this is an elegantly presented manuscript that presents a wealth of data obtained from state-of-the-art transcriptomics and bioinformatics, some issues considerably weaken the main conclusions. These weaknesses entail an almost complete absence of details as to the stimuli being used for the in vitro activation of B cells (e.g., were they exposed to IL-4, TGF-beta and/or CD40L?). In the absence of this information, the discussion of intrinsic biases towards specific cell fates cannot be fully interpreted and understood, as different stimuli induce different B cell fates. In addition, there seems to be some misunderstanding about IgE class switching and somatic hypermutation, which differ in healthy and allergic individuals. Also, the novelty of this work is reduced by the fact that some of its conclusions are either largely expected or already known from studies published previously. Altogether, these issues make some statements and conclusions confusing and, sometimes, misleading. The following additional major comments are provided.

1. Which B cell activation protocol was used? No information is provided in the main text or supplementary information. Yet, this information is key to fully understand many of the conclusions of this work (e.g., ... memory B cells are intrinsically two-fold more persistent in vitro (2A)), which largely depend on the nature of the stimuli used in the in vitro B cell culture.
2. It would be informative to use more than one B cell activation program, e.g., CD40L with or without a cytokine as well as CD40L vs. CpG-DNA. Authors make broad statements about B cell fates without discussing the impact of a given signal on a given B cell fate. For instance, do memory B cells follow the same differentiation program upon stimulation with CD40L, IL-4 or a combination of CD40L and IL-4? How about differences between a TD signaling program such as that provided by CD40L and IL-10 and TI signaling program such as that provided by CpG-DNA and IL-10?
3. Page 3, first line: After low quality and non-B cells (Fig S1A & B). What does this statement mean? The sentence seems incomplete.
4. First, we noted that non-B cells present in the input rapidly became undetectable by day 4, which shows the specificity of the cytokines for B cell expansion. Which cytokines are we talking about? No detail is provided.
5. Plasmablasts were not distinguished from plasma cells.
6. Critically, we observed no appreciable evidence of hypermutation in vitro (S2C), consistent with prior literature (Bergthorsdottir et al. 2001). This statement is vague, misleading and likely inaccurate for the following reasons. (a) The B cell culture conditions used by the authors are completely unknown. (b) It was shown that SHM can be achieved under specific in vitro B cell culture conditions that include the presence of activated CD4+ T cells (PMID: 9052835; PMID: 10092799; PMID: 10878357; PMID: 12145648). Did authors try to recapitulate those culture conditions?

7. Some of the reported findings are repetitive of previously published results and provide no additional new information. For example:

   • a) "Interestingly, we found mutated B cells were far more likely to express genes involved in T cell interaction (2B), suggesting Memory B cells are intrinsically licensed to enter an inflammatory state which activates T cells". This evidence is already published (PMID: 7535180 among many other published studies).
   • b) "Instead, Naive B cells were biased toward expressing lectins and CCR7, suggesting Naive B cells are intrinsically primed to home into the lymphatic system and germinal centers (2B)".

This evidence is already published (PMID: 9585422 among many other published studies). 8. We quantified the in vitro dynamics of CSR through the lens of mutation status, which revealed strongly different fate biases between germline and mutated cells (2D). Most strikingly, B cells which switched to IGHE were almost exclusively derived from germline progenitors: the ratio of germline IGHE cells to mutated IGHE cells was (8-fold - inf, 95 % CI). Also this evidence is not novel (PMID: 34050324 among other published studies) and, again, must reflect the presence of specific culture conditions that remain completely undisclosed. This is incredibly confusing. 9. Authors should mention that non-switched memory B cells include IgDlowIgM+CD27+ and IgD-IgM+CD27+ memory B cells. Some authors define these distinct memory B cell fractions as marginal zone (MZ) or MZ-like B cells (please, notice that splenic MZ B cells recirculate in humans) and IgM-only B cells, respectively (PMID: 28709802; PMID: 9028952; PMID: 10820234; PMID: 11158612; PMID: 26355154; PMID: 15191950; and PMID: 24733829 among many other published studies). 10. Thus, CSR from IGHM cells did not meaningfully contribute to the abundance of IGHA+ cells in the population. Also this conclusion may be misleading and/or inaccurate. Indeed, an efficient class switching to IgA requires the exposure of naïve B cells to the cytokine TFG-beta in addition to a robust TD (CD40L) or TI (CpG DNA or BAFF or APRIL) co-signal. Was TGF-beta present in this culture? 11. In contrast, we noted that many intraclonal class-switching events appeared to be directly from IGHM to IGHE. Explanations involving unobserved cells with intermediate isotypes notwithstanding, these data illustrate the relative ease with which B cells can switch directly to IGHE. It is very difficult to interpret this statement, as no information regarding the B cell-stimulating conditions used is provided. In addition, relevant literature is not quoted (e.g., PMID: 34050324). 12. Our data for IGHE cells contrasts with in vivo data which show IgE B cells to be: (1) very rare, (2) apparently derived from sequential switching (e.g. from IgG1 to IgE) (Horns et al., 2016; Looney et al., 2016), and (3) often heavily hypermutated (Croote et al., 2018). While this reviewer agrees with the first comment (switching to IgE is relatively rare in vivo, at least in healthy individuals), the other statements are quite inaccurate. Indeed, unmutated extrafollicular naïve B cells from tonsils and possibly other mucosal districts directly class switch from IgM to IgE in healthy individuals, thereby generating a low-affinity IgE repertoire. In principle, low-affinity IgE antibodies may protect against allergy by competing with high-affinity IgE specificities. In allergic patients, high-affinity IgE clones emerge from class-switched and hypermutated memory B cells that sequentially switch from IgG1 or IgA1 to IgE as a result of specific environmental conditions, including an altered skin barrier (PMID: 22249450; PMID: 30814336; PMID: 32139586). Moreover, in contrast to what stated by authors, sequential IgG1/IgA1-to-IgE class switching mostly occurs in allergic patients but is less frequent in healthy individuals, where IgE specificities are less mutated (PMID: 30814336). Along the same lines, IgE is heavily mutated only in allergic individuals with significant molecular evidence of sequential IgG1/IgA1-to-IgE class switching (PMID: 30814336; PMID: 32139586). Overall, the data provided by Swift M. et al. are largely confirmatory of previously published evidence.

Taken together, these data suggest that while direct switching to IGHE from Naive progenitors is trivial in vitro, niche factors or intrinsic death programs efficiently limit their generation or lifetime in vivo." I cannot understand this conclusion, which seems to contradict earlier statements. 13. I am not sure I learned much regarding the "cell-intrinsic" fate bias and transcriptional memory of B cells after reading this elegantly presented but confusing and superficially discussed manuscript (please, see also comments 15-23).

Minor comments

1. Figures 1B, S1C and S1D are not referred to in the text.
2. 2B in the Text is 2D in the Figure.
3. 2D in the Text is 2E in the Figure.
4. Figure 2D seems to show only 10 genes. Please, clarify.
5. 2E in the text is 2F in the Figure.
6. Figure S3B is not indicated in the text.
7. Figure 3E is not indicated in the text.
8. Figure S4A is not indicated in the text.
9. In some sections of the text, Figure panels are not sequentially discussed, which makes the text very difficult to follow.

Significance

NATURE AND SIGNIFICNCE OF THE ADVANCE

I could not grasp a significant advance in this study as provided.

COMPARE TO EXISTING PUBLISHED KNOWLEDGE

As detailed in my specific comments, many of the conclusions of this work are largely expected or already published. It is difficult to evaluate the substance and originality of other conclusions in manuscript as provided.

AUDIENCE

If adequately and extensively revised, this work could be interesting to a broad audience.

MY EXPERTISE

B cell biology; B cell subsets; regulation of antibody production, including class switching; signals skewing antibody responses to a specific isotype.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Reply to the Reviewers

I thank the Referees for their...

Referee #1

1. The authors should provide more information when...

Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...

Response: We expanded the comparison

Minor comments:

1. The text contains several...

Response: We added...

Referee #2

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

This study addresses the newly appreciated role of E-cadherin in tumor cell invasion. A strength of this study is their inducible RasV12 Drosophila model of transformation, which allows them to follow cell dissemination at the basal midgut. The authors demonstrate, convincingly, that depletion of E-cadherin in this model impairs cell dissemination. However, their efforts to demonstrate a role for two Ca2+ signaling pathways mediated by IP3R and Piezo channels are not convincing given the use of blunt and non-specific pharmacological tools, lack of Ca2+ monitoring, and generally descriptive observations.

A significant weakness in this study is the sole and unsubstantiated use of Arm (the Drosophila b-catenin ortholog) as a proxy for e-cadherin localization and abundance. This leads to unsubstantiated statements such as, "disassembly of adherens junctions rather than E-cad levels" is involved in dissemination of cells. The role of E-cad is complicated by the observation that both knockdown and overexpression of E-cad perturb cell dissemination in their model.

In Figure 4, the role of Piezo in Arm distribution is presented in confocal images of a few cells. Is this statistically significant? This experiment needs to be quantified with more cells to substantiate the claim that Arm signals redistribute from cell boundary to cytosol in Piezo and Calpain knockdowns. Piezo channels are non-selective for cations. This means that the results of the knockdown cannot be assigned to changes in cytosolic Ca2+. At the least, cytosolic Ca2+ levels should be monitored. The authors suggest that calpains work with Piezo through Ca2+ signaling on the basis of a single experiment in which calpain knockdown has a similar effect on Arm localization to Piezo knockout. This conclusion seems speculative and is not adequately supported.

Thapsigargin is a blunt tool that causes a large, non-physiological and irreversible increase in cytosolic Ca2+. The observation that high Ca2+, resulting from Tg treatment, mediates disassembly of cadherins junction is not new, and indeed has been known for at least 20 years. Therefore, the effect of Tg in causing relocation of Arm signals from the adherens junctions is not insightful.

Use of GdCl3 is a non-specific inhibitor of many ion channels, including voltage gated Ca2+ channels, TRP channels, leak K+ channels, etc. It cannot be used to infer the role of Piezo when used as described in this study. The observation that Tg + GdCl3 somewhat preserves basal/junction ratios of Arm, relative to Tg alone, is interesting but uninterpretable without additional experiments, including measurements of cytoplasmic Ca2+ levels. None of the findings related to Figure 5 are mechanistically insightful and the imputed role of Piezo is not convincing.

Significance

Overall, although this study addresses a topic of high significance and interest, the limitations in experimental approach do not allow mechanistic insights that advance our understanding of the role of E-cadherin in tumor invasion.

Referee Cross-commenting

Dear fellow reviewers,

My expertise is in calcium signaling and ion transporters/channels. Although I was excited to review this work based on the abstract, I do not think that the link to calcium signaling (figs 4-5) is insightful or mechanistic, as I explain in my review. I realize that experimental approaches may be limited by the fly model, but the conclusions made by the authors would not be persuasive in other models.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In this study, Cabrera et. al. examine the role of DE-cadherin in cell dissemination, using a model of RasV12-transformed cells in the Drosophila hindgut. They show that E-cad relocates in the transformed cells from cell-cell junctions to basal invasive protrusions, and this relocation depends on calcium release from the ER by the PLC-IP3R-CAMK pathway. DE-cad is not required for the formation of actin-rich protrusions. However, upon RNAi-mediated DE-cad depletion (or depletion of the PLC-IP3R pathway), the basement membrane is not degraded and cell dissemination is inhibited. Furthermore, piezo and calpain are also shown to be required for DE-cad to assemble at invasive protrusions.

Major comments:

The data in figures 1-3 is overall convincing, although some of the points are missing quantification. The data supporting a role for piezo in regulating DE-cad assembly at adherens junctions is weaker. The data derived from ex-vivo pharmacological treatments (figure 5) is difficult to interpret or draw conclusions from.

Specific comments:

1. The data on DE-cad overexpression is puzzling. When DE-Cad is overexpressed in Ras-transformed cells it relocates from AJ to invadopodia and the ECM is degraded just like in Ras-transformed cells, yet cell dissemination is reduced by 50%. Why? The authors don't offer any explanation and in the absence of one I don't see how the DE-cad overexpression data adds to the story.
2. DE-cad is knocked down by RNA interference using two different sequences. Admittedly, I am not a fly person, but isn't there a way for fly geneticists to show how well the KD worked? What percentage of WT DE-cad is left? This same question can be applied to all the RNAi experiments.
3. Arm redistribution and co-localization with F-actin should be quantified. While the "representative" images are of high quality and their presentation in XY and XZ or basal, middle, ortho views is very helpful, the data from multiple cells, from multiple flies, from at least 3 different experiments must be quantified to show by how much does the level of E-cad decrease at AJ and by how much does it increase at the basal protrusions. Also, the degree of co-localization of F-actin and E-cad at invadopodia should be quantified. Perhaps F-actin is not the best marker for invadopodia since it is also present in other structures.
4. The data presented in Figure 3c-h also needs to be properly quantified for level of DE-cad at lateral sides of cells and on basal side from multiple cells in multiple animals and at least 3 experiments.
5. The connection made between piezo and DE-cadherin (figure 4) is tenuous. Piezo is known (based on the authors previous work as well as others) to be upstream of invadopodia formation, so it is not surprising that E-cad does not localize to invadopodia when they don't exist. The authors do not provide any evidence to directly link piezo activity or calcium entry to DE-cadherin localization at invadopodia or at AJ and therefore their claim that "calcium signaling mediated by the Piezo-calpain pathway plays a distinct role in the DE-cad/Arm remodeling process" is purely speculative. The images of fuzzy DE-cadherin in figure 4b are hardly proof.
6. The experiment in fig 5a,b showing that calcium release from the ER leads to AJ disassembly, nicely complements the in vivo data in fig 3c-h. However, the remaining of figure 5, dealing with piezo, is entirely unconvincing. The images in 5f don't resemble invadopodia and have nothing to do with them. The data in 5i regarding apical delamination is interesting, but does not support their point about piezo and AJ. They don't even show DE-cadherin.

Minor comments:

Have a figure "for the reviewer" should be avoided. The authors must write the paper and include diagrams or images so that any reader is able to understand the model system just as well as the reviewer.

Significance

In my assessment of significance I am only taking into account the conclusions that I feel are well supported. Depending on when the preprint they cite (ref. 52) will be published, this could be the first report of E-cad localizing in invadopodia and, importantly, being essential for ECM degradation and cell dissemination. Also of significance is the identification of intracellular calcium release as a signal for AJ disassembly and relocation of E-cad to invasive protrusions. While the molecular mechanism through which E-cad regulates ECM degradation is not elucidated, the paper still represents a significant advance for the fields of cancer cell biology and cell and developmental biology and would be of interest to a wide audience.

I am a cell and developmental biologist with expertise in cell adhesion and morphogenesis. I am not an expert on Drosophila.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

This interesting manuscript examines how intracellular calcium signaling and cation channels (piezo) may affect the remodelling of adherens junctions in tumor cells. The authors use the Drosophila midgut model to examine the dissemination of RasV12 cells. Authors show that at day 2 of Ras expression, DE-cadherin/b-catenin complex redistributes from adherens junction to basal invasive protrusions. Depletion of DE-cadherin attenuates the dissemination and local invasion of RasV12 cells, as manifest in damage to underlying visceral muscles. The authors attribute disassembly of cadherin from cell-cell junctions to IP3R-dependent release of calcium from ER stores and subsequent activation of CaMKs. Surprisingly, the authors found an apparently separate role for Piezo (potentially mediating calcium signaling) to support the assembly of DE-cad at the invasive protrusions.

Overall, this is a well-presented report that outlines the basic features of the phenomena with data of good quality. Over the years, there have been intermittent efforts to characterize the impact of calcium signaling on cell-cell junctions but much remains to be learnt. This manuscript is informative for focusing attention on a model of cadherin downregulation (and redistribution) in early tumors. However, it is somewhat descriptive and lacks depth of mechanistic analysis. For example, how might the cadherin in invasive protrusions be contribution to matrix degradation? What are the targets of either the IP3/CamK pathway or piezo/calpain signaling? Clearly, these foundational observations could be taken in many different directions. But some development would enrich the current package. Some suggestions follow.

Major comments:

1. What is the role of E-cadherin in invasive protrusions? The authors use DE-cadherin depletion to support a role for this relocated pool of cadherin in tumor dissemination. However, this doesn't distinguish between a direct action of the cadherin complex at the protrusions from some indirect effect. Further analysis of the "protrusion" cadherin could be informative. For example, one possibility is that E-cadherin is essential to localise other proteins to the invasive protrusion. Authors have previously reported (Lee et al. 2020) that cortactin is essential for dissemination of the RasV12 cells. Considering that the phenotype of cortactin KD (presented in Lee et al. 2020) and DE-cad KD mostly overlap, and literature suggests that E-cadherin and cortactin colocalise and can influence each others' localisation in cultured cells (e.g. Ren et al. 2009; PMCID: PMC2707247). Could KD of DE-cad perturb cortactin localisation at the invasive protrusions? This could be doable in a reasonable time, as according to Lee et al. 2020 authors already have a relevant Drosophila strain, that lets visualisation of cortactin.
2. Another route to explore in greater depth is the impact of DE-cadherin depletion on the protrusion. The authors show that upon DE-cad KD, some actin-rich puncta are still able to form at the basal surface of the Ras cells. This should be characterised in more details to assess whether DE-cad KD affects the number, size and dynamics of forming protrusions. At current stage, it's not obvious why DE-cad depleted Ras cells do not disseminate, even though they are able to form the invasive protrusions. As authors may potentially use movies/images they already have, this should be doable within reasonable time frames.
3. Authors could expand the discussion section to address the issue of the unusual DE-cad localisation. The open, basic question that the authors haven't addressed at any stage is "what does the DE-cad bind to at the invasive protrusions?". Do authors speculate that DE-cad is able to interact with some components of ECM/VM (as would be suggested by the cartoon Fig S5)? Have authors considered that E-cadherin transinteractions forming eg. between small adjacent protrusions may serve to stabilise the initial structures and let them develop into more prominent invasive protrusions?
4. Given the rapid intracellular diffusivity of calcium, it is interesting that two different sources of calcium have such different effects on the cadherin pool. Why does calcium released from ER by IP3R is enough to disassemble the cadherin complex at AJs, but signaling from Piezo is needed to regulate assembly of the cadherin complex at the invasive protrusions. Can authors comment on why calcium released by ER can't control both processes? Is it possible to visualize the dynamic subcellular distribution of calcium in these cells to test if there are different pools involved?
5. Further, the discrepancy between the influence of the piezo1 KD and GdCl3 treatment on the localisation of the DE-cad/Arm is quite striking. As literature suggests an existence of a physical interaction between Piezo channel and E-cadherin, blocking Piezo vs depleting it may potentially affect E-cadherin differently. To address this, can authors e.g. add GdCl3 treatment (covering same timeframes as the Piezo KD) to data presented in Fig 4b (this would also eliminate the ambiguity caused by possible differences between in vivo vs ex vivo conditions - Fig 4 vs Fig 5). Also, is the phenotype of loss of junctional DE-cad/Arm upon Piezo KD limited to the Ras cells or does it also happen in GFP control cells?
6. Is the observed phenotype (dissemination and involvement of DE-cad) limited to RasV12 oncogene? Do authors have any experience with other oncogenes (e.g. Src)? If an adequate Drospihila strain is available to authors, it would be worth confirming that the presented findings are not limited to only one oncogene.
7. Finally, the paper feels like a small, follow up story that is hugely dependent on author's 2020 paper (Lee et al. 2020). It may be quite hard to follow if the reader is not familiar with the 2020 paper, as eg. the current paper does not describe what the "dissemination process" actually is, instead it directs the reader to the 2020 paper (similarly the figure for reviewers consists of images taken from the 2020 paper, rather than the current one).

Minor comments:

1. How was junctional intensity of proteins quantified in samples that don't have obvious junctional staining (eg. Fig5b images and quantification for TG/ TG+GdCl3 in c and d or Image 5f and quantification in h). Do authors use an additional junctional marker to define where the junction is? If not, how is the position of junctions determined when the measured protein is dispersed from junctions?
2. Is the calculation for IP3R-iGD1676 in Fig S3a correct? It doesn't match representative images presented in Fig3a or data in Fig3b, where IP3R-iGD1676 is not any different from other IR3R knock downs. If the quantification in Fig S3a is correct, the image presented in Fig3a is not representative of the majority of phenotype in this condition.
3. Can delamination of Ras cells (Fig S1b and S3b) be quantified? It's quite challenging to assess the number of delaminated cells (especially in S3b).
4. It would be good to quantitate the change in Arm (Fig 3d-h), in addition to showing representative images.
5. Can authors describe the names used for labelling graphs in the figure captions? Some of the names used are not obvious for readers not already familiar with them eg. labels presented FigS3c - it's not obvious what norpA is, and the figure captions don't help at all.
6. Is the antibody used for DE-cad able to distinguish between DE-cad and other cadherins (especially N-cadherin, which is commonly upregulated during EMT).

Significance

We believe that after addressing the above concerns, the paper may provide an informative advance to current knowledge about dissemination of early cancer cells leading to cancer metastasis. As authors mentioned, literature is not clear about involvement of E-cadherin in cancer metastasis. It was first suggested that loss of E-cadherin increases the metastatic potential of tumourigenic cells (Berx et al. 1995; Bogenrieder et al. 2003), however, more recent work indicated that the loss of E-cadherin, while increasing invasiveness, decreases metastatic potential, as well as cell proliferation and survival of circulating tumour cells (Padamanaban et al. 2019), indicating that the role of E-cadherin in cancer is much more complicated than previously appreciated. Thus, assessing the role of E-cadherin during cancer cell delamination in vivo may be a powerful and informative tool to bring some clarity to the cancer biology field.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reply to the reviewers

We sincerely thank the reviewers for their thoughtful and helpful input. Below were have included our response to the points raised by our reviewers.

*Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: Reichling et al. used flow-injection time-of-flight mass spectrometry (FIA-MS), a chromatography-free high-throughput method of untargeted metabolomics, to assess the temporary changes in the concentrations of polar metabolites within budding yeast exposed to the TORC1 (target of rapamycin complex 1) inhibitor rapamycin. The water-soluble metabolomes of the rapamycin-treated wild-type (WT) strain, many mutants in the non-essential genes lacking known protein components of the TORC1-upstream and -downstream signaling and numerous gene-deletion mutants missing the redundant small-molecule protein receptors that might participate in TORC1 signaling were analyzed by FIA-MS. The quality of the quantitative metabolome profiling performed by the FIA-MS method was validated with the help of a time-consuming liquid chromatography/mass spectrometry-based metabolomics of WT cells. *

* Using the FIA-MS-based metabolome profiling, the authors revealed that rapamycin treatment upregulates or downregulates the polar metabolomes specific to a distinct set of cellular processes. Similar patterns of the temporal dynamics of rapamycin-induced changes in the metabolomes characteristic of these cellular processes were observed in WT cells and mutants deficient in known protein components of TORC1 signaling. Remarkably, the authors found that three mutants (i.e., BCK1, CLA4 and CFF1) impaired in the small-molecule protein receptors that were unknown for their roles in TORC1 signaling exhibit the temporal dynamics of rapamycin-induced metabolome changes comparable to the mutants deficient in the known positive protein regulators of TORC1 signaling. Furthermore, the comparative metabolome-based analyses of relationships between rapamycin-treated cells defective in the many known and several unknown protein regulators of TORC1 signaling allowed authors to conclude that such analyses objectively reflect the functional connectivity of these protein regulators. Moreover, the authors provided evidence that comparing the metabolome profiles in rapamycin-treated WT and TORC1 signaling mutant cells enables the identification of new metabolic reactions and pathways affected by and/or contributing to the TORC1-dependent nutrient signaling network. *

* In this proof-of-principle study, the authors also employed the liquid chromatography-tandem mass spectrometry for a quantitative proteomic comparison of rapamycin-treated WT cells and mutants impaired in the known and previously unknown protein components of TORC1 signaling. They found that rapamycin induces comparable changes in the proteomes of all these cells. This proteomic analysis confirmed that the small-molecule protein receptors previously unknown as TORC1 signaling components and identified as such components only with the help of dynamic metabolome are integrated into the TORC1 signaling network. The authors further confirmed the essential contribution of the novel protein components of the TORC1 signaling to this type of nutrient signaling in the experiments on measuring growth rate changes following a nitrogen source upshift and assessing metabolome and proteome alterations after a nitrogen source downshift.

Comments: The manuscript is clearly written and of high technical quality. All claims are convincing, fully supported by the experimental data and appropriately discussed in the context of previous literature. The authors have been fair in their treatment of previous literature. They provided the methodological detail sufficient for others to reproduce the experiments. No additional experiments are needed to support the claims made in the manuscript. The experiments are adequately replicated and statistical analysis is appropriately performed. *

* Reviewer #1 (Significance (Required)):

This pioneering study represents a major conceptual advancement in the fields of using condition-specific, dynamic metabolome profiling for the deep understanding of relationships between genes integrated into a signaling pathway, discovering novel genes incorporated into a cellular signaling network and its small-molecule regulators, and identifying the metabolic pathways governed by a signal transduction network. *

* The treatment of the existing literature on the research topic by the manuscript's authors is balanced and fair. *

* This well-organized and clearly written manuscript is a must-read for anyone interested in the molecular mechanisms of cellular signaling. *

* My field of expertise involves exploring the molecular dynamics of complex cellular processes using advanced genetic, cell biological and biochemical approaches (including the mass spectrometry-based analyses of the cellular proteomes, lipidomes and water-soluble metabolomes). *

* I recommend accepting this manuscript for publication in any journal affiliated with Review Commons. **

*

We thank reviewer 1 heartily for their comments and for the time they spent reviewing our work.*

** Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this manuscript, Rechiling et al., have used a unique approach by exploiting a temporal dynamic high-throughput metabolome profiling (using flow-injection time-of-flight mass spectrometry) to measure the metabolome profiles of many mutants in yeast, which allow them to newly annotate 3 genes in the TOR signaling pathway. This work demonstrate elegantly that dynamic perturbation of the cell allows inferring gene function when using a metabolomics-based guilt-by-association scheme. They were able to successfully find genes like CFF1, BCK1 and CLA4 which might act as positive regulators in the TOR pathway. This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. Overall, the manuscript was concisely well written, and the finding has a great potential to improve our understanding of gene function and genetic determinism of metabolism in model organisms.

Major comments: As the cellular response to rapamycin is not restricted to changes at the level of the metabolome, the authors investigated the proteomic response of each mutant to reaffirm their functional relationship to the TOR pathway. In this regard, it is not clear why the author did not consider a time-course analysis of the proteome as they did for the metabolome. The measurable steady-state proteomic signature might also reflect a buffered cellular state which might hide other response.

*

A time course analysis of the proteome for all mutants would be extremely interesting. However, due to the time and cost of proteomic analysis we instead focused on one time point for rapamycin treatment in the context of proteomics analysis. The proteome response data from mutants treated with rapamycin for 1 hour was used to identify the altered proteome responses of the mutants presented in figure 3A. This allowed us to explore the proteome responses of the mutants to rapamycin, and overcome the cellular buffering that our reviewer correctly points to.

* **Minor comments: Many typos in the methodology section (e.g., potassium phosphate ...) *

The typos in the methods section have been corrected.

* **Supplementary Fig 1: Not clear what this figure show and how it supports the author's claim that FIA-MS was validated by LC-MS. *

The positive linear relationship between the values for the measured metabolites by FIA-MS and LC-MS indicate that similar results were obtained with both methods. We have altered the text to make this clearer.*

**Reviewer #2 (Significance (Required)):

This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. The finding has a great potential to improve our understanding of gene function and genetic determinism of metabolic adaptation in model organisms. *

We thank reviewer 2 for their helpful comments, and the time they spent reviewing our work.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In this manuscript, Rechiling et al., have used a unique approach by exploiting a temporal dynamic high-throughput metabolome profiling (using flow-injection time-of-flight mass spectrometry) to measure the metabolome profiles of many mutants in yeast, which allow them to newly annotate 3 genes in the TOR signaling pathway. This work demonstrate elegantly that dynamic perturbation of the cell allows inferring gene function when using a metabolomics-based guilt-by-association scheme. They were able to successfully find genes like CFF1, BCK1 and CLA4 which might act as positive regulators in the TOR pathway.

This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. Overall, the manuscript was concisely well written, and the finding has a great potential to improve our understanding of gene function and genetic determinism of metabolism in model organisms.

Major comments:

As the cellular response to rapamycin is not restricted to changes at the level of the metabolome, the authors investigated the proteomic response of each mutant to reaffirm their functional relationship to the TOR pathway. In this regard, it is not clear why the author did not consider a time-course analysis of the proteome as they did for the metabolome. The measurable steady-state proteomic signature might also reflect a buffered cellular state which might hide other response.

Minor comments:

Many typos in the methodology section (e.g., potassium phosphate ...) Supplementary Fig 1: Not clear what this figure show and how it supports the author's claim that FIA-MS was validated by LC-MS.

Significance

This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. The finding has a great potential to improve our understanding of gene function and genetic determinism of metabolic adaptation in model organisms.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

Reichling et al. used flow-injection time-of-flight mass spectrometry (FIA-MS), a chromatography-free high-throughput method of untargeted metabolomics, to assess the temporary changes in the concentrations of polar metabolites within budding yeast exposed to the TORC1 (target of rapamycin complex 1) inhibitor rapamycin. The water-soluble metabolomes of the rapamycin-treated wild-type (WT) strain, many mutants in the non-essential genes lacking known protein components of the TORC1-upstream and -downstream signaling and numerous gene-deletion mutants missing the redundant small-molecule protein receptors that might participate in TORC1 signaling were analyzed by FIA-MS. The quality of the quantitative metabolome profiling performed by the FIA-MS method was validated with the help of a time-consuming liquid chromatography/mass spectrometry-based metabolomics of WT cells.

Using the FIA-MS-based metabolome profiling, the authors revealed that rapamycin treatment upregulates or downregulates the polar metabolomes specific to a distinct set of cellular processes. Similar patterns of the temporal dynamics of rapamycin-induced changes in the metabolomes characteristic of these cellular processes were observed in WT cells and mutants deficient in known protein components of TORC1 signaling. Remarkably, the authors found that three mutants (i.e., BCK1, CLA4 and CFF1) impaired in the small-molecule protein receptors that were unknown for their roles in TORC1 signaling exhibit the temporal dynamics of rapamycin-induced metabolome changes comparable to the mutants deficient in the known positive protein regulators of TORC1 signaling. Furthermore, the comparative metabolome-based analyses of relationships between rapamycin-treated cells defective in the many known and several unknown protein regulators of TORC1 signaling allowed authors to conclude that such analyses objectively reflect the functional connectivity of these protein regulators. Moreover, the authors provided evidence that comparing the metabolome profiles in rapamycin-treated WT and TORC1 signaling mutant cells enables the identification of new metabolic reactions and pathways affected by and/or contributing to the TORC1-dependent nutrient signaling network.

In this proof-of-principle study, the authors also employed the liquid chromatography-tandem mass spectrometry for a quantitative proteomic comparison of rapamycin-treated WT cells and mutants impaired in the known and previously unknown protein components of TORC1 signaling. They found that rapamycin induces comparable changes in the proteomes of all these cells. This proteomic analysis confirmed that the small-molecule protein receptors previously unknown as TORC1 signaling components and identified as such components only with the help of dynamic metabolome are integrated into the TORC1 signaling network. The authors further confirmed the essential contribution of the novel protein components of the TORC1 signaling to this type of nutrient signaling in the experiments on measuring growth rate changes following a nitrogen source upshift and assessing metabolome and proteome alterations after a nitrogen source downshift.

Comments:

The manuscript is clearly written and of high technical quality. All claims are convincing, fully supported by the experimental data and appropriately discussed in the context of previous literature. The authors have been fair in their treatment of previous literature. They provided the methodological detail sufficient for others to reproduce the experiments. No additional experiments are needed to support the claims made in the manuscript. The experiments are adequately replicated and statistical analysis is appropriately performed.

Significance

This pioneering study represents a major conceptual advancement in the fields of using condition-specific, dynamic metabolome profiling for the deep understanding of relationships between genes integrated into a signaling pathway, discovering novel genes incorporated into a cellular signaling network and its small-molecule regulators, and identifying the metabolic pathways governed by a signal transduction network.

The treatment of the existing literature on the research topic by the manuscript's authors is balanced and fair.

This well-organized and clearly written manuscript is a must-read for anyone interested in the molecular mechanisms of cellular signaling.

My field of expertise involves exploring the molecular dynamics of complex cellular processes using advanced genetic, cell biological and biochemical approaches (including the mass spectrometry-based analyses of the cellular proteomes, lipidomes and water-soluble metabolomes).

I recommend accepting this manuscript for publication in any journal affiliated with Review Commons.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1 CROSS-CONSULTATION COMMENTS

All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).

Response: In our fist submission, our full PDF file did not use the high-resolution figures due to large file size of some images. For re-submission, we uploaded our original, high-resolution figures. We also re-performed several immunostaining experiments and took confocal images to increase the quality of our figures. Please see below for detailed response.

Reviewer #1 (Evidence, reproducibility, and clarity (Required)):

Summary:

In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for beta-cell maturation.While the effect of Lats1 and 2 in beta-cell development has never been investigated, the outcome of the study is largely confirmative; namely YAP's necessity to switch off whenever an endocrine cell is formed while it also balances inflammation.

The study depends on cell-specific Lats1 and 2-KO mouse models and in-vitro assessments of mechanisms how loss of LATS leads to beta-cell derangement at the time of ngn3 expression and how the inflammatory pathway is activated are missing. Observations are mostly based on stainings of quite low quality and it is unclear how authors performed quantitative evaluations and how many cells from how many mice were counted. Figures show high background, e.g. CD45 in Fig.3 which would make a robust evaluation impossible. Also YAP staining, usually well-expressed in ductal cells, shows low quality and high background. Usually the antibody is well-know for its weak performance in fluorescence and should only be used with chromogenic labels.

Response: ____ To show the co-localization of multiple proteins, immunofluorescence staining is necessary. ____We added and described, in detail, how we performed macrophage quantification in the Methods section.

Reviewer #2 (Evidence, reproducibility, and clarity (Required)):

Summary:

Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.

Major comments:

Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway participation in pancreatic endocrine development. This sentence is too vague. Previous study has already reported that Ngn3 expression and Yap loss occur in parallel within the same cell during development of the endocrine pancreas (Mol Endocrinol Baltim Md. 2015;29: 1594-607. doi:10.1210/me.2014-1375).

Response: ____ As reviewer pointed out, the previous study only reported that Ngn3 expression and YAP1 loss occur in parallel within the same cells, and NGN3 can turn off Yap1 transcription in cell culture setting. However, our study showed that NGN3 alone is not sufficient to turn off Yap1 gene in vivo. Using our genetic model, we revealed that the Hippo pathway controling the nuclear localization of YAP1 is essential at the initiation of endocrine differentiation and allows YAP1 to be turned off at the transcriptional level. ____Our rescue model (NLTY mice) confirmed that the endocrine development defects observed in NL pancreas are caused by YAP1/TAZ, suggesting the necessity of controlling YAP1/TAZ by the Hippo pathway during endocrine development. Failure to sequester YAP1/TAZ outside of nuclei by the Hippo pathway in Ngn3-expressing endocrine progenitors halts the development of these cells.

Reviewer #2:

In the present study, removal of YAP1/TAZ rescued the defect in endocrine specification and differentiation in LATS1/2-null pancreas. These results indicate that both of LATS1/2 and YAP/TAZ are not essential for the normal endocrine specification and differentiation. Furthermore, these results suggest that Hippo pathway-mediated YAP1/TAZ inhibition is also unnecessary for proper pancreatic endocrine specification and differentiation. Additionally, these results suggest that just a deletion of YAP/TAZ is sufficient for endocrine specification and differentiation. How is the endocrine specification and differentiation in Ngn3creyap1fl/flTazfl/fl mice?

Response: ____ Our NL model (deletion of Lats1/2 in Ngn3-expressing cells) showed endocrine development defects, suggesting that Lats1/2 are essential for normal endocrine specification and differentiation. Removal of YAP1/TAZ in Lats1/2 null cells rescued the endocrine defects of NL pancreas. Genetic removal of YAP1/TAZ mimics Hippo pathway-mediated sequestration/degradation of YAP1/TAZ by LATS1/2. Uncontrolled YAP1/TAZ due to genetic removal of Lats1/2 blocked the endocrine specification and differentiation. This further demonstrates the necessity for functional Lats1/2 for inhibition of YAP1/TAZ in Ngn3-expressing cells during endocrine development. Ngn3CreYap1fl/flTazfl/fl mice have no endocrine defects, which is consistent with previous reports that Ngn3 expression and YAP1 loss occur in parallel within the same cells. Thus, we did not present this result.

Reviewer #2:

In Figure 2B and 3B, YFP expression (an indicator for LATS1/2 deletion) is more detectable in control compared to LATS1/2 null mice, suggesting that the LATS1/2 expression is more decreased in control mice. Is this true?

Response:____ YFP expression shows Cre activity which can be used as an indicator for Lats1/2 deletion. However, the expression level of YFP does not correlate with the expression level of Lats1/2 because YFP is controlled by the Rosa26 gene promoter. We did observe a higher level of YFP expression in endocrine cells compared with non-endocrine cells in NL pancreas. It is possible that the Rosa26 promoter is more active in endocrine cells. However, this is beyond the scope of our research.

Reviewer #2:

Please show the expression of YFP, NGN3, and YAP/TAZ in figure 6A to confirm their expression status.

Response:____ In Figure 6, we intend to show that loss of YAP1/TAZ can rescue NL mice defects at P1 where Ngn3 expression is low. The NLTY pancreas showed much more endocrine cells compared to NL pancreas, suggesting that Lats1&2 functions to control YAP1/TAZ. Genetic ablation of YAP1/TAZ in Ngn3-expressing endocrine cells equals sequestering of YAP1/TAZ outside of nuclei by LATS1/2. Thus, we did not show expression of YFP, NGN3, and YAP1/TAZ in Figure 6A. Instead, we reperformed immunostaining for Figure 5B and took high-magnification confocal images to show expression pattern of NGN3 and YAP1 in endocrine progenitors.

Reviewer #2:

Please present the sequential changes of LATS1/2, YAP/TAZ, NGN3 expressions in the control mice and LATS1/2 null mice at least E12.5, E16.5, and P1, to make ii easy to understand them for general readers.

Response:____ At E12.5, the Hippo pathway plays important roles in pancreas development. See reference 11. Ngn3 peaks at E15.5 and only expresses in endocrine progenitors. We have published a review paper (ref. 7) “Wu Y, Aegerter P, Nipper M, Ramjit L, Liu J, Wang P. Hippo Signaling Pathway in Pancreas Development. Front Cell Dev Biol. 2021;9: 663906. doi:10.3389/fcell.2021.663906” as a detailed background for this manuscript, including pancreas development, up-to-date publication on Hippo pathway in pancreas development, and a model for YAP1 function in endocrine development. Thus, we did not include this background information in this manuscript.

Reviewer #2:

Minor comments: Please describe the affiliation of authors (number 3 and 4).

Response:____ It has been included in the manuscript: 3Department of Molecular Medicine, 4Department of Population Health Sciences.

Reviewer #2:

In table 1, the details of secondary antibodies were not described.

Response:____ We have added tables to show all primary and secondary antibodies used in the paper.

Reviewer #2 (Significance (Required)): Previous studies have already reported that Ngn3 expression and Yap loss occur in parallel manner during development of the endocrine pancreas. The primary aim of the present study is whether the YAP loss is mediated by LATS1/2 in Ngn3 positive cells.

Response:____ The primary aim of our study is to understand if the Hippo pathway plays an important function in endocrine pancreas development.

Reviewer #2:

Furthermore, although authors concluded that Hippo pathway (LATS1/2)-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation, the null condition both of LATS1/2 and YAP/TAZ in Ngn3 positive cells provided the normal endocrine specification and differentiation (Figure 6). These findings do not support the conclusion.

Response:____ The defects in our NL (Lats1/2 null) pancreas in endocrine development strongly suggest the essential role of Hippo pathway in endocrine development. The null conditions of both Lats1/2 and Yap1/Taz (NLTY mice) mimic the effects of LATS1/2-mediated control of YAP1/TAZ in endocrine progenitors via genetic ablation rather than through the canonical Hippo kinase cascade. This further demonstrates the importance of proper Hippo signaling during endocrine development to inhibit YAP1/TAZ via the Hippo kinase cascade in Ngn3-expressing endocrine progenitors.

Reviewer #3 (Evidence, reproducibility, and clarity (Required)):

Summary: In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo pathway is essential for endocrine progenitor specification and differentiation but not for pancreatic beta cell function in mice. Their results are in line with prior studies, but some major issues ensue largely due to the lack of data quantification to substantiate the authors' claims, the use of ambiguous/imprecise terminologies, and making unsubstantiated claims.

Major comments:

Immunofluorescence was used to evaluate the expression, co-localization, and subcellular localization of proteins of interest (Figures 2~7). However, except for the estimation of macrophage densities (4D and 6C), none of the other immunofluorescence experiments are accompanied by quantifications and statistical analyses to substantiate the authors' claims. In addition, the red channel in several figure panels (eg. 2A and 4A) was suboptimal making interpretations very difficult. Quantification of immunofluorescence data can be done by manual counting or automated counting using software such as ImageJ or QuPath, followed by statistical analyses to provide objective evidence.

Response:____ We did not upload high resolution images for Bioxriv publication. We now uploaded the high resolution images. We also repeated a few immunostaining experiments and have taken confocal images to increase clarity.

Reviewer #3:

The endocrine component constitutes only a portion of the pancreas. The authors use whole pancreases to compare the expression levels of cell type-specific or Yap1 target genes between the knockout and control mice through qPCR. While there may be technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells, the caveats of the experiments (eg. inferring cell type-specific gene expression changes from whole organ) should be highlighted along with any inconsistent results. For example, among the three ductal genes tested in Figure 1C, only Krt19 has significantly higher mRNA expression in NL vs control, but the possible reasons for such discrepancy between ductal markers were not discussed. The authors used immunofluorescence, which is only semi-quantitative, to determine that Yap1 protein abundance is increased in Lats1&2 knockout cells. qPCR should also be performed for Yap1 in addition to Yap1 target genes to augment their claim of Yap1 expression being increased. More details on how the relative mRNA expression was computed is also necessary for readers to accurately interpret the results.

Response:____ As reviewer pointed out that there are technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells. We can only quantify gene expression level through RT-qPCR. We have discussed the high level of Krt19 and unchanged level of Sox9 in Figure 3B and 3C when we performed immunostaining. The results from the two experiments are consistent. We observed KRT19 positive staining but no SOX9 staining in Lats1/2 null Ngn3-expressing cells. This result is consistent with RT-qPCR result. We have postulated in the Discussion section that Krt19 may be directly controlled by YAP1.

Furthermore, Lats1/2 controls the protein level and localization of YAP1/TAZ, thus immunostaining can show the cellular localization.

In addition, we added the n’s used to perform RT-qPCR experiments to our manuscript and figures. We also added details to provide clarity on calculations made for relative mRNA expression from RT-qPCR experiments in the Methods section.

Reviewer #3:

The increased immunofluorescent detection of Yap1 in the NL pancreases at E16.5 is interesting but warrants further investigation. Is the increase restricted to endocrine progenitor cells or all endocrine compartments? George et al. (Mol Endocrinol. 2015) used RNA in situ hybridization and found that Yap1 mRNA expression is undetectable in the endocrine pancreas at E16.5, but here the authors observe increased Yap1 protein detected by immunofluorescence in the pancreases of animals with Lats1&2 knockout at E16.5. Although the authors speculate on a possible mechanism that may explain the discrepancies, it is important to evaluate whether the knockout really results in reactivation of Yap1 transcription and whether Yap1 auto-regulates on the transcriptional level.

Response:____ The increase of YAP1 staining is restricted to endocrine progenitor cells and blocks the endocrine differentiation. The endocrine cells in NL pancreas are escapers of Lats1/2 deletion and have no YAP1 expression, which is consistent with George et al. findings. The model we proposed is that in Ngn3-expressing cells, Lats1/2 are required to keep YAP1/TAZ out of nuclei so that Ngn3 can repress YAP1 expression. Loss of Lats1/2 led to high nuclear YAP1 which may block Ngn3’s ability to suppress YAP1. ChIP-seq or ChIP-PCR on YAP1 promoter will answer the question whether YAP1 auto-regulates on the transcriptional level. However, the small number of cells in mouse pancreas limits us to perform this experiment.

Reviewer #3:

The numbers of animals used are unclear except for those depicted by the barplots. There is also no mention of the number of cells or fields analyzed for immunofluorescence experiments, which is essential for any quantitative comparisons and claims. The n's should be added throughout.

Response:____ We added n’s to all figures and in our manuscript.

Reviewer #3:

The authors repeatedly refer to the dataset from Cebola et al. (Nat Cell Biol 2015), which was generated from human embryonic pancreatic progenitor cells, to speculate that TEAD1 binds to the promoter regions of YAP1, CDH1 and KRT19 and therefore may promote YAP1 autoregulation or expression of CDH1 and KRT19, explaining some of their immunofluorescence observations. However, they fail to acknowledge potential differences that may ensue due to the different species examined. Co-staining of Yap1 and Cdh1/Krt19 would indicate whether co-expression of Yap1 and Cdh1/Krt19 is indeed evident in the context of their study and provide further evidence to support their speculations.

Response:____ Reviewer is correct that the dataset of TEAD1 ChIP-seq from Cebola et al. was generated from human embryonic pancreatic progenitor cells. These data are in line with our mouse experimental results where cells with high YAP1, due to loss of Lats1&2, continue to have high YAP1, CDH1 and KRT19. We do not have evidence to point out the potential differences between mouse and human.

Reviewer #3:

Several observations are over-interpreted or over-stated, and should be qualified as preliminary or speculative with proper wording. For example, P14: "differentiation...was blocked by lack of expression of ISL1 and NKX2.2". Although the authors observe low Isl1 and Nkx2.2 staining in NL vs control pancreases, no experiments were done to substantiate the claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in this context.

Response:____ We did not claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in NL pancreas. ISL1 and NKX2.2 are markers for endocrine differentiation. The lack of ISL1 and NKX2.2 expression indicates that endocrine differentiation has been blocked.

Reviewer #3:

P15: "...KRT19 expression is not controlled by SOX9, but instead by YAP1". The authors observe that Krt19 proteins are increased in Lats1&2-null Ngn3+ cells, whereas Sox9 proteins were unchanged. However, they do not provide evidence that Yap1 controls Sox9 expression.

__Response:____ No Sox9 expression was observed in Ngn3-expressing cells in both Control and NL pancreases (Figure 3B and 3C) while we observed YAP1 nuclei staining in NGN3-positive cells (Figure 5B), suggesting that YAP1 does not control Sox9 expression.

__

Reviewer #3:

Minor comments:

It is mentioned that deletion of Lats1&2 in Ngn3+ cells results in fewer acinar cells and smaller islets, evident by reduction in Ins+ or Gcg+ cells, whereas such genetic ablation in pancreatic beta cells does not result in any phenotype. Did the deletion of Lats1&2 in Ngn3+ cells similarly lead to reduction in other endocrine cell types?

Response:____ We showed that there was no positive staining for ISL1 and NKX2.2 in progeny of Lats1&2 null Ngn3-expressing cells, suggesting the block of endocrine differentiation including all endocrine cells. The INS+ and GCG+ cells in NL pancreas are the escapers in which Lats1&2 were not deleted. Other endocrine cells should be affected too. We observed smaller sized NL mice with low blood glucose levels. We have postulated that brain expression of Ngn3Cre may contribute to these phenotypes.

Reviewer #3:

The authors use the word "expression" without specification to refer to both mRNA expression and marker fluorescence levels throughout the text. This is inaccurate and potentially confusing. More specific terminologies should instead be used to avoid ambiguity.

Response:____ We have added mRNA in the appropriate places where we discuss results from RT-qPCR experiments. All other places are protein expression results by immunostaining. We followed the general guideline for formatting gene and protein name throughout the manuscript: mouse gene symbols are italicized, with only the first letter in upper-case; protein symbols are not italicized, and all letters are in upper-case.

Reviewer #3:

Figure S1D is mis-referred to as S1E in the text. Figure 7G is missing. Table S1 is mis-referred to as S Table 2 in the text. Typo on page 19: "Controlcontrol" Figure S6B is mis-referred to as Figure S6A in the text.

Response:____ We have made all appropriate changes to the manuscript to correct these mistakes.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary

In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo pathway is essential for endocrine progenitor specification and differentiation but not for pancreatic beta cell function in mice. Their results are in line with prior studies, but some major issues ensue largely due to the lack of data quantification to substantiate the authors' claims, the use of ambiguous/imprecise terminologies, and making unsubstantiated claims.

Major comments:

1. Immunofluorescence was used to evaluate the expression, co-localization, and subcellular localization of proteins of interest (Figures 2~7). However, except for the estimation of macrophage densities (4D and 6C), none of the other immunofluorescence experiments are accompanied by quantifications and statistical analyses to substantiate the authors' claims. In addition, the red channel in several figure panels (eg. 2A and 4A) was suboptimal making interpretations very difficult. Quantification of immunofluorescence data can be done by manual counting or automated counting using software such as ImageJ or QuPath, followed by statistical analyses to provide objective evidence.
2. The endocrine component constitutes only a portion of the pancreas. The authors use whole pancreases to compare the expression levels of cell type-specific or Yap1 target genes between the knockout and control mice through qPCR. While there may be technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells, the caveats of the experiments (eg. inferring cell type-specific gene expression changes from whole organ) should be highlighted along with any inconsistent results. For example, among the three ductal genes tested in Figure 1C, only Krt19 has significantly higher mRNA expression in NL vs control, but the possible reasons for such discrepancy between ductal markers were not discussed. The authors used immunofluorescence, which is only semi-quantitative, to determine that Yap1 protein abundance is increased in Lats1&2 knockout cells. qPCR should also be performed for Yap1 in addition to Yap1 target genes to augment their claim of Yap1 expression being increased. More details on how the relative mRNA expression was computed is also necessary for readers to accurately interpret the results.
3. The increased immunofluorescent detection of Yap1 in the NL pancreases at E16.5 is interesting but warrants further investigation. Is the increase restricted to endocrine progenitor cells or all endocrine compartments? George et al. (Mol Endocrinol. 2015) used RNA in situ hybridization and found that Yap1 mRNA expression is undetectable in the endocrine pancreas at E16.5, but here the authors observe increased Yap1 protein detected by immunofluorescence in the pancreases of animals with Lats1&2 knockout at E16.5. Although the authors speculate on a possible mechanism that may explain the discrepancies, it is important to evaluate whether the knockout really results in reactivation of Yap1 transcription and whether Yap1 auto-regulates on the transcriptional level.
4. The numbers of animals used are unclear except for those depicted by the barplots. There is also no mention of the number of cells or fields analyzed for immunofluorescence experiments, which is essential for any quantitative comparisons and claims. The n's should be added throughout.
5. The authors repeatedly refer to the dataset from Cebola et al. (Nat Cell Biol 2015), which was generated from human embryonic pancreatic progenitor cells, to speculate that TEAD1 binds to the promoter regions of YAP1, CDH1 and KRT19 and therefore may promote YAP1 autoregulation or expression of CDH1 and KRT19, explaining some of their immunofluorescence observations. However, they fail to acknowledge potential differences that may ensue due to the different species examined. Co-staining of Yap1 and Cdh1/Krt19 would indicate whether co-expression of Yap1 and Cdh1/Krt19 is indeed evident in the context of their study and provide further evidence to support their speculations.
6. Several observations are over-interpreted or over-stated, and should be qualified as preliminary or speculative with proper wording. For example, a. P14: "differentiation...was blocked by lack of expression of ISL1 and NKX2.2". Although the authors observe low Isl1 and Nkx2.2 staining in NL vs control pancreases, no experiments were done to substantiate the claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in this context. b. P15: "...KRT19 expression is not controlled by SOX9, but instead by YAP1". The authors observe that Krt19 proteins are increased in Lats1&2-null Ngn3+ cells, whereas Sox9 proteins were unchanged. However, they do not provide evidence that Yap1 controls Sox9 expression.

Minor comments:

1. It is mentioned that deletion of Lats1&2 in Ngn3+ cells results in fewer acinar cells and smaller islets, evident by reduction in Ins+ or Gcg+ cells, whereas such genetic ablation in pancreatic beta cells does not result in any phenotype. Did the deletion of Lats1&2 in Ngn3+ cells similarly lead to reduction in other endocrine cell types?
2. The authors use the word "expression" without specification to refer to both mRNA expression and marker fluorescence levels throughout the text. This is inaccurate and potentially confusing. More specific terminologies should instead be used to avoid ambiguity.
3. Figure S1D is mis-referred to as S1E in the text.
4. Figure 7G is missing.
5. Table S1 is mis-referred to as S Table 2 in the text.
6. Typo on page 19: "Controlcontrol"
7. Figure S6B is mis-referred to as Figure S6A in the text.

Significance

The exact roles of the Hippo pathway in pancreas organogenesis are far from being delineated. The current study proposes a new model wherein the Hippo pathway kinase cascade acts to sequester and inhibit Yap1 in the cytosol for Ngn3 to drive endocrine specification, including inhibition of Yap1 expression. This work is potentially valuable to further our understanding of the physiological roles of the Hippo pathway in the context of endocrine pancreas development.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary:

Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.

Major comments:

• Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway participation in pancreatic endocrine development. This sentence is too vague. Previous study has already reported that Ngn3 expression and Yap loss occur in parallel within the same cell during development of the endocrine pancreas (Mol Endocrinol Baltim Md. 2015;29: 1594-607. doi:10.1210/me.2014-1375).
• In the present study, removal of YAP1/TAZ rescued the defect in endocrine specification and differentiation in LATS1/2-null pancreas. These results indicate that both of LATS1/2 and YAP/TAZ are not essential for the normal endocrine specification and differentiation. Furthermore, these results suggest that Hippo pathway-mediated YAP1/TAZ inhibition is also unnecessary for proper pancreatic endocrine specification and differentiation. Additionally, these results suggest that just a deletion of YAP/TAZ is sufficient for endocrine specification and differentiation. How is the endocrine specification and differentiation in Ngn3creyap1fl/flTazfl/fl mice?
• In Figure 2B and 3B, YFP expression (an indicator for LATS1/2 deletion) is more detectable in control compared to LATS1/2 null mice, suggesting that the LATS1/2 expression is more decreased in control mice. Is this true?
• Please show the expression of YFP, NGN3, and YAP/TAZ in figure 6A to confirm their expression status.
• Please present the sequential changes of LATS1/2, YAP/TAZ, NGN3 expressions in the control mice and LATS1/2 null mice at least E12.5, E16.5, and P1, to make ii easy to understand them for general readers.

Minor comments:

Please describe the affiliation of authors (number 3 and 4).

In table 1, the details of secondary antibodies were not described.

Significance

Previous studies have already reported that Ngn3 expression and Yap loss occur in parallel manner during development of the endocrine pancreas. The primary aim of the present study is whether the YAP loss is mediated by LATS1/2 in Ngn3 positive cells.

Furthermore, although authors concluded that Hippo pathway (LATS1/2)-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation, the null condition both of LATS1/2 and YAP/TAZ in Ngn3 positive cells provided the normal endocrine specification and differentiation (Figure 6). These findings do not support the conclusion.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for beta-cell maturation.

While the effect of Lats1 and 2 in beta-cell development has never been investigated, the outcome of the study is largely confirmative; namely YAP's necessity to switch off whenever an endocrine cell is formed while it also balances inflammation. The study depends on cell-specific Lats1 and 2-KO mouse models and in-vitro assessments of mechanisms how loss of LATS leads to beta-cell derangement at the time of ngn3 expression and how the inflammatory pathway is activated are missing. Observations are mostly based on stainings of quite low quality and it is unclear how authors performed quantitative evaluations and how many cells from how many mice were counted. Figures show high background, e.g. CD45 in Fig.3 which would make a robust evaluation impossible. Also YAP staining, usually well-expressed in ductal cells, shows low quality and high background. Usually the antibody is well-know for its weak performance in fluorescence and should only be used with chromogenic labels.

Referees cross-commenting

All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).

Significance

Largely confirmative. Single model approach with relatively low quality of the IHC analyses.

My expertise: inflammation, beta-cell, islets, Hippo pathway

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: The authors compared the various multinucleated cells, osteoclasts, LCG and FBGC. Overall, the manuscript shows rigor in the analyses, and also very interesting approaches for retrieving mononuclear cells, for instance using DC-STAMP siRNA. This work adds very much to understanding the biological differences, as summarized in figure 6h. A lot of work in osteoclast field with for instance qPCR is hampered because, inevitably, a mix of mononuclear and multinucleated cells is always measured. Here, a solid attempt to separate those mixes with cell sorting and subsequent analysis on the mononuclear and multinucleated isolates, really adds. Choice of figures is good, also the extra info of the supplementary figures is relevant and makes it easy to read.

Major and minor concerns:

1. For osteoclasts, various markers exist for their biological characterization, for instance the ability to resorb bone. What, apart from the arrangement and number of nuclei, were the biological parameters that confirmed that the cells made by addition of IFN or IL-4 were LCG and FBGC? [Authors’ reply]. In order to address this point, we focused on gene sets that characterize LCGs and FBGCs. By doing so, we aimed to identify (i) lineage dependent factors and (ii) markers of LGCs and FBGCs. (See new Supplementary Figure 1B and C, New Supplementary Table 1 and highlighted text in Results). As expected, and in line with the lineage-determining factors, the transcriptomics comparison between mononucleated/multinucleated IFN-γ and IL-4-differentiated macrophages showed predominance of IFN-γ and IL-4-related pathways, respectively (Supplementary Figure 1B and C and Supplementary Table 1). Among known LGC and FBGC markers, we confirmed up-regulation of CCL7 [1] and CD86 [2], respectively.* As per the biological parameters, we indeed confirm that FBGCs show enhanced phagocytosis properties (Figure 5C) while LGCs can form granuloma-like clusters in vitro (Figure 4D and E). Altogether, we characterize LGCs and FBGCs with (i) polykaryon-specific nuclear arrangement, (ii) polykaryon-specific gene expression markers, (iii) previously shown and new phenotypic characteristics such as LGCs’ unique ability to form in vitro clusters containing CD3+ cells. *

In fig 2c: did the authors perform stainings with isotype control antibodies? In my experience, quite often, antibodies stain mononuclear cells much intenser, since the cytoplasm is much more condense, less spread over a large area.

[Authors’ reply]. According to the reviewer’s suggestion, we provide isotype control staining for MRC1 in IFN-g-stimulated mononucleated/multinucleated cells by ImageStream (left panel) and immunofluorescence in LGCs, FBGCs and osteoclasts (right panel). There was negligible staining with the isotype control antibody for MRC1 in both settings (Figure provided to the journal).

*We did not observe a potential artefact of staining in multinucleated cells when compared to mononuclear cells. In fact, some markers of multinucleation such as B7-H3 is augmented in LGCs (Figure 4E). *

Resorption assay in 6 is not clear. It is weird that osteoclasts apparently display so limited resorption? Also the traces are not typical for osteoclasts. Please explain.

[Authors’ reply]. Human osteoclasts are cultured for 2 days on hydroxyapatite-coated plates and the amount of resorption is dependent on the healthy donor the peripheral blood is derived from. In addition to genetic variability, the support (hydroxyapatite) is different from dentine, which is also widely used for measuring osteoclast resorptive activity. The visualization of the human osteoclast resorption is made by transparency (area not coated by hydroxyapatite due to its resorption) on image J.

Provide a better image Supplementary 2A, even at 250% the lettering is vague. What do the colours in 2A mean?

[Authors’ reply]. *According to the reviewer’s suggestion, we now provide the Supplementary Figure 2A with better resolution. In STRING protein-protein interaction analysis, there is no particular meaning of the node color itself. *

CROSS-CONSULTATION COMMENTS

I have read the comments of the other two reviewers, and together. I absolutely agree with their additions, Indeed, supplementary tables are lacking, as well as there could be a bit more emphasis on the fact that it is all in vitro work. Together, I think the three of us are complementary in our comments, with good overlap as well. Any effort to stain for instance pathology material with the markers that have been found, would be great, especially for the LGC and the FBGC, that are much less studied in the field of MNGs. Having said that ,I can also live without this addition, but then it could be highlighted in the discussion that these are the future avenues that should be considered. Collaborate with Pathology!

[Authors’ reply]. We appreciate that the reviewer provides cross-consultation comments which we address in our revised manuscript. As such, we discuss future avenues regarding the translatability of these results to human pathology involving MGCs.

Reviewer #1 (Significance (Required)):

This manuscript is particularly interesting to those who are interested in the BIOLOGY of MNCs. In essence, three types of MNCs were cultured and compared, with each of them a specific function.

I am an osteoclast expert (76 publications), and have two publications on FBGCs

[Authors’ reply]. *We sincerely thank the reviewer for his/her pertinent comments, enthusiasm for our findings and for providing us an overall summary of our findings in view of all other reviewer comments. *

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary:

In this manuscript, the authors performed a comparative transcriptome analysis of mononuclear and multinuclear human osteoclasts, LGCs and FBGCs. They found that multinucleation triggers a significant downregulation of macrophage identity in all three types of MGCs. Furthermore, RNA-seq data and in-vitro functional analysis of multinucleated cells showed that macrophage cell-cell fusion and multinucleation enhance phagocytosis and contribute to lysosome-dependent intracellular iron homeostasis. Furthermore, multinucleation of osteoclasts promoted mitochondrial activity and oxidative phosphorylation, resulting in maximal respiration. This unique and interesting study addresses the fundamental question of how cell-cell fusion and multinucleation contribute to cellular activity and biological homeostasis.

Major comments

1 The authors generated mature multinucleated cells by stimulating human PBMC-derived macrophages with either IFN-g, IL-4, or RANKL. However, no quantitative data have been presented to determine how effectively IL-4, IFN-g, and RANKL can induce multinucleated giant cells from mononuclear macrophages. Quantitative data showing induction efficiency would provide a more detailed picture of the overall experiment.

[Authors’ reply]. According to the reviewer’s suggestion, quantitative data showing the efficiency of these cytokines to induce multinucleation (i.e. fusion index) is now provided as part of the revised Supplementary Figure 1A (right panel).

2 The authors mentioned, "The distinct morphological appearance of these three types of MGCs (Figure 1B) suggested cell type-specific functional properties and shared mechanisms underlying macrophage multinucleation". However, there is no discussion or data showing how the nuclear arrangement and intracellular location affect the biological function of multinucleated cells.

[Authors’ reply]. This is good point and is now discussed in the revised manuscript (see highlighted text in revised manuscript and below).

Whether MGC-specific nuclear arrangements and/or numbers are indicative of specialized function is currently unclear. Intracellular nuclei arrangement is likely to be important for the sealing zone formation in a polarized bone-resorbing osteoclast. Furthermore, whether distinct transcriptional activities are assigned to different nuclei of the MGC also remain to be tested. Recent elegant work performed in multinucleated skeletal myofibers suggest transcriptional heterogeneity among the different nuclei of the polykaryon [3].

3 Based on the results of DC-stamp knockdown experiments, the authors concluded that cell-cell fusion and multinucleation suppress the mononuclear phagocytic gene signature. However, to strengthen this hypothesis, it would be necessary to provide at least data showing that DC-stamp knockdown reduces the number of multinucleated cells.

[Authors’ reply]. According to the reviewer’s suggestion, we provide data showing that DCSTAMP knockdown reduces multinucleation in LGCs and FBGCs (see below and new Supplementary Figure 2F). For human osteoclasts, the data was included in our previously published paper ([4] and figure provided to the journal).

4 In Figure4, the authors showed that transcripts in LGCs were enriched for antigen presentation and adaptive immune system pathways. In addition, multinucleation of LGCs increased the surface expression of B7-H3 (CD276) and colocalized with CD3+ cells, suggesting that LGC multinucleation potentiates T cell activation. However, the authors did not present enough data to demonstrate the antigen-presenting ability of LGCs or their specific T cell activating capacity.

[Authors’ reply]. We agree with the reviewer that our data on a potential role of LGCs’ on T cell activation is based on increased surface expression of B7-H3 and the unique CD3+ cluster forming ability of LGCs. In order to check for further markers of antigen presentation, we have performed MHC-1 and MHC-2 quantification by ImageStream in 3 types of MGCs (figure provided to the journal).

Although there was no difference in MHC-I/MHC-2 between the mononucleated and multinucleated macrophages, the mean fluorescent intensity (MFI) range was the highest in IFN-g-stimulated macrophages, suggesting that LGCs may be better equipped for antigen presentation than the other 2 types of MGCs. A more comprehensive analysis of antigen presentation requires enzymatic digestion and isolation and phenotyping of LGCs from clusters in vitro and human tissues in vivo. This is a program of research that we have initiated as part of a separate study, which will focus on the in vivo relevance of the current findings such as the unique Ag presentation ability of LGCs in a non-sterile tissue environment.

5 Figure 6 clearly shows that mature multinucleated osteoclasts exhibit increased ATP production and maximal respiration. However, the glycolytic pathway did not differ between mononuclear and multinuclear osteoclasts. No explanation for this observation has been provided. It is easy to understand that osteoclasts acquire ATP through aerobic respiration during multinucleation. But how NADPH, which is essential for its redox reaction, is produced? Is it by acquiring αKG from the glutamine pathway?

[Authors’ reply]. This is a point worth expending (see also discussion; highlighted text). Osteoclast multinucleation is characterized by increased mitochondrial gene expression which also translates into increased spare respiratory capacity (SRC or maximal respiration). This metabolic rewiring does not modify glycolysis and basal respiration rate. As the reviewer correctly states, increased SRC may be a way to supply more ATP to the energy-demanding polykaryon.

As per the production of NAD(P)H as an electron source for ETC, it could indeed be through glutamine rather than glucose usage in multinucleated osteoclasts. Furthermore, as iron is an essential cofactor for ETC activity through activity of iron-sulfur clusters, the mitochondrial concentration of iron is likely to be critical for the mitochondrial activity of multinucleated osteoclasts (see also discussion).

Minor comments:

6 Supplementary tables 1-6 were not provided.

[Authors’ reply]. We apologize for this. The revised versions of supplementary tables are provided as part of the revised manuscript.

7 Figure 2D right panel, difficult to see DAPI+ nuclei.

[Authors’ reply]. Thanks for pointing this out. We have now replaced Figure 2D with a more pronounced DAPI+ nuclei.

Reviewer #2 (Significance (Required)):

Although it is well known that multinucleation of cells constantly occurs, especially in osteoclasts, skeletal muscle, and trophoblasts of the placenta, the biological significance of multinucleation and the intracellular functions of multinucleation are not well understood. In this unique study, three types of multinucleated cells were generated from human peripheral blood to elucidate the genetic and functional differences between mononucleated and multinucleated cells. Furthermore, by demonstrating the possibility that the morphological peculiarity of multinucleation can regulate cell function, this paper provides clues to understanding the underlying biology of multinucleated cells and how they maintain cell function in homeostatic and pathological settings.

[Authors’ reply]. We thank the reviewer for finding our study unique and biologically meaningful. We also thank the reviewer for all the suggestions that improved significantly the overall message of the manuscript.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary:

The manuscript of Ahmadzadeh and Pereira et al is an interesting study of the fusion process key to the formation of multinucleated giant cells (MGCs). Our current ability to discriminate between different types of MGCs is limited, and there are gaps in our understanding of the molecular determinants of cell fusion. In this study, the authors isolated different MGC variants - osteoclasts, Langhans giant cells (LGCs) and foreign body giant cells (FBGCs) and identified common, as well as MGC-specific genes and pathways involved in the process of cell fusion. The approach of isolating and comparing different types of MGCs is novel, and the manuscript is well presented and written. However, due to the in vitro nature of the study, the physiological significance of the findings is unclear. I have further minor and major points for the authors to address, as detailed below.

Minor comments:

1. The approach to isolate the different MGCs using FACS and imaging technique is highly novel. However the difference between MGC subtypes isolated isn't immediately apparent beyond the morphological comparisons. In my opinion some of the results of MGC-specific assays from Figures 4, 5 and 6 can be included in Figure 1, e.g. TRAP staining and hydroxyapatite resorption for osteoclasts, to provide evidence of purity and specificity of each MGC subtype early on in the manuscript. Classical or canonical genes associated with each MGC subtype can also be highlighted in the volcano plots in Figure 1C, e.g ACP5, CTSK, TNFRSF11A for osteoclasts. [Authors’ reply]. We thank the reviewer for this point and we agree it is important to highlight markers for each polykaryon early in the manuscript. In accordance with this reviewers’ comment (and also with Reviewer 1’s point), we first verified existence of lineage-dependent factors and markers of LGCs and FBGCs as these cells are relatively less well-defined compared to osteoclasts. (New Supplementary Figure 1B and C and New Supplementary Table 1). As expected, and in line with the lineage-determining effects, the transcriptomics comparison between mononucleated/multinucleated IFN-γ and IL-4-differentiated macrophages showed predominance of IFN-γ and IL-4-related pathways, respectively (New Supplementary Figure 1B and C and New Supplementary Table 1). Among known LGC and FBGC markers, we confirmed up-regulation of CCL7 [1] and CD86 [2], respectively (New Supplementary Table 1). We have added this information in the revised manuscript (see highlighted text). Osteoclast phenotyping is provided by TRAP staining and resorption assay (Figure 6C) and we also confirm that CTSK is indeed significantly up-regulated upon multinucleation (LogFc=1.69; P=9.2 x 10E-6; highlighted in the revised manuscript).

The overall decrease in phagocytic identity of all the MGCs, and the specific upregulation of phagocytic pathways in the FBGCs are conflicting. Are there subsets of phagocytic pathways that were down and upregulated during the formation of FBGCs?

[Authors’ reply]. This is a very good point. As the reviewer indicates, the results suggest that subsets of phagocytic pathways are changed upon multinucleation. All three types of MGCs show a downregulation of transcripts that belong to Fc receptors and complement C1Q family. However only FBGCs show an up-regulation of S. Aureus bioparticle-mediated phagocytosis. Hence the exact surface receptors responsible for this pathogen clearance remain to be identified. FBGC phagocytosis is a complex process including non-canonical phagocytosis pathways and participation of increased membrane area and endoplasmic reticulum [5, 6]*. Whether these pathways are specifically induced in human FBGCs remain to be identified. We now discuss this point in the revised manuscript (see highlighted text in Discussion). *

What are the identities of the mononuclear cells in each of the MGC experiment? They appeared to be quite heterogeneous based on the DEGs identified, beyond the common phagocyte signature. Can the authors comment on the difference between the mononuclear cells and whether this will affect the DEG analysis?

[Authors’ reply]. This is also a very relevant point that we now address in the revised manuscript (New Supplementary Figure 1B and C; New Supplementary Table 1 and highlighted revised text in Results). The reviewer is correct that MGC-specific pathways are in line with the known function of each polykaryon (Figure 4A, 5A and 6A). To what extent lineage-dependent effects (e.g. IFN-g and IL-4) are conserved between the mononucleated and multinucleated state is yet to be determined. In order to address this point, we compared DEG in IFN-g and IL-4-differentiated mononucleated macrophages to the ones obtained in multinucleated macrophages (New Supplementary Figure 1B and C; New Supplementary Table 1). The results showed that the multinucleated cell state preserves the majority of the lineage-dependent pathways which are very significantly represented at the mononucleated cell state (e.g. IFN-g and IL-4-related pathways). Interestingly, although less significant, this analysis also showed pathways that were specific to the mononucleated or multinucleated state in IFN-γ-differentiated macrophages when compared to IL-4-differentiated ones and vice versa. (Supplementary Figure 1B and C). For instance, TRAF3-dependent IRF activation pathway is specific to mononucleated IFN-g-differentiated macrophages (Supplementary Figure 1B).

The authors should also frame/discuss the findings in the context of diagnostic and therapeutic potentials to highlight the clinical significance of this study.

[Authors’ reply]. We thank the reviewer for this point and we now discuss our results from a clinical/diagnostic perspective (see highlighted text in the Discussion and below).

From a clinical perspective, since lysosome-regulated intracellular iron homeostasis appears to be a general condition for macrophage multinucleation across different tissues, its blockade may hold therapeutic potential. However, it is still unclear whether granulomatous disease can benefit from targeting LGC fusion. For non-granulomatous inflammatory diseases, inhibiting MGC formation by targeting lysosomes may be a therapeutic avenue. This approach would avoid FBGC-related adverse effects during foreign body reaction or inhibit the formation of MGCs of white adipose tissue during obesity. v-ATPase inhibitors have been previously proposed to inhibit osteoclast activity and bone resorption [7]* so their selective targeting in the lysosomal compartment may be generalized to other MGCs such as FBGCs. In addition to potential clinical translation, the results presented in this study require confirmation in tissues originating from human pathology involving MGCs. *

Major comments:

• As mentioned before, the physiological significance of the findings is unclear. Some form of in vivo data is needed to support some of the key conclusions of the study, e.g validating some of the markers of the pathways identified (common and MGC subtype-specific), and the role of lysosome-mediated iron homeostasis in multinucleation. The authors can make use of the FACs and imaging approaches they developed to look at MGCs in relevant tissues. [Authors’ reply]. This is an important point that we would like to explore in a comprehensive way. We have initiated a 2-year program to undertake a Multiplexed Immunohistochemistry (mIHC) using MILAN (Multiple Iterative Labeling by Antibody Neodeposition) https://www.lpcm.be/multiplex-ihc-milan approach in human biopsies using >100 antibodies. The current study is pivotal in selecting the gene targets (i.e. common and MGC-specific markers) for prioritization. We foresee to gain critical pathophysiological information about the tissue characteristics of MGCs. The reviewer would acknowledge that these high-throughput and biopsy-based initiatives are lengthy and not the primary scope of our current findings which set the foundation of major cellular events governing multinucleation in macrophages.

Reviewer #3 (Significance (Required)):

Significance:

• The approach of isolating and comparing different types of MGCs is novel, and the findings certainly improved our understanding of the fusion processes of MGCs. However, the physiological role of these processes in health and disease that involve MGCs is still unclear due to the lack of in vivo data. The findings were discussed in quite a bit of detail in the context of current literature, though clinical impact was not explored. [Authors’ reply]. *We are grateful to Reviewer 3 for raising relevant and constructive points regarding the main findings. His/her review significantly improved the clarity of the overall manuscript. *

We recognize our study lacks human clinical association, but we highlight the prospective translatability of our findings and the usage of donor-based human macrophages throughout the manuscript. As also recommended by Reviewer 1 in his/her cross-consultation, we discuss the potential clinical impact of our findings in the Discussion of our revised manuscript.

• My background is bone biology with a very keen interest in osteoclast biology so arguably my knowledge on other MGCs eg LGCs and FBGCs is limited. References

• Chen Y, Jiang H, Xiong J, Shang J, Chen Z, Wu A, Wang H: Insight into the Molecular Characteristics of Langhans Giant Cell by Combination of Laser Capture Microdissection and RNA Sequencing. J Inflamm Res 2022, 15:621-634.

• McNally AK, Anderson JM: Foreign body-type multinucleated giant cells induced by interleukin-4 express select lymphocyte co-stimulatory molecules and are phenotypically distinct from osteoclasts and dendritic cells. Exp Mol Pathol 2011, 91(3):673-681.
• Petrany MJ, Swoboda CO, Sun C, Chetal K, Chen X, Weirauch MT, Salomonis N, Millay DP: Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers. Nat Commun 2020, 11(1):6374.
• Pereira M, Ko JH, Logan J, Protheroe H, Kim KB, Tan ALM, Croucher PI, Park KS, Rotival M, Petretto E et al: A trans-eQTL network regulates osteoclast multinucleation and bone mass. Elife 2020, 9.
• McNally AK, Anderson JM: Multinucleated giant cell formation exhibits features of phagocytosis with participation of the endoplasmic reticulum. Exp Mol Pathol 2005, 79(2):126-135.
• Milde R, Ritter J, Tennent GA, Loesch A, Martinez FO, Gordon S, Pepys MB, Verschoor A, Helming L: Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction. Cell Rep 2015, 13(9):1937-1948.
• Qin A, Cheng TS, Pavlos NJ, Lin Z, Dai KR, Zheng MH: V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption. Int J Biochem Cell Biol 2012, 44(9):1422-1435.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary:

The manuscript of Ahmadzadeh and Pereira et al is an interesting study of the fusion process key to the formation of multinucleated giant cells (MGCs). Our current ability to discriminate between different types of MGCs is limited, and there are gaps in our understanding of the molecular determinants of cell fusion. In this study, the authors isolated different MGC variants - osteoclasts, Langhans giant cells (LGCs) and foreign body giant cells (FBGCs) and identified common, as well as MGC-specific genes and pathways involved in the process of cell fusion. The approach of isolating and comparing different types of MGCs is novel, and the manuscript is well presented and written. However, due to the in vitro nature of the study, the physiological significance of the findings is unclear. I have further minor and major points for the authors to address, as detailed below.

Minor comments:

• The approach to isolate the different MGCs using FACS and imaging technique is highly novel. However the difference between MGC subtypes isolated isn't immediately apparent beyond the morphological comparisons. In my opinion some of the results of MGC-specific assays from Figures 4, 5 and 6 can be included in Figure 1, e.g. TRAP staining and hydroxyapatite resorption for osteoclasts, to provide evidence of purity and specificity of each MGC subtype early on in the manuscript. Classical or canonical genes associated with each MGC subtype can also be highlighted in the volcano plots in Figure 1C, e.g ACP5, CTSK, TNFRSF11A for osteoclasts.

• The overall decrease in phagocytic identity of all the MGCs, and the specific upregulation of phagocytic pathways in the FBGCs are conflicting. Are there subsets of phagocytic pathways that were down and upregulated during the formation of FBGCs?

• What are the identities of the mononuclear cells in each of the MGC experiment? They appeared to be quite heterogeneous based on the DEGs identified, beyond the common phagocyte signature. Can the authors comment on the difference between the mononuclear cells and whether this will affect the DEG analysis?

• The authors should also frame/discuss the findings in the context of diagnostic and therapeutic potentials to highlight the clinical significance of this study

Major comments:

• As mentioned before, the physiological significance of the findings is unclear. Some form of in vivo data is needed to support some of the key conclusions of the study, e.g validating some of the markers of the pathways identified (common and MGC subtype-specific), and the role of lysosome-mediated iron homeostasis in multinucleation. The authors can make use of the FACs and imaging approaches they developed to look at MGCs in relevant tissues.

Significance

Significance:

• The approach of isolating and comparing different types of MGCs is novel, and the findings certainly improved our understanding of the fusion processes of MGCs. However the physiological role of these processes in health and disease that involve MGCs is still unclear due to the lack of in vivo data. The findings were discussed in quite a bit of detail in the context of current literature, though clinical impact was not explored.

• My background is bone biology with a very keen interest in osteoclast biology so arguably my knowledge on other MGCs eg LGCs and FBGCs is limited.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary:

In this manuscript, the authors performed a comparative transcriptome analysis of mononuclear and multinuclear human osteoclasts, LGCs and FBGCs. They found that multinucleation triggers a significant downregulation of macrophage identity in all three types of MGCs. Furthermore, RNA-seq data and in-vitro functional analysis of multinucleated cells showed that macrophage cell-cell fusion and multinucleation enhance phagocytosis and contribute to lysosome-dependent intracellular iron homeostasis. Furthermore, multinucleation of osteoclasts promoted mitochondrial activity and oxidative phosphorylation, resulting in maximal respiration. This unique and interesting study addresses the fundamental question of how cell-cell fusion and multinucleation contribute to cellular activity and biological homeostasis.

Major comments:

1. The authors generated mature multinucleated cells by stimulating human PBMC-derived macrophages with either IFN-g, IL-4, or RANKL. However, no quantitative data have been presented to determine how effectively IL-4, IFN-g, and RANKL can induce multinucleated giant cells from mononuclear macrophages. Quantitative data showing induction efficiency would provide a more detailed picture of the overall experiment.

2. The authors mentioned, "The distinct morphological appearance of these three types of MGCs (Figure 1B) suggested cell type-specific functional properties and shared mechanisms underlying macrophage multinucleation". However, there is no discussion or data showing how the nuclear arrangement and intracellular location affect the biological function of multinucleated cells.

3. Based on the results of DC-stamp knockdown experiments, the authors concluded that cell-cell fusion and multinucleation suppress the mononuclear phagocytic gene signature. However, to strengthen this hypothesis, it would be necessary to provide at least data showing that DC-stamp knockdown reduces the number of multinucleated cells.

4. In Figure4, the authors showed that transcripts in LGCs were enriched for antigen presentation and adaptive immune system pathways. In addition, multinucleation of LGCs increased the surface expression of B7-H3 (CD276) and colocalized with CD3+ cells, suggesting that LGC multinucleation potentiates T cell activation. However, the authors did not present enough data to demonstrate the antigen-presenting ability of LGCs or their specific T cell activating capacity.

5. Figure 6 clearly shows that mature multinucleated osteoclasts exhibit increased ATP production and maximal respiration. However, the glycolytic pathway did not differ between mononuclear and multinuclear osteoclasts. No explanation for this observation has been provided. It is easy to understand that osteoclasts acquire ATP through aerobic respiration during multinucleation. But how NADPH, which is essential for its redox reaction, is produced? Is it by acquiring αKG from the glutamine pathway?

Minor comments:

1. Supplementary tables 1-6 were not provided.

2. Figure 2D right panel, difficult to see DAPI+ nuclei.

Significance

Although it is well known that multinucleation of cells constantly occurs, especially in osteoclasts, skeletal muscle, and trophoblasts of the placenta, the biological significance of multinucleation and the intracellular functions of multinucleation are not well understood. In this unique study, three types of multinucleated cells were generated from human peripheral blood to elucidate the genetic and functional differences between mononucleated and multinucleated cells. Furthermore, by demonstrating the possibility that the morphological peculiarity of multinucleation can regulate cell function, this paper provides clues to understanding the underlying biology of multinucleated cells and how they maintain cell function in homeostatic and pathological settings.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

The authors compared the various multinucleated cells, osteoclasts, LCG and FBGC. Overall, the manuscript shows rigor in the analyses, and also very interesting approaches for retrieving mononuclear cells, for instance using DC-STAMP siRNA. This work adds very much to understanding the biological differences, as summarized in figure 6h. A lot of work in osteoclast field with for instance qPCR is hampered because, inevitably, a mix of mononuclear and multinucleated cells is always measured. Here, a solid attempt to separate those mixes with cell sorting and subsequent analysis on the mononuclear and multinucleated isolates, really adds. Choice of figures is good, also the extra info of the supplementary figures is relevant and makes it easy to read.

Major and minor concerns:

For osteoclasts, various markers exist for their biological characterization, for instance the ability to resorb bone. What, apart from the arrangement and number of nuclei, were the biological parameters that confirmed that the cells made by addition of IFN or IL-4 were LCG and FBGC? In fig 2c: did the authors perform stainings with isotype control antibodies? In my experience, quite often, antibodies stain mononuclear cells much intenser, since the cytoplasm is much more condense, less spread over a large area. Resorption assay in 6 is not clear. It is weird that osteoclasts apparently display so limited resorption? Also the traces are not typical for osteoclasts. Please explain. Provide a better image Supplementary 2A, even at 250% the lettering is vague. What do the colours in 2A mean?

CROSS-CONSULTATION COMMENTS

I have read the comments of the other two reviewers, and together. I absolutely agree with their additions, Indeed, supplementary tables are lacking, as well as there could be a bit more emphasis on the fact that it is all in vitro work. Together, I think the three of us are complementary in our comments, with good overlap as well. Any effort to stain for instance pathology material with the markers that have been found, would be great, especially for the LGC and the FBGC, that are much less studied in the field of MNGs. Having said that ,I can also live without this addition, but then it could be highlighted in the discussion that these are the future avenues that should be considered. Collaborate with Pathology!

Significance

This manuscript is particularly interesting to those who are interested in the BIOLOGY of MNCs. In essence, three types of MNCs were cultured and compared, with each of them a specific function.

I am an osteoclast expert (76 publications), and have two publications on FBGCs

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This paper provides a detailed step by step protocol of the CUT&RUN technique, which enables high-resolution chromatin mapping and probing, adapted to the malaria parasite Plasmodium falciparum. In particular, Kafsack and colleagues apply the CUT&RUN protocol to infected red blood cells from in-vitro culture and obtain very good quality genome-wide profiles of two histone modifications, H3K4me3 and H3K9me3. The results are congruent with previous ChIP-seq data with a substantial improvement in terms of coverage and chip-to-input noise. The protocol is very detailed and the figures are great.

Major comments: 1. Authors successfully adapted the CUT&RUN protocol in P. falciparum. First, the binding profiles obtained by CUT&RUN for H3K4me3 and H3K9me3 are very similar to those reported by previous ChIP-seq studies. Secondly, by down-sampling 4X and 16X the test samples, authors demonstrate that 1M PE reads of sequencing depth would be enough to obtain accurate profiling of these histone modifications.

Despite this data is convincing, only one region in chr. 8 is shown as an example in figures 2, 3 and 4.

Different regions should be included, at least as supplementary figures, to reinforce their conclusions.

__Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states. We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.

Related to this, there is evidence of the impact of chromatin structure on ChIP-seq analysis. Specifically, heterochromatin is typically depleted in ChIP input controls because of technical and experimental issues and this can result in a false enrichment of heterochromatic regions in the tested sample. How represented is heterochromatin (i.e. sub-telomeric and telomeric regions) in the test and control samples using the cut&run protocol?

__Response: __The reviewer is correct that chromatin structure may alter accessibility which may bias absolute measurements but since the accessibility biases based to chromatin-structure are identical for both the histone PTM-specific antibody and the isotype control and cancel out in the enrichment score.

How biased is the cut&run sample compare to the ChIP-seq sample?

__Response: __We have included this in Supplementary Figure 1. The H3K4me3 and H3K9me3 enrichment scores are strongly correlated both between CUT&RUN replicates and between CUT&RUN and previously published ChIP-seq results.

In this sense, it would be desirable if authors provide more information about the quality analysis results, for example the chip to input signal ratio and the coverage for heterochromatic (telomeric, centromeric and subtelomeric) regions.

__Response: __We agree that this would be of interest to the reader. For this reason, the full genome-wide enrichment tracks were made available for all datasets in this study. We have added language to draw further attention to this availability.

Additionally, loci typically biased in ChIP-seq samples, i.e. clonally variant gene families in sub-telomeric regions, should be shown as examples.

__Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states, including 2 var genes (PF3D7_0808600 and PF3D7_0808700) and two rifin genes (PF3D7_0808800 and PF3D7_0808900). We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7, which also include subtelomeric and non-subtelomeric heterochromatin loci containing these genes. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.

1. For P. falciparum WGS a PCR-free library preparation is strongly recommended. We wonder if it would be possible to try to integrate this step in their CUT&RUN protocol.

__Response: __Since such biases are sequence dependent, they would impact raw coverage but cancel out in the enrichment plots since the sequence-based biases are identical in both samples. While PCR-free amplification may be desirable for some applications, we feel this is outside the scope of this study to implement these changes.

It would have been desirable to have tried the CUT&RUN protocol on other type of proteins, different to hPTMs which are highly abundant, for example one of the Pf Api-AP2 transcription factors. Assaying the CUT&RUN protocol on a different type of protein shouldn't be cost/time consuming and would provide evidence of the versatility of the approach.

Response: As indicated by the title, this protocol was optimized specifically for profiling of histone modifications. CUT&RUN has been used in other systems to profile genome-wide binding of other proteins but this was not our aim and outside the scope of this study.

1. The step by step protocol is very detailed, however there are some parts that need to be better explained:

In the section "Binding cells to Concanavalin A-coated beads": it's not mentioned the harvest time and the stage of the parasites used. In addition, several methods are proposed for iRBCs enrichment, but is not mentioned which method was used and the life stage of the parasites. In this part of the protocol authors state "resuspend cells containing 1-5x107 nuclei to a cell density to 1x107 cells/mL".

According to our calculations, to guarantee this nuclei number it would be necessary to enrich in iRBCs and late stages. Otherwise the red blood cells density should be much larger. Could you please clarify this point?

Response: For this study we enriched for trophozoites using a percoll/sorbitol density gradient, which we have now clarified in step 8. However, whether and which enrichment strategy is employed will vary based on the desired parasite stages and experimental design.

In the section "P. falciparum culturing and synchronization of erythrocytic stages" the authors indicate that the method used for synchronization was double-synchronization with sorbitol treatment to achieve a {plus minus} 6 h synchrony. The details provided appear insufficient to replicate the procedure. E.g. it's not explained how the double step synchronization was performed and for how long the culture was incubated after the synchronization.

The number of parasite cells and the life-stage used is mentioned at the end (in the section of expected outcomes). It would be more useful if this information is specified at the beginning together with the most appropriate procedure to get an iRBC culture well synchronised and enriched in late stages.

Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.

• With regards to reproducibility, all experiments were done in replicate (3 Rs) and the statistics appear adequate.

Minor comments: - Abstract. A closing bracket is missing.

Response: Corrected - Step 11: Split each sample into 1mL aliquots at ?

Response: Corrected

• The affinity of proteins A/G to IgG antibodies varies based on host species and IgG subtype (see link). This link does not seem to work

Response: Corrected

• Low bind tubes are mentioned several times. Please clarify whether it refers to low bind protein or low bind DNA. Step 77.

Response: The vendor and catalog number for the low-bind tubes are specified in the Reagents, Materials & Equipment list.

Which was the desired sequencing depth per library? It could be mentioned here. It is mentioned later in "Quantification and statistical analysis" that the initial desired depth was of 40M read pairs, but what was the real depth obtained? from the Figure 4 seems to be less than 17M read pairs per sample.

Response: Thank you for catching that error. The target was 10M read pairs per library but since CUT&RUN is so specific the isotype controls release less DNA and the resulting libraries produces fewer clusters than aimed for leading to slight over sequencing of the remaining samples.

• Step 79. Please clarify/justify why 50 bp paired-end reads were chosen as sequence length. Response: After excluding the telomere repeats 100 bp (50+50) are sufficient for uniquely mapping 98.3% of the nuclear. Paired-end sequencing was chosen over single-end because it provides the actual size of each fragment.

In the section "Quantification and statistical analysis", references to Figure 3 and 4 are inverted or do not correspond with the actual figures 3 and 4.

Response: Corrected

• Figure 2. Among the replicates, sample 2 seems to have higher background, could you comment why? Response: It is inherent in biological replicates that one would have the greatest amount of noise but we unfortunately have no further insight into why Sample 2 had a higher elevated background that Samples 1 and 3. Furthermore, even with this slightly higher background the relative enrichment of signal to noise ratio enrichment peaks are readily identifiable.

• Below some suggestions that may help the authors improve the presentation of their data and conclusions:

The limitations and potential shortcomings of the protocol are mentioned along the text (e.g. the use of different antibodies, different targets, weak interactions..), but could be good if they are included in a different section, preferably at the end.

Response: A “Limitations” section was added.

Also in this section it would be good if they develop further (or at least speculate) on the differences in the protocol or things to consider if other type of proteins are assayed (i.e. TFs).

Response: As mentioned above, we have not applied to CUT&RUN to profile chromatin other than Histone PTMs, as this was not the aim of our study. Since chromatin-bound histones always occur within a nucleosomal context, we are hesitant to make claims to the utility of this specific protocol for profiling DNA-binding proteins with smaller DNA-binding footprints. That said, CUT&RUN has been used to great success in other systems to profile a wide range of chromatin-bound proteins. We have included mention of this at the end of the introduction.

Authors should better comment on the potential impact of chromatin structure and DNA sequence (i.e. AT richness) on the biased representation of heterochromatic regions in the data, the level of background and the peak calling analysis.

Response: For the enrichment scores, sequence and accessibility biases cancel out since they are the same for both the PTM-specific antibody and the isotype controls.

The coverage of critical loci, like those belonging to clonally variant gene families, should be calculated and examples of tracks included as supplemental figures.

Response: Gene Expression Omnibus under accession number GSE210062 as indicated in the Quantification & Statistical Analysis and data availability sections.

Authors claim that the CUT&RUN protocol has exceptionally low background and has been successfully used to profile chromatin interactions from very small numbers of cells. But it is not specified how many. That is, which is the standard in other fields and how it compares with the number of cells used here.

Response: As stated in the note following step 10, we did not optimize the minimum number of parasites required in this study since at the 1e7 iRBC required for each sample correspond as little as 1mL of bloodstage culture 2% parasitemia and 5% hematocrit. The down-sampling analysis in figure 3 suggests that the number of input cells can likely be reduced at least 10-fold.

Information about synchronisation, estimation of iRBCs density and nuclear content appears insufficiently described and has been fragmented in different sections so it is difficult to replicate. For example, within the section "Binding cells to Concanavalin A-coated beads" different alternative protocols for iRBCs synchronisation and enrichment are mentioned but it is not clear whether authors actually perform that step. It could be convenient to describe it and include it in the step-by-step protocol.

Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.

Significance (Required) The CUT&RUN is a novel technique to profile chromatin modifications genome-wide that has been successfully adapted to P. falciparum by the authors. This technique overcomes important limitations of the traditional ChIP-seq and provides better quality data. First, fewer cells and lower sequencing depths are required which is fundamental for the analysis of certain parasite life stages. Second, the binding step is carried out in-situ using unfixed and intact cells. This allows to avoid crosslinking, which can interfere with target recognition that results in unspecific background, and also avoids the random fragmentation of the chromatin, that can bias in the analysis.

This work is significant since it represents the first CUT&RUN step by step protocol adapted to P. falciparum. The results are important for researchers from the malaria field and parasitologists in general who could eventually leverage this protocol to other Apicomplexa.

Our expertise is on transcriptional regulation, molecular parasitology, genomics and epigenomics, of malaria parasites. We hope the comments above will help the authors to improve the ms. Congratulations on the work.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): ____ In general the paper is very clear and convincing and I have only minor comments for the authors to address.

Introduction The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement? Response: Additional references (Baranello et al, O’Neill et al) were added.

In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.

Response: The reference was added.

Figure 2 A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.

Response: Thank you for the suggestion. We have made these changes.

Figure 3 An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.

Response: Correct, no HP1 CUT&RUN was performed, we are just trying to show that H3K9me3 CUT&RUN recapitulates ChIP-seq of both H3K9me3 and HP1, which binds to H3K9me3.

Figure 4 The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.

Response: The reason we included the down-sampling of CUT&RUN sequence reads was to explore whether were over-sequencing our CUT&RUN libraries not to provide a comparison to ChIP-seq. For simplicity we have therefore kept the figure as is.

DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?

Response: RNAse A is already present since it was included in the STOP buffer at step 35.

The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.

Response: TE buffer recipe was added.

Clean-up of PCR amplified library Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure. Response: Corrected

Typos and writing It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there. Response: It is defined in the 3rd paragraph of the introduction

I've mostly seen ChIP-seq with a dash between the IP and the seq.

Response: Corrected

Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.' Response: Corrected

Figure 2 legend. “(purple) in of three biological replicates” should be “(purple) in three biological replicates” Response: Corrected

Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'. Response: Corrected

Post-ligation Clean-up “Wash twice with 200µl of 80% Ethanol freshly prepared” should be “Wash twice with 200µl of freshly prepared 80% Ethanol” Response: Corrected

Library PCR amplification “Fragments are PCR amplified using Kapa polymerase, which it is more efficient” should be “Fragments are PCR amplified using Kapa polymerase, which is more efficient” Response: Corrected

Figure 7 legend “Indicated in the tope left panel” Should be “Indicated in the top left panel” Response: Corrected

Expected outcomes “to dismiss any sort of contamination” should be “To dismiss contamination” Response: Corrected

Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate. Response: Corrected

CROSS-CONSULTATION COMMENTS Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.

Reviewer #3 (Significance (Required)):

This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In general the paper is very clear and convincing and I have only minor comments for the authors to address.

Introduction

The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement?

In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.

Figure 2

A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.

Figure 3

An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.

Figure 4

The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.

DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?

The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.

Clean-up of PCR amplified library

Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure.

Typos and writing

It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there.

I've mostly seen ChIP-seq with a dash between the IP and the seq.

Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.'

Figure 2 legend.

(purple) in of three biological replicates

Should be

(purple) in three biological replicates

Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'.

Post-ligation Clean-up Wash twice with 200µl of 80% Ethanol freshly prepared

Should be

Wash twice with 200µl of freshly prepared 80% Ethanol

Library PCR amplification Fragments are PCR amplified using Kapa polymerase, which it is more efficient

Should be

Fragments are PCR amplified using Kapa polymerase, which is more efficient

Figure 7 legend Indicated in the tope left panel

Should be

Indicated in the top left panel

Expected outcomes to dismiss any sort of contamination

Should be

To dismiss contamination

Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate.

CROSS-CONSULTATION COMMENTS

Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.

Significance

This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

This paper provides a detailed step by step protocol of the CUT&RUN technique, which enables high-resolution chromatin mapping and probing, adapted to the malaria parasite Plasmodium falciparum. In particular, Kafsack and colleagues apply the CUT&RUN protocol to infected red blood cells from in-vitro culture and obtain very good quality genome-wide profiles of two histone modifications, H3K4me3 and H3K9me3. The results are congruent with previous ChIP-seq data with a substantial improvement in terms of coverage and chip-to-input noise. The protocol is very detailed and the figures are great.

Major comments:

• Authors successfully adapted the CUT&RUN protocol in P. falciparum. First, the binding profiles obtained by CUT&RUN for H3K4me3 and H3K9me3 are very similar to those reported by previous ChIP-seq studies. Secondly, by down-sampling 4X and 16X the test samples, authors demonstrate that 1M PE reads of sequencing depth would be enough to obtain accurate profiling of these histone modifications. Despite this data is convincing, only one region in chr. 8 is shown as an example in figures 2, 3 and 4. Different regions should be included, at least as supplementary figures, to reinforce their conclusions. Related to this, there is evidence of the impact of chromatin structure on ChIP-seq analysis. Specifically heterochromatin is typically depleted in ChIP input controls because of technical and experimental issues and this can result in a false enrichment of heterochromatic regions in the tested sample. How represented is heterochromatin (i.e. sub-telomeric and telomeric regions) in the test and control samples using the cut&run protocol? How biased is the cut&run sample compare to the ChIP-seq sample? In this sense, it would be desirable if authors provide more information about the quality analysis results, for example the chip to input signal ratio and the coverage for heterochromatic (telomeric, centromeric and subtelomeric) regions. Additionally, loci typically biased in ChIP-seq samples, i.e. clonally variant gene families in sub-telomeric regions, should be shown as examples.

• For P. falciparum WGS a PCR-free library preparation is strongly recommended. We wonder if it would be possible to try to integrate this step in their CUT&RUN protocol. It would have been desirable to have tried the CUT&RUN protocol on other type of proteins, different to hPTMs which are highly abundant, for example one of the Pf Api-AP2 transcription factors. Assaying the CUT&RUN protocol on a different type of protein shouldn't be cost/time consuming and would provide evidence of the versatility of the approach.

• The step by step protocol is very detailed, however there are some parts that need to be better explained: In the section "Binding cells to Concanavalin A-coated beads": it's not mentioned the harvest time and the stage of the parasites used. In addition, several methods are proposed for iRBCs enrichment, but is not mentioned which method was used and the life stage of the parasites. In this part of the protocol authors state "resuspend cells containing 1-5x107 nuclei to a cell density to 1x107 cells/mL". According to our calculations, to guarantee this nuclei number it would be necessary to enrich in iRBCs and late stages. Otherwise the red blood cells density should be much larger. Could you please clarify this point? In the section "P. falciparum culturing and synchronization of erythrocytic stages" the authors indicate that the method used for synchronization was double-synchronization with sorbitol treatment to achieve a {plus minus} 6 h synchrony. The details provided appear insufficient to replicate the procedure. E.g. it's not explained how the double step synchronization was performed and for how long the culture was incubated after the synchronization. The number of parasite cells and the life-stage used is mentioned at the end (in the section of expected outcomes). It would be more useful if this information is specified at the beginning together with the most appropriate procedure to get an iRBC culture well synchronised and enriched in late stages.

• With regards to reproducibility, all experiments were done in replicate (3 Rs) and the statistics appear adequate.

Minor comments:

• Abstract. A closing bracket is missing.

• Step 11: Split each sample into 1mL aliquots at ?

• The affinity of proteins A/G to IgG antibodies varies based on host species and IgG subtype (see link). This link does not seem to work

• Low bind tubes are mentioned several times. Please clarify whether it refers to low bind protein or low bind DNA. Step 77. Which was the desired sequencing depth per library? It could be mentioned here. It is mentioned later in "Quantification and statistical analysis" that the initial desired depth was of 40M read pairs, but what was the real depth obtained? from the Figure 4 seems to be less than 17M read pairs per sample.

• Step 79. Please clarify/justify why 50 bp paired-end reads were chosen as sequence length. In the section "Quantification and statistical analysis", references to Figure 3 and 4 are inverted or do not correspond with the actual figures 3 and 4.

• Figure 2. Among the replicates, sample 2 seems to have higher background, could you comment why?

• Below some suggestions that may help the authors improve the presentation of their data and conclusions: The limitations and potential shortcomings of the protocol are mentioned along the text (e.g. the use of different antibodies, different targets, weak interactions..), but could be good if they are included in a different section, preferably at the end. Also in this section it would be good if they develop further (or at least speculate) on the differences in the protocol or things to consider if other type of proteins are assayed (i.e. TFs). Authors should better comment on the potential impact of chromatin structure and DNA sequence (i.e. AT richness) on the biased representation of heterochromatic regions in the data, the level of background and the peak calling analysis. The coverage of critical loci, like those belonging to clonally variant gene families, should be calculated and examples of tracks included as supplemental figures. Authors claim that the CUT&RUN protocol has exceptionally low background and has been successfully used to profile chromatin interactions from very small numbers of cells. But it is not specified how many. That is, which is the standard in other fields and how it compares with the number of cells used here. Information about synchronisation, estimation of iRBCs density and nuclear content appears insufficiently described and has been fragmented in different sections so it is difficult to replicate. For example, within the section "Binding cells to Concanavalin A-coated beads" different alternative protocols for iRBCs synchronisation and enrichment are mentioned but it is not clear whether authors actually perform that step. It could be convenient to describe it and include it in the step-by-step protocol.

Significance

The CUT&RUN is a novel technique to profile chromatin modifications genome-wide that has been successfully adapted to P. falciparum by the authors. This technique overcomes important limitations of the traditional ChIP-seq and provides better quality data. First, fewer cells and lower sequencing depths are required which is fundamental for the analysis of certain parasite life stages. Second, the binding step is carried out in-situ using unfixed and intact cells. This allows to avoid crosslinking, which can interfere with target recognition that results in unspecific background, and also avoids the random fragmentation of the chromatin, that can bias in the analysis.

This work is significant since it represents the first CUT&RUN step by step protocol adapted to P. falciparum. The results are important for researchers from the malaria field and parasitologists in general who could eventually leverage this protocol to other Apicomplexa.

Our expertise is on transcriptional regulation, molecular parasitology, genomics and epigenomics, of malaria parasites. We hope the comments above will help the authors to improve the ms. Congratulations on the work.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

This reviewer did not leave any comments

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): *In the manuscript " Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans" the authors show that a hormetic heat shock requires the peroxidase catalase ctl-2 for longevity and thermotolerance. Furthermore the authors characterize the hormetic stress response in ctl-2 mutants and show that ctl-2 is required for the proper formation of HSF-1 stress granules, and that ctl-2 mutants have a changed transcriptional response to heat shock and a changed induction of heat-stress induced oxidative stress. The authors go on to characterize peroxisomes, and peroxidase-regulated fatty acids, as well as mitochondria in both ctl-2 mutants and upon heat shock. The authors conclude that functional peroxisomes play an important role in the hormetic heat stress response.

This is an interesting study, that demonstrates that the loss of the peroxidase catalase ctl-2 plays an important role in the heat stress response. The authors need to provide more details on experimental repeats and experiments strengthening the conclusion that peroxisomes are generally important for the hormetic stress response would also improve this manuscript.

Furthermore, some textual changes would clarify some specifics in this manuscript.*

**Major issues:** *1. Throughout the manuscript it is unclear how many times the experiments were conducted (e.g. Lifespan, thermotolerance) or how many biological replicates were used (qRT-PCR, peroxisome characterization etc). This critical information should be included. A table with the individual lifespan experiments and thermotolerance experiments is expected. *

We are grateful to the Reviewer for drawing our attention to this and we have now included this information throughout the manuscript.

• The authors are characterizing the ctl-2 mutant upon heat stress and find some compelling differences to wild-type animals. The authors state "We measured the expression levels of two peroxisomal transport proteins, PRX-5 and PRX-11, and found no differences in expression between WT and Δctl-2 strains, suggesting that morphogenesis should not be impaired and that proteins needed for their proper function should be present, with the exception of ctl-2 in the Δctl-2 strain (Figure 3A)." The authors however conclude that functional peroxisomes are required for the benefits of a hormetic HS. What the authors are in fact demonstrating is that ctl-2 specifically is required for the hormetic HS response. To demonstrate that peroxisomes in general are required they should disrupt peroxisome function by additional means. What the authors also demonstrate is that heat shock has an effect on peroxisomes. These differences need to be clarified. It is also not fully clear whether peroxisomes are functional in ctl-2 mutants? Further peroxisome-relevant enzymes should be tested for levels and functionality in ctl-2 mutants and upon HS. *

This is correct and we have now addressed the above-mentioned issues throughout the manuscript. As the Reviewer noted, the peroxisomes are functional if monitored as the transcript levels of two peroxisomal transport proteins, implying that they have the potential to import their components. On the other hand, they are lacking the catalase, so they do not have a complete set of their components. Our phrasing in the original manuscript indeed was not optimal and we have now modified the conclusions, stating the peroxisomal catalase is important for the proper heat shock response.

Moreover, it is partially correct to say that a thorough explanation is missing as to what heat shock does to peroxisomes. We would like to emphasize that we measured the transcript levels of three enzymes from the very long chain fatty acid oxidation: straight-chain acyl-CoA oxidase, ACOX-1, MAO-C-like dehydratase domain protein, MAOC-1, and propanoyl-CoA C-acyltransferase, DAF-22. We have now measured the transcript levels of several other peroxisomal enzymes in the WT and the ctl-2 mutant under optimal conditions and heat shock and reported about it in the manuscript.

• The TORC1 experiments are too indirect. The authors should perform WesternBlots with a S6Kinase -Phospho antibody to determine whether TORC1 is inhibited or not. Alternatively, the authors may choose to remove this experiment from the manuscript without disrupting the main message of the manuscript, since no link between TORC1 and peroxisome function nor with HS has been established.

*

We are aware that the classical experiment to measure the activity of TORC1 is the Western Blot based assay to observe the phosphorylation of S6 kinase. However, we find the available antibodies not good enough to perform this experiment. We, therefore, conducted a different experiment to estimate the TORC1 activity, and it is the one described in our manuscript. We suggest that, as the phosphorylation of S6 is a downstream readout of TORC1 activity, so is the transcript level of the group of genes we measured, as reported previously (Das, Melo et al. 2017; Kenyon 2010; Kenyon 2011; Robida-Stubbs, Glover-Cutter et al. 2012; from the manuscript). While we are aware of the shortcomings of the assay we used, we find it valuable to report that the heat shock has an inhibitory effect on the TORC1 activity in WT C. elegans but not in the peroxisomal mutant. We would like to ask the Reviewer to reconsider this comment.

**Textual changes:** *1. The authors should adhere to C. elegans nomenclature convention (at least once), by referring to the ctl-2 deletion with the specific allele name [ctl-2(xx)] (unlike in yeast, ∆ is not commonly used). Furthermore a more thorough description of the specific allele (complete gene deletion? Point mutation? Truncation?) should be included. *

We have now taken care of this issue.

• Throughout the manuscript the authors should refer back to their specific hormetic heat shock paradigm, since several studies (also cited here) have shown differences in the specific physiological changes (e.g. HS on day 1 of adulthood has profound effects on broodsize, whereas the authors show here that a HS at L4 does not, other differences in terms of activation of other stress reporters has also been reported). *

Since our data regarding brood size were not put into context of the present study and were not studied in-depth, we have decided to remove these results from the present manuscript.

• The abbreviation OGT (original growth temperature??) should be defined *

OGT stands for optimal growth temperature and we have now defined it in the manuscript.

• The authors should change the sentence "The binding of HSF-1 to the HSEs can be visualized by a HSF-1::GFP fusion protein". This is not technically true. While HS has been shown to induce HSF-1 stress granules and these stress granules have been indirectly shown to correlate with transcription, it has however not been demonstrated that HSF-1 is bound to HSEs or whether it is bound to other DNA stretches or what is in fact transcribed by HSF-1 when it localizes to these foci. *

We are grateful to the Reviewer for this clarification, which we have included into the manuscript.

• The authors describe the morphological changes of peroxisomes upon HS, however they missed the opportunity to fully interpret these results in the discussion. Again, their conclusion that peroxisomes may be impaired in ctl-2 mutants is very vague. *

We agree with this comment and we have now taken care of this issue by introducing additional clarifications.

---

Reviewer #1 (Significance (Required)):

*Hormesis and the role of peroxisomes in stress responses is an important and interesting topic. *

The role of peroxisomes in stress responses has not been addressed.

*Researchers with interest in stress responses will be interested in this work.

My expertise lies in C.elegans stress and longevity with a specific focus on hormetic mechanisms.*

**Referee Cross-commenting**

While I agree with most comments from the other referees, I don't believe it is feasible to ask the authors to generate c. Elegans cell cultures for any follow up experiments. I would be satisfied with a more thorough comparison of the HS response between WT and Ctl-2 mutants, I.e: compare preconditiöning: 1h and 4h HS at L4 and d1 and then do thermotolerance experiments

We thank the Reviewer for this comment. We agree that to generate C. elegans cell cultures is not feasible. We have, however, performed the HS treatments in mammalian cells in culture.

HEK cells were transfected with Lipofectamine 3000 for 48h with either negative siRNA control (siCont), siPEX5 or siCAT at 100 nM, according to the manufacturer’s protocol. For HSR induction, cells were moved for 1h to a humidified incubator containing 5% CO2 at 42,5ºC, or maintained at 37ºC. After, RNA was immediately isolated with NucleoSpin RNA Columns for RNA purification (Macherey-Nagel), and 1000 ng of total RNA was converted to cDNA with iScriptTM cDNA Synthesis Kit (Biorad). Relative gene expression of PEX5, catalase (CAT), HSP90AA1, HSP70 (Hspa1b) and small HSPs (Hspb1, Hsph1 and Hspe1) were determined by qPCR, which were performed in a CFX Opus 384 Real-Time PCR System (Biorad) in a final volume of 5 µL using 2× SYBR Green PCR master mix and 0.3 μM of each primer pair. Relative gene expressions were calculated according to the Pfaffl method, with the results being normalized to the three most stable housekeeping genes in this experimental setting (HPRT, B2M and RPL7), as determined with NormFinder. The Ct averages of the four siCont samples at 37ºC were used as the calibrators to determine ΔCt for each gene. Data comparisons were conducted with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests and data is presented as mean ± SEM (Figures 1 and 2).

Although the siRNA-mediated silencing of CAT and PEX5 was successful (93% and 60% respectively), as determined by qPCR (Figure 1), the expression of several HSPs (HSP70, HSP90 and the small HSPs Hspb1, Hsph1 and Hspe1) remained similar following HS between either CAT- or PEX5- silenced cells and siCont-transfected cells (Figure 2). Preliminary data from SH-SY5Y cells treated as described above yielded similar results (data not shown). These observations may suggest that catalase and/or functional peroxisomes are not necessary for HSR induction and hormesis, but these experiments were limited to two cell lines and one form of HS (1h at 42,5ºC). Therefore, it is possible that different protocols may yet unravel a still undescribed link between peroxisomes, HSR, and life extension, but we to pursue this will take a long time and would be far outside the scope of this manuscript.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Summary:** In the manuscript entitled "Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans", Musa et al. explored the role of functional peroxisomes in the heat shock (HS)-induced hormesis in C. elegans using peroxisomal catalase ctl-2 deletion mutants. They showed that ctl-2 deletion abolished HS-induced longevity and suppressed the HS-induced upregulation of small heat shock proteins and HS-induced HSF-1 nuclear accumulation. Furthermore, the authors analyzed the differences in HS-induced phenotypes between wild type animals and ctl-2 mutants: the activation of the antioxidant response, the pentose phosphate pathway, and increased triglyceride content, which are the most prominent changes observed during heat shock in wild-type animals. However, there is only weak evidence supporting their claim that functional peroxisomes are required for HS-induced hormesis. * **Major comments:** 1. The authors conclude that the functional peroxisomes are essential for the HS-induced hormesis based only on one observation that the transient heat shock treatment did not increase the lifespan in the short-lived ctl-2 mutants. *

This is only partially correct. In addition to the absence of lifespan extension in the ctl-2 mutants, our conclusion was based also on the lower extent of the heat shock protein expression during HS in ctl-2 mutant. In the revision, we have reinforced this conclusion by adding the measurements of the kinetics of the heat shock response activation in the WT and the ctl-2 mutant, whereby the possible differences in the kinetics between the strains have been excluded as a possible interpretation. The results are now reported in the manuscript.

*Because the ctl-2 mutants are short-lived, the author should have carefully examined the relevant role of functional peroxisomes in hormesis response by conducting lifespan measurements using ctl-2 RNAi-treated animals or mutants deficient in other genes essential for peroxisome function. *

• *

We entirely agree with this comment. Since we are unable to perform such an elaborate study in this moment, and due to other similar comments from other Reviewers, we have decided to modify the conclusion, pointing out that it is the lack of the peroxisomal catalase that is associated to the lower extent of the heat shock response activation.

• The authors examined the difference between wild-type animals and ctl-2 mutants in many aspects; however, there is no rational explanation or examination that their observations have a role in HS-induced hormesis. *

We acknowledge that we are lacking the mechanistic insight into the reported phenomenon. This will be the focus of future studies, while in our present study, we have modified the conclusions to match the presented results.

• There is a severe shortage in the explanation of their experiments, especially for the methods of the experiments and statistical procedures. The authors should clarify the details of the experiments with proper statistical analyses. Follows are the points to be clarified for Figure 1 as insufficiency examples in the explanation in their study.

*

We have now taken care of this issue throughout the manuscript.

*Fig. 1A, B: Is this the representative survival curve of several experiments? The authors should clarify this point. The authors should include a table with the statistical numbers: the number of animals they used, the number of animals they measure in each experiment, and the p-value. *

• *

We have now clarified this issue in the manuscript.

*Fig. 1C: Is this a representative brood size of one animal, the mean brood size of some animals of one representative experiment, or the mean brood sizes from some experiments? The authors should clarify this point. Also, the authors should perform statistical analysis. *

We have removed the results related to the brood size from the present version of the manuscript.

*Fig. 1D-F: The authors should state the number of experiments they conducted. The author should perform multiple comparison statistical analyses instead of t-test without any correction. *

Done.

*Fig. 1H: Is this a representative plot of a representative experiment or the plot of the mean values from some experiments? The authors should clarify this point. The author should also clarify the tissue they analyzed and the exact number of the experiment (the number of nuclei in each animal they analyze, the number of animals they analyze in each experiment). The author should also clarify the detailed statistical procedure: one-way ANOVA or two-way ANOVA; which multiple comparison method they used in their analyses. *

We have now clarified all these issues.

**Minor comments:** *The author should explain the details for the experiments in either the Methods section or the figure legends. The authors should integrate the figures 6B-J (6B-D -> one figure, 6E-G -> one figure, and 6H-J -> one figure) into three figures because there are redundancies in these figures. In addition, the authors should perform the statistical analyses with multiple comparison procedures instead of a simple t-test. *

We have now done as suggested by the Reviewer.

*The authors should perform statistical analyses on Figs. 6N and 6O. *

Done.

*The authors would better cite relevant articles when referring to representative target genes of UPRER, UPRmt, and TORC1. *

Done.

Reviewer #2 (Significance (Required)): *Understanding the mechanism underlying the HS-induced hormesis in a multicellular organism is essential in the research field. Their finding that functional peroxisomes play a pivotal role in the HS-induced hormesis, if properly demonstrated, would provide us with the significant progress in this field; however, much more proper experiments are required to support their conclusion. Nevertheless, their finding can stimulate the attention of researchers who study the aging process, stress response, and especially peroxisome function.

I am an expertise in the study of aging in C. elegans. *

Reviewer #3 (Evidence, reproducibility and clarity (Required)): In this MS, the authors aim at the understanding of the role of peroxisome related-anti-oxidant capacity for the effect of a heat conditioning treatment on the HSR and associated longevity. Despite the finding that ctl-2 mutants they used show reduced resistance upon heat pre-conditioning, neither the mechanism (Ctl-2 is required for HSR) nor the claim that functional peroxisomes are required for heat shock-induced hormesis are, in my view, fully proven by the data in this MS. What the data basically show is that, related to a fragile status of the ctl-2 mutants, pre-conditioning was either to severe (toxic) or/and lead to development defects such that it was no longer effective in priming organismal resistance, likely to HSR-independent features. * **Major comments:** Figure 1: The level of pre-conditioning induced resistance (as opposed to intrinsic sensitivity) that can be induced in a given genetic background is dependent on a number of things, one of which is the severity of the priming dose. A more severe heat shock (that initially causes more damage) leads to a slower rate of tolerance development but the level of tolerance (of the surviving cells) is much higher. However, if too toxic, the priming treatment will result is loss of cells, which - at the organismal level- not or less reveal the resistance of the surviving primed and thus tolerant cells. *

Agreed.

Furthermore, such intrinsic sensitivity (un-primed) is determined by many more factors that only the capacity to induce the HSR. It is thus important to better evaluate the relevance of differences in intrinsic sensitivity of wildtype and ctl-2 mutants to the priming heat shock (figure 1F). Albeit interesting that Ctl-2 strains are hypersensitive to heat, this data also could imply that the real mechanism of being able to build up induced-resistance and longevity is not mechanistically due to an altered regulation of the HSR, but merely a reflection of that intrinsic difference in the sensitivity to the damage inflicted by the priming heat treatment. *

Also, as the treatment was given during the L4 stage, many of the effects may be blurred by differences in the sensitivity to heat treatment on developmental processes, conditions under which also many HSP are differently regulated (also HSF-1 independently). Whilst still interesting, this is even more complex to interpret mechanistically.

*

We chose the late L4 stage as opposed to adults since worms are mostly developed at L4, and have no embryos, which is a big advantage. The presence of embryos may confound the measurements, especially in the qPCR experiments since they may express HSPs due to their role in developmental processes as opposed to heat stress. It was also reported elsewhere that L4 stage is the most sensitive to HS in terms of HSR, while exhibiting higher survival rate than younger or older worms following HS. We have included this explanation in the manuscript.

*In fact, to conclude on the mechanistic involvement on peroxisome redox status for resistance inducting by heat priming, one of would require to e.g. derive cell lines from wildtype and ctl-2 mutant worms and perform an induced-thermotolerance / survival experiments (a iso-toxic and iso-dose heat priming treatments) to see whether ctl-2 truly have an impaired HSR due to cell autonomous features.

*

We are not able to establish cell lines from C. elegans, however, we have performed the suggested experiment in the mammalian cells in culture.

HEK cells were transfected with Lipofectamine 3000 for 48h with either negative siRNA control (siCont), siPEX5 or siCAT at 100 nM, according to the manufacturer’s protocol. For HSR induction, cells were moved for 1h to a humidified incubator containing 5% CO2 at 42,5ºC, or maintained at 37ºC. After, RNA was immediately isolated with NucleoSpin RNA Columns for RNA purification (Macherey-Nagel), and 1000 ng of total RNA was converted to cDNA with iScriptTM cDNA Synthesis Kit (Biorad). Relative gene expression of PEX5, catalase (CAT), HSP90AA1, HSP70 (Hspa1b) and small HSPs (Hspb1, Hsph1 and Hspe1) were determined by qPCR, which were performed in a CFX Opus 384 Real-Time PCR System (Biorad) in a final volume of 5 µL using 2× SYBR Green PCR master mix and 0.3 μM of each primer pair. Relative gene expressions were calculated according to the Pfaffl method, with the results being normalized to the three most stable housekeeping genes in this experimental setting (HPRT, B2M and RPL7), as determined with NormFinder. The Ct averages of the four siCont samples at 37ºC were used as the calibrators to determine ΔCt for each gene. Data comparisons were conducted with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests and data is presented as mean ± SEM (Figures 1 and 2).

Although the siRNA-mediated silencing of CAT and PEX5 was successful (93% and 60% respectively), as determined by qPCR (Figure 1), the expression of several HSPs (HSP70, HSP90 and the small HSPs Hspb1, Hsph1 and Hspe1) remained similar following HS between either CAT- or PEX5- silenced cells and siCont-transfected cells (Figure 2). Preliminary data from SH-SY5Y cells treated as described above yielded similar results (data not shown). These observations may suggest that catalase and/or functional peroxisomes are not necessary for HSR induction and hormesis, but these experiments were limited to two cell lines and one form of HS (1h at 42,5ºC). Therefore, it is possible that different protocols may yet unravel a still undescribed link between peroxisomes, HSR, and life extension, but we to pursue this will take a long time and would be far outside the scope of this manuscript.

*Related to the actual data in figure 1A-c, also relative effects need to be taken into account as the Ctl-2 mutants are short lived. E.g., if one looks at maximum life span, the differences between the strains seem minimal (20/17 = 1,17 fold for wildtype and 15/13 = 1,15 fold for Ctl-2), so to conclude on no effect of heat pre-conditioning in ctl-2 strains seems an overstatement. *

This is not entirely correct. Usually, in lifespan measurements in C. elegans, we compare the median lifespan. For WT worms, we measured increased median and maximum lifespan; median lifespan was increased from 11 to 14 days post HS (≈20% increase), and maximum lifespan from 17 to 20 days (15% increase) (Figure 1A). In contrast, while maximum lifespan of the ctl-2(ua90)II strain after HS was increased from 13 to 15 days (≈13% increase), the median lifespan was unchanged and was 10 days for both HS and OGT ctl-2(ua90)II worms (Figure 1B). Overall, mild HS did not significantly affect ctl-2(ua90)II strain lifespan.

*Regarding to the brood size, it is not only true that these are smaller for clt-2 worms, but also that there was no effect of the pre-conditioning treatment in wildtype whereas a reduction was caused by the pre-conditioning of the ctl-2 worms. What does this imply?

*

Since the results of brood size were not sufficiently understood and out of context of the present study, we have removed them from the current version of the manuscript.

*Regarding the HSF/HSP data. First of all, a better visual insight in the quantitative differences in basal transcription levels between the strains should be provided. It looks as if they could be significantly lower for at least HSP16.1 in clt-2 strains. *

We have now included description of these results in the manuscript.

*Next, it would then be essential to evaluate the responses relative to these basal levels in the ctl-2 lines themselves (and not relative to that in wildtype animals). Second, looking at Hsp70, the HSP being most dependent of HSF1 upon a heat shock, the data imply that there is nothing wrong with the heat shock activated HFS-1 response in ctl-2 as such. As stated above, magnitude differences might also be a matter of kinetics, so measuring this at a single time point (4h after HS) may e.g. too early for being at its peak in ctl-2 cells. *

• *

We agree with this comment. Therefore, we have performed the experiment where we evaluated the kinetics of the heat shock response in the WT and the ctl-2 mutant worm. The measurements of the kinetics of the heat shock response activation in the WT and the ctl-2 mutant reveal that there are no differences in the kinetics between the strains, whereby the possible differences in the kinetics between the strains have been excluded as a possible interpretation. The results are now reported in the manuscript.

*Third, it must be emphasized that HSF-1 foci/granules formation is a well-known feature of the response of HSF-1 to heat shock, but these granules are not the site of Hsp transcription, i.e they are not functionally related to HSP expression and not necessarily correlate quantitatively to HSR activation. So, the conclusion that HSF-1 activation/the HSR is truly attenuated in ctl-2 strains is -in my view- not fully proven. In fact, there is an intricate related between small HSP and oxidative stress and the lower (if correct) Hsp16.1 and Hsp16.2 expression could rather be related to such features.

*

We thank the Reviewer for drawing our attention to this issue, which we have now addressed in the manuscript by modifying the conclusion of a specific set of results related to the HSF-1 foci formation during heat shock. We have also discussed more extensively the connection with the oxidative stress.

*Figure 2: As for the HSR, kinetics may be different for wildtype and ctl-2 strains for all these endpoints, reflecting the higher intrinsic, non-primed heat sensitivity of the ctl-2 strain. Again, whilst interesting phenotypically and maybe relevant physiologically (i.e. being able to be primed as weak animal to show organismal resistance), this means that the data are elusive in terms of mechanisms.

*

We acknowledged from the beginning that we are missing mechanistic details of the presented result, and we therefore agree with this comment. As for the possible differences in the kinetics of the heat shock response activation in the WT and the ctl-2 worms: the measurements of the kinetics of the heat shock response activation in the WT and the ctl-2 mutant reveal that there are no differences in the kinetics between the strains, whereby the possible differences in the kinetics between the strains have been excluded as a possible interpretation. The results are now reported in the manuscript.

*In panel 2E, the lack of elevation in CellROX fluorescence by heat shock in wildtype cells in explained as due to the result in activation of the antioxidant defenses. Whereas this may sound OK, it is contradictory the reasoning given above that heat stress induced oxidative stress and hence cause G6PD upregulation (data panel 2D). In addition, whist the authors suggest they are the same, to me the CellROX data for ctl-2 strains appear to be lower (rather than, if anything, higher) for unstressed ctl-2 strains than wildtype strains. Is this not surprising given they are used as model for an impaired oxidation status? And does this not (also) indicate that the knockout lines have developed compensating strategies? Anyway: I got confused here.

*

First, there is no significant difference between the CellROX signal of the unstressed WT and the ctl-2 mutants. Why exactly the WT and ctl-2 mutant worms have the same CellROX signal is hard to say; the absence of the peroxisomal catalase could have triggered the activation of the cytosolic antioxidant defenses in optimal conditions already, or other compensatory strategies. It is also possible that the absence of the peroxisomal catalase has its consequences only in stressful conditions. In addition, our results during heat shock suggest that the WT strain is able to neutralize the ROS produced during heat shock, unlike the ctl-2 mutant. The increase in the G6PD expression may have helped with that, since it fed the activation of the pentose phosphate pathway. Our reasoning is that the ctl-2 mutant was for some reason not able to respond in the same way as the WT, and not that there was no need. The anti-ROS protection seems to have been insufficient during heat shock in the ctl-2 strain.

*Figure 3: First of all, it seems dangerous to conclude anything on peroxisome NUMBER here as what is measured is the presence of an imported FGP-tagged protein into peroxisomes and hence difference may be (also) due to import related effects. In fact, EM data would be required to make firmer conclusions on peroxisomal morphology, size and numbers. *

• *

In principle we agree with this comment, however, we demonstrate in Figure 3 that Prx-5 and Prx-11 expression levels do not display any differences in the WT and ctl-2 worms. Therefore, we remain confident about the analysis of the peroxisome number. We hesitate to perform an EM analysis due to the lack of specific markers; anything even slightly resembling a peroxisome could falsely be counted as one. In other words, every methodological approach comes with its own imperfections.

*In panel C, it is unclear what is significantly different from what; is the signal in clt-2 strains truly lower in ctl-2 strains that in wildtype strains? *

We thank the Reviewer for drawing our attention to this. In Figure 3C, for the significance analysis, everything was compared to the WT in optimal growth conditions. We have now clarified this in the text. The difference between the ctl-2 mutant and the WT is debatable: while the medians of the data sets are significantly different, this difference may not have any biological significance. Still, we reported the result of the statistical analysis.

*Figure 4 - 6: I sympathize with the comprehensive analysis presented in these figures. It is clear to me that different things are either (not) up or down in unprimed ctl-2 strains and that heat shock does or does not cause similar effects on these endpoints in wildtype and ctl-2 strains. Whilst this indeed shows that they do respond differently, I do not understand from these that what it all means and, in particular if and how it related causally or consequentially to the impaired priming effects (if true) of the heat shock in ctl-2 strains. *

The results presented in Figure 4-6 have not been put into a mechanistic context in the present study. In the mentioned figures, we do not report any causal or consequential relationships, but we do wish to report on the differential phenotypes of lipid metabolism and storage, as well as mitochondrial morphology in the WT and ctl-2 mutant worms. However, the presented analyses were performed well and we believe that these results may benefit to other researchers working in related topics.

**Minor additional comments (textual only)**

*Whereas this paper discusses the possibility of pre-heat conditioning to induce (long term) resistance, the generality of this as being a hormesis response (or general stress responses) related to other challenges is not warranted and all text concerning that should be deleted. Stresses damaging primarily DNA (ionizing radiation) or proteins & lipid (heat shock) are fundamentally different and each of them requires entirely different and largely independent systems to respond to. *

Done.

*Moreover, whilst the induced HSR is clearly an established hermetic response, this is still far less clear for e.g DNA damage responses. Therefore, it is also relevant define the types of stress to which is referred to e.g. when mentioning the envorinmental stimuli to which ctl-2 mutants are apparently hypersensitive. *

Done.

Also, the text related to cell non-autonomous response is irrelevant to this study and should be deleted. *

*

Done.

*Page 3 lines 1-4: It is incorrect to write that the role HSP has been put forward as most relevant to heat-induced hormesis. This is how they were discovered, but it is now clear that they play a role in many other pre-conditioning induced resistant phenotypes as well as in the cell-intrinsic sensitivity to proteotoxic stresses in general. Please also be aware that for unprimed, intrinsic resistance to e.g. heat shock, pre-existing levels of HSP are more relevant than the ability to activate the HSR. Activating the HSR is more relevant to resistance to more chronic temperature elevations and acquired resistance via the priming (hormesis).

*We thank the Reviewer for drawing our attention to this. We agree about the relevance of these points and have modified the manuscript accordingly.

Reviewer #3 (Significance (Required)): * Regulation of the cell intrinsic heat shock response is an important item to understand how cels may be primed to become resilient to (certain) other stresses. Besides the main studied regulator (HSF-1), many other levels of regulation likely exist and intra-organellar communication and proteostasis might be an important aspect for controlling such regulation.

As such, peroxisome proteostasis (that, unlike other organelles, are not (also) controlling a organellar unfolded protein response) is an interesting organelle that could co-control the cytosolic heat shock response. So, the aim of this study per se is quite interesting, However, although for evaluation of physiological relevance c. elegance is a good model of choice, for the mechanistic studies, cellular experiments would have been better suited. *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In this MS, the authors aim at the understanding of the role of peroxisome related-anti-oxidant capacity for the effect of a heat conditioning treatment on the HSR and associated longevity. Despite the finding that ctl-2 mutants they used show reduced resistance upon heat pre-conditioning, neither the mechanism (Ctl-2 is required for HSR) nor the claim that functional peroxisomes are required for heat shock-induced hormesis are, in my view, fully proven by the data in this MS. What the data basically show is that, related to a fragile status of the ctl-2 mutants, pre-conditioning was either to severe (toxic) or/and lead to development defects such that it was no longer effective in priming organismal resistance, likely to HSR-independent features.

Major comments:

Figure 1: The level of pre-conditioning induced resistance (as opposed to intrinsic sensitivity) that can be induced in a given genetic background is dependent on a number of things, one of which is the severity of the priming dose. A more severe heat shock (that initially causes more damage) leads to a slower rate of tolerance development but the level of tolerance (of the surviving cells) is much higher. However, if too toxic, the priming treatment will result is loss of cells, which - at the organismal level- not or less reveal the resistance of the surviving primed and thus tolerant cells.

Furthermore, such intrinsic sensitivity (un-primed) is determined by many more factors that only the capacity to induce the HSR. It is thus important to better evaluate the relevance of differences in intrinsic sensitivity of wildtype and ctl-2 mutants to the priming heat shock (figure 1F). Albeit interesting that Ctl-2 strains are hypersensitive to heat, this data also could imply that the real mechanism of being able to build up induced-resistance and longevity is not mechanistically due to an altered regulation of the HSR, but merely a reflection of that intrinsic difference in the sensitivity to the damage inflicted by the priming heat treatment.

Also, as the treatment was given during the L4 stage, many of the effects may be blurred by differences in the sensitivity to heat treatment on developmental processes, conditions under which also many HSP are differently regulated (also HSF-1 independently). Whilst still interesting, this is even more complex to interpret mechanistically.

In fact, to conclude on the mechanistic involvement on peroxisome redox status for resistance inducting by heat priming, one of would require to e.g. derive cell lines from wildtype and ctl-2 mutant worms and perform an induced-thermotolerance / survival experiments (a iso-toxic and iso-dose heat priming treatments) to see whether ctl-2 truly have an impaired HSR due to cell autonomous features.

Related to the actual data in figure 1A-c, also relative effects need to be taken into account as the Ctl-2 mutants are short lived. E.g., if one looks at maximum life span, the differences between the strains seem minimal (20/17 = 1,17 fold for wildtype and 15/13 = 1,15 fold for Ctl-2), so to conclude on no effect of heat pre-conditioning in ctl-2 strains seems an overstatement. Regarding to the brood size, it is not only true that these are smaller for clt-2 worms, but also that there was no effect of the pre-conditioning treatment in wildtype whereas a reduction was caused by the pre-conditioning of the ctl-2 worms. What does this imply?

Regarding the HSF/HSP data. First of all, a better visual insight in the quantitative differences in basal transcription levels between the strains should be provided. It looks as if they could be significantly lower for at least HSP16.1 in clt-2 strains. Next, it would then be essential to evaluate the responses relative to these basal levels in the ctl-2 lines themselves (and not relative to that in wildtype animals). Second, looking at Hsp70, the HSP being most dependent of HSF1 upon a heat shock, the data imply that there is nothing wrong with the heat shock activated HFS-1 response in stl-2 as such. As stated above, magnitude differences might also be a matter of kinetics, so measuring this at a single time point (4h after HS) may e.g. too early for being at its peak in ctl-2 cells. Third, it must be emphasized that HSF-1 foci/granules formation is a well-known feature of the response of HSF-1 to heat shock, but these granules are not the site of Hsp transcription, i.e they are not functionally related to HSP expression and not necessarily correlate quantitatively to HSR activation. So, the conclusion that HSF-1 activation/the HSR is truly attenuated in ctl-2 strains is -in my view- not fully proven. In fact, there is an intricate related between small HSP and oxidative stress and the lower (if correct) Hsp16.1 and Hsp16.2 expression could rather be related to such features.

Figure 2: As for the HSR, kinetics may be different for wildtype and ctl-2 strains for all these endpoints, reflecting the higher intrinsic, non-primed heat sensitivity of the ctl-2 strain. Again, whilst interesting phenotypically and maybe relevant physiologically (i.e. being able to be primed as weak animal to show organismal resistance), this means that the data are elusive in terms of mechanisms.

In panel 2E, the lack of elevation in CellROX fluorescence by heat shock in wildtype cells in explained as due to the result in activation of the antioxidant defenses. Whereas this may sound OK, it is contradictory the reasoning given above that heat stress induced oxidative stress and hence cause G6PD upregulation (data panel 2D). In addition, whist the authors suggest they are the same, to me the CellROX data for ctl-2 strains appear to be lower (rather than, if anything, higher) for unstressed ctl-2 strains than wildtype strains. Is this not surprising given the are used as model for an impaired oxidation status? And does this not (also) indicate that the knockout lines have developed compensating strategies? Anyway: I got confused here.

Figure 3: First of all, it seems dangerous to conclude anything on peroxisome NUMBER here as what is measured is the presence of an imported FGP-tagged protein into peroxisomes and hence difference may be (also) due to import related effects. In fact, EM data would be required to make firmer conclusions on peroxisomal morphology, size and numbers.

In panel C, it is unclear what is significantly different from what; is the signal in clt-2 strains truly lower in ctl-2 strains that in wildtype strains?

Figure 4 - 6: I sympathize with the comprehensive analysis presented in these figures. It is clear to me that different things are either (not) up or down in unprimed ctl-2 strains and that heat shock does or does not cause similar effects on these endpoints in wildtype and ctl-2 strains. Whilst this indeed shows that they do respond differently, I do not understand from these that what it all means and, in particular if and how it related causally or consequentially to the impaired priming effects (if true) of the heat shock in ctl-2 strains.

Minor additional comments (textual only)

Whereas this paper discusses the possibility of pre-heat conditioning to induce (long term) resistance, the generality of this as being a hormesis response (or general stress responses) related to other challenges is not warranted and all text concerning that should be deleted. Stresses damaging primarily DNA (ionizing radiation) or proteins & lipid (heat shock) are fundamentally different and each of them requires entirely different and largely independent systems to respond to.

Moreover, whilst the induced HSR is clearly an established hermetic response, this is still far less clear for e.g DNA damage responses. Therefore, it is also relevant define the types of stress to which is referred to e.g. when mentioning the envorinmental stimuli to which ctl-2 mutants are apparently hypersensitive.

Also, the text related to cell non-autonomous response is irrelevant to this study and should be deleted.

Page 3 lines 1-4: It is incorrect to write that the role HSP has been put forward as most relevant to heat-induced hormesis. This is how they were discovered, but it is now clear that they play a role in many other pre-conditioning induced resistant phenotypes as well as in the cell-intrinsic sensitivity to proteotoxic stresses in general. Please also be aware that for unprimed, intrinsic resistance to e.g. heat shock, pre-existing levels of HSP are more relevant than the ability to activate the HSR. Activating the HSR is more relevant to resistance to more chronic temperature elevations and acquired resistance via the priming (hormesis).

Significance

Regulation of the cell intrinsic heat shock response is an important item to understand how cels may be primed to become resilient to (certain) other stresses. Besides the main studied regulator (HSF-1), many other levels of regulation likely exist and intra-organellar communication and proteostasis might be an important aspect for controlling such regulation.

As such, peroxisome proteostasis (that, unlike other organelles, are not (also) controlling a organellar unfolded protein response) is an interesting organelle that could co-control the cytosolic heat shock response. So, the aim of this study per se is quite interesting, However, although for evaluation of physiological relevance c. elegance is a good model of choice, for the mechanistic studies, cellular experiments would have been better suited.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary:

In the manuscript entitled "Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans", Musa et al. explored the role of functional peroxisomes in the heat shock (HS)-induced hormesis in C. elegans using peroxisomal catalase ctl-2 deletion mutants. They showed that ctl-2 deletion abolished HS-induced longevity and suppressed the HS-induced upregulation of small heat shock proteins and HS-induced HSF-1 nuclear accumulation. Furthermore, the authors analyzed the differences in HS-induced phenotypes between wild type animals and ctl-2 mutants: the activation of the antioxidant response, the pentose phosphate pathway, and increased triglyceride content, which are the most prominent changes observed during heat shock in wild-type animals. However, there is only weak evidence supporting their claim that functional peroxisomes are required for HS-induced hormesis.

Major comments:

1. The authors conclude that the functional peroxisomes are essential for the HS-induced hormesis based only on one observation that the transient heat shock treatment did not increase the lifespan in the short-lived ctl-2 mutants. Because the ctl-2 mutants are short-lived, the author should have carefully examined the relevant role of functional peroxisomes in hormesis response by conducting lifespan measurements using ctl-2 RNAi-treated animals or mutants deficient in other genes essential for peroxisome function.
2. The authors examined the difference between wild-type animals and ctl-2 mutants in many aspects; however, there is no rational explanation or examination that their observations have a role in HS-induced hormesis.
3. There is a severe shortage in the explanation of their experiments, especially for the methods of the experiments and statistical procedures. The authors should clarify the details of the experiments with proper statistical analyses. Follows are the points to be clarified for Figure 1 as insufficiency examples in the explanation in their study.

<Points for Figure 1: they should also carefully consider the clarification on other figures> Fig. 1A, B: Is this the representative survival curve of several experiments? The authors should clarify this point. The authors should include a table with the statistical numbers: the number of animals they used, the number of animals they measure in each experiment, and the p-value. Fig. 1C: Is this a representative brood size of one animal, the mean brood size of some animals of one representative experiment, or the mean brood sizes from some experiments? The authors should clarify this point. Also, the authors should perform statistical analysis. Fig. 1D-F: The authors should state the number of experiments they conducted. The author should perform multiple comparison statistical analyses instead of t-test without any correction. Fig. 1H: Is this a representative plot of a representative experiment or the plot of the mean values from some experiments? The authors should clarify this point. The author should also clarify the tissue they analyzed and the exact number of the experiment (the number of nuclei in each animal they analyze, the number of animals they analyze in each experiment). The author should also clarify the detailed statistical procedure: one-way ANOVA or two-way ANOVA; which multiple comparison method they used in their analyses.

Minor comments:

The author should explain the details for the experiments in either the Methods section or the figure legends. The authors should integrate the figures 6B-J (6B-D -> one figure, 6E-G -> one figure, and 6H-J -> one figure) into three figures because there are redundancies in these figures. In addition, the authors should perform the statistical analyses with multiple comparison procedures instead of a simple t-test. The authors should perform statistical analyses on Figs. 6N and 6O. The authors would better cite relevant articles when referring to representative target genes of UPRER, UPRmt, and TORC1.

Significance

Understanding the mechanism underlying the HS-induced hormesis in a multicellular organism is essential in the research field. Their finding that functional peroxisomes play a pivotal role in the HS-induced hormesis, if properly demonstrated, would provide us with the significant progress in this field; however, much more proper experiments are required to support their conclusion. Nevertheless, their finding can stimulate the attention of researchers who study the aging process, stress response, and especially peroxisome function.

I am an expertise in the study of aging in C. elegans.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In the manuscript " Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans" the authors show that a hormetic heat shock requires the peroxidase catalase ctl-2 for longevity and thermotolerance. Furthermore the authors characterize the hormetic stress response in ctl-2 mutants and show that ctl-2 is required for the proper formation of HSF-1 stress granules, and that ctl-2 mutants have a changed transcriptional response to heat shock and a changed induction of heat-stress induced oxidative stress. The authors go on to characterize peroxisomes, and peroxidase-regulated fatty acids, as well as mitochondria in both ctl-2 mutants and upon heat shock. The authors conclude that functional peroxisomes play an important role in the hormetic heat stress response.

This is an interesting study, that demonstrates that the loss of the peroxidase catalase ctl-2 plays an important role in the heat stress response. The authors need to provide more details on experimental repeats and experiments strengthening the conclusion that peroxisomes are generally important for the hormetic stress response would also improve this manuscript.

Furthermore, some textual changes would clarify some specifics in this manuscript.

Major issues:

1. Throughout the manuscript it is unclear how many times the experiments were conducted (e.g. Lifespan, thermotolerance) or how many biological replicates were used (qRT-PCR, peroxisome characterization etc). This critical information should be included. A table with the individual lifespan experiments and thermotolerance experiments is expected.
2. The authors are characterizing the ctl-2 mutant upon heat stress and find some compelling differences to wild-type animals. The authors state "We measured the expression levels of two peroxisomal transport proteins, PRX-5 and PRX-11, and found no differences in expression between WT and Δctl-2 strains, suggesting that morphogenesis should not be impaired and that proteins needed for their proper function should be present, with the exception of ctl-2 in the Δctl-2 strain (Figure 3A)." The authors however conclude that functional peroxisomes are required for the benefits of a hormetic HS. What the authors are in fact demonstrating is that ctl-2 specifically is required for the hormetic HS response. To demonstrate that peroxisomes in general are required they should disrupt peroxisome function by additional means. What the authors also demonstrate is that heat shock has an effect on peroxisomes. These differences need to be clarified. It is also not fully clear whether peroxisomes are functional in ctl-2 mutants? Further peroxisome-relevant enzymes should be tested for levels and functionality in ctl-2 mutants and upon HS.
3. The TORC1 experiments are too indirect. The authors should perform WesternBlots with a S6Kinase -Phospho antibody to determine whether TORC1 is inhibited or not. Alternatively, the authors may choose to remove this experiment from the manuscript without disrupting the main message of the manuscript, since no link between TORC1 and peroxisome function nor with HS has been established.

Textual changes:

1. The authors should adhere to C. elegans nomenclature convention (at least once), by referring to the ctl-2 deletion with the specific allele name [ctl-2(xx)] (unlike in yeast, ∆ is not commonly used). Furthermore a more thorough description of the specific allele (complete gene deletion? Point mutation? Truncation?) should be included.
2. Throughout the manuscript the authors should refer back to their specific hormetic heat shock paradigm, since several studies (also cited here) have shown differences in the specific physiological changes (e.g. HS on day 1 of adulthood has profound effects on broodsize, whereas the authors show here that a HS at L4 does not, other differences in terms of activation of other stress reporters has also been reported).
3. The abbreviation OGT (original growth temperature??) should be defined
4. The authors should change the sentence "The binding of HSF-1 to the HSEs can be visualized by a HSF-1::GFP fusion protein". This is not technically true. While HS has been shown to induce HSF-1 stress granules and these stress granules have been indirectly shown to correlate with transcription, it has however not been demonstrated that HSF-1 is bound to HSEs or whether it is bound to other DNA stretches or what is in fact transcribed by HSF-1 when it localizes to these foci.
5. The authors describe the morphological changes of peroxisomes upon HS, however they missed the opportunity to fully interpret these results in the discussion. Again, their conclusion that peroxisomes may be impaired in ctl-2 mutants is very vague.

Significance

Hormesis and the role of peroxisomes in stress responses is an important and interesting topic.

The role of peroxisomes in stress responses has not been addressed.

Researchers with interest in stress responses will be interested in this work.

My expertise lies in C.elegans stress and longevity with a specific focus on hormetic mechanisms.

Referee Cross-commenting

While I agree with most comments from the other referees, I don't believe it is feasible to ask the authors to generate c. Elegans cell cultures for any follow up experiments. I would be satisfied with a more thorough comparison of the HS response between WT and Ctl-2 mutants, I.e: compare preconditiöning: 1h and 4h HS at L4 and d1 and then do thermotolerance experiments

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

The authors do not wish to provide a response at this time. We aim to provide a revised version of the manuscript within two month.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In recent years, significant progress has been made in defining the molecular details of many structural features of the nuclear pore complex (NPC). However, one area that remains ill-defined is the interface between the core structures of the NPC and the pore membrane domain. This is an especially intriguing area when one considers that the NPC contains several integral proteins and numerous peripheral membrane proteins contain amphipathic helices whose functions and interactions with the membrane, as well as with one another, remain largely undefined.

In this manuscript by Amm et al., the authors have examined the functional role of the integral membrane Nup Ndc1 and its interactions with various peripheral membrane Nups, including members of the Nup84 complex (termed the Y-complex) and the linker Nups Nup53 and Nup59. The authors show that Ndc1 interacts with specific members of the Nup84 complex, namely Nup120 and Nup133, supporting the idea that Ndc1 functions, in part, to anchor this NPC substructure to the pore membrane. In addition, they identified an amphipathic helix (AH) within the C-terminal half of Ndc1, and they showed that it can directly bind to membranes. Importantly, they have used genetic assays to show that the Ndc1-AH functionally interacts with AHs present at the C-terminus of Nup53 and Nup59. Strikingly, they show that the lethal phenotype detected in strains lacking Ndc1 can be suppressed by the deletion of NUP53, but not NUP59, and, more specifically, only the loss of the C-terminal AH Nup53 was required to suppress the lethal phenotype of the ndc1 null mutation. Further ultrastructural analysis of these mutants revealed that, while these mutants were viable, they exhibited extensive NE expansion phenotypes.

Overall, the data presented in this manuscript are of high quality, and the experiments are well controlled. My specific comments are relatively minor and listed below.

Minor points

1) The authors state "Serial ultrathin sections of fixed yeast cells overexpressing ProtA-CtNdc1 revealed that these unusual extranuclear membrane proliferations exhibited pore-like structures with diameters similar to the diameter of NPCs within the nuclear membrane (Fig. 2C)." This is not entirely clear from the data. I suggest the authors provide direct measurements that support their statement.

2) The authors examined the total cellular lipid content following overexpression of Ndc1-AH-containing constructs, as well as ProtA-ScHmg1. There is little discussion of the significance of these results, which would provide a clear justification for including these data in the manuscript.

3) There are numerous typographical and grammatical errors throughout the manuscript that need to be addressed.

Significance

The results presented in this manuscript provide further insight into the molecular interactions between Nups and the pore membrane. They suggest that AHs present in a subset of Nups perform linked functions and contribute, in part, to nuclear membrane biogenesis. As such, these results are an important advance in our knowledge of NPC structure and function. They will be of general interest to those studying the function of NPCs and, more generally, NE and organelle biogenesis.

Reviewer expertise: NPC structure and function, NE biogenesis, yeast model system.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Amm et al report on the role of new motifs and interactions between the essential and conserved integral nuclear pore membrane protein Ndc1 and other key components of the yeast nuclear pore complex. They show that members of the Y-subcomplex that coats the pore membrane bind directly to Ndc1 and identify an amphipathic helix at the C-terminus of Ndc1 that displays genetic interactions with other nucleoporins carrying analogous amphipathic helices. The authors find that cells can survive without Ndc1 when these related amphipathic helices from other nups are coincidentally deleted.

Despite significant recent advances in our structural understanding of the nuclear pore complex, how the NPC associates with the curved nuclear membrane remains poorly understood. Previous studies in yeast have uncovered significant redundancy in this association but again the basis for this remains unclear. Therefore, I find this study on the amphipathic helix of Ndc1 and its interaction with other membrane binding components of the NPC an important and timely contribution to the field. Technically, the paper is solid and I find that most of the authors' conclusions are well supported by the evidence they provide (but see below for few experimental issues). Overall, the paper is well written, and despite the use of several mutants and methodologies, it is easy to read. I think the paper's significance would improve if the authors could present some "larger picture" view on how the Ndc1 helix and/or domains they describe interact with the Nup84 complex and the pore membrane or other elements of the NPC. For example, the authors make the remarkable finding that removal of Nup53 makes ndc1 nulls able to survive. Would it be possible to use existing models of the yeast NPC and provide some structural explanation of why that is? However, I would like to emphasize that this is not required to support the main claims of the paper and should only be considered if the authors wish to provide a more "molecular" view of their findings.

Specific experimental issues and clarifications:

• A major part of the manuscript describes a detailed structure-function analysis of Ndc1. The link between the two domains of ScNdc1 studied and their effects on membrane proliferation could be better defined: specifically, can the authors exclude that the N-domain of Ndc1 that includes its transmembrane domain, is not also involved in the membrane proliferation phenotype shown in Fig2A and C? It also seems as if GAL-ProtA-ScNdc1 (1-260) also causes growth inhibition (Fig. 2F). How do cells with GAL-ProtA-ScNdc1 (1-260) look like? Finally, although the authors convincingly show that overexpression of 261-655 inhibits growth, from the EM it seems as its effects on membrane proliferation is not the same as that of the overexpression of full-length Ndc1 (compare Fig. 3D vs Fig. 2D).

• Figure 1A: Do the CtNups shown under "Input" represent 100% of what used in the binding reaction? If so, please indicate at the figure.

• CtNup120 and CtPom133 would migrate close to CtPom152, which could make visualization by Coomassie stain a bit tricky - if the authors could provide SDS PAGE gels with lower %, that would be helpful. Along similar lines, how do the authors know that CtNup120beta does not bind the CtNdc1 if these two appear to migrate at the same size (Fig. 1D)?

• Figure 1B, GUVs: Why do the authors use CtNup85 for the GUV experiment instead of CtNup84 that was used in Fig. 1A?

• Moreover, CtNup120 and CtNup133 ...BC08/SCL1 (Fig. 1C)" Don't see this in Fig. 1C

• The imaging of ProtA-AHNdc1-eGFP (Fig. 3C) is not great and the localization of the AH does not look very clear - can the authors provide better micrographs? Perhaps co-expression of a red ER reporter or similar reporter would also help.

• The ndc1 nup53 double mutant appears to display a striking cold-sensitive growth defect (Supplemental Figure 6A, compare 23 vs 30C). Can the authors comment on this?

Significance

Despite significant recent advances in our structural understanding of the nuclear pore complex, how the NPC associates with the curved nuclear membrane remains poorly understood. Previous studies in yeast have uncovered significant redundancy in this association but again the basis for this remains unclear. Therefore, I find this study on the amphipathic helix of Ndc1 and its interaction with other membrane binding components of the NPC an important and timely contribution to the field. Technically, the paper is solid and I find that most of the authors' conclusions are well supported by the evidence they provide (but see below for few experimental issues). Overall, the paper is well written, and despite the use of several mutants and methodologies, it is easy to read. I think the paper's significance would improve if the authors could present some "larger picture" view on how the Ndc1 helix and/or domains they describe interact with the Nup84 complex and the pore membrane or other elements of the NPC. For example, the authors make the remarkable finding that removal of Nup53 makes ndc1 nulls able to survive. Would it be possible to use existing models of the yeast NPC and provide some structural explanation of why that is? However, I would like to emphasize that this is not required to support the main claims of the paper and should only be considered if the authors wish to provide a more "molecular" view of their findings.

Audience: Mostly the following - Nuclear pore complex, nuclear envelope, and possibly some membrane biologists.

My field of expertise: Cell biology, Nuclear envelope.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Ndc1 is a transmembrane nucleoporin, essential for insertion of the nuclear pore complex (NPC) and spindle pole body (SPB) into the nuclear envelope (NE). How NE-associated proteins contribute to the bending and fusion of membranes during NPC insertion has not been fully elucidated. Here, the authors report a number of loosely connected, interesting observations related to Ndc1 function. Their main findings are the following: (i) The N-terminal transmembrane domain of Ndc1 mediates the membrane recruitment of two Y-complex nucleoporins. Therefore, these interactions are likely to contribute to NPC biogenesis. (ii) Over-expression of a novel amphipathic helix (AH) in the non-essential C-terminus of Ndc1, and of a similar AH in the non-essential nucleoporin Nup53, alters the lipid composition and nuclear morphology of yeast cells, although the underlying mechanisms remain unknown. (iii) The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53, or by deleting the transmembrane nucleoporin POM34. Surviving strains have altered nuclear morphology (NE expansions), and are sensitive to membrane-fluidizing drugs, suggesting that NPC assembly is somehow linked to lipid homeostasis.

Overall, the experiments are of high technical quality, are presented in a clear way, and the conclusions are well-supported by the data. I have some minor suggestions for clarifications, which can be addressed by textual changes or by additional experiments.

1. When overexpressed in budding yeast, the C-terminal domain of Ndc1 is toxic and induces membrane expansion with NPC-like openings, which the authors describe as enlarged ER membranes (Figure 2). Could these be NE expansions instead? ER and NE membranes are continuous but perhaps this issue could be addressed by examining the distribution of fluorescent markers specific for each compartment.

2. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53 or by deleting POM34. These experiments are done using a plasmid shuffle strategy, in which Ndc1 is temporarily expressed from a low copy plasmid. I wonder if surviving strains are stable, or whether they survive for a limited time only due to stabilisation of the Ndc1 protein in the absence of Nup53 or Pom34. Could the authors discard this possibility, for example by checking whether viable double mutants are recovered after backcrossing of the survivor strains?

3. Cells over-expressing Ndc1, and surviving ndc1-delta strains display ER and/or NE expansions. It would be interesting to discuss these observations in the context of nuclear morphology studies by the Cohen-Fix and Liakopoulos labs, among others, showing NE expansion is partially dependent on the coordination between lipid synthesis, cell growth rate, and cell cycle progression (doi: 10.1091/mbc.E18-04-0204, 10.1091/mbc.e05-09-0839, 10.1016/j.cub.2012.04.022).

4. Related to the previous point: nuclear membrane expansions caused by metaphase arrest usually overlap with the nucleolus, and appear DAPI-negative. Did the authors examine nucleolar distribution relative to NE expansion in cells shown in figure 4C? Along the same lines, what is the cell cycle distribution of cells with ER/NE expansion? If they are delayed in mitosis, nuclear morphology defects may be a secondary consequence of cell cycle progression defects, themselves due to NPC and/or SPB insertion problems.

5. I suggest to rephrase the last sentence of the abstract: "nuclear membrane biogenesis dependent on a balanced ratio between amphipathic motifs in diverse nucleoporins is essential for interphase NPC biogenesis". This study does not directly assess NPC biogenesis and therefore, the interesting link between lipids and NPC biogenesis remains correlative.

6. It would be useful to include some information on the number of cells observed in the EM figures.

7. Results, first page: "Moreover, CtNup120 and CtNup133 did not associate with GUVs containing the unrelated inner nuclear membrane protein BC08/SCL1 (Fig. 1C)" should be Figure S1C.

8. P. 19: "Prompted by the finding that Ndc1 and Nup53/Nup59 amphipathic motifs may (modify?) the nuclear ... "

I am an expert in yeast genetics and cell cycle progression.

Significance

Significance: This report describes novel functional motifs in the Ndc1 protein that may be important for NPC assembly, and intriguing genetic interactions between NPC assembly and lipid homeostasis pathways. Although the mechanisms linking Ndc1 motifs with NE expansion and lipid composition remain unclear, these observations will be interesting for researchers working on NPC biogenesis and nuclear morphology.

Reviewer Expertise: yeast genetics, cell cycle progression and NPCs.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reviewer #1

Major #1

This study primarily uses the genetic mouse model in which LSD1 gene is inactivated after tamoxifen injection in 8 weeks old mice, as shown in supplemental figure 1 B and C. 8 weeks after birth postnatal growth of muscle is not complete and the contribution of satellite cells to muscle growth is still significant. Therefore the timing of tamoxifen injection used cannot discriminate if the observed phenotype involves the function of LSD1 during the post-natal growth of the muscle or in the muscle fibers or both. One way to demonstrate the real contribution of LSD1 in the maintenance of muscle fibers plasticity under environmental stress would be to inject Tamoxifen later (around 10-12 weeks of age), in order to remove a possible bias caused by the contribution of satellite cells during the post-natal growth. At least key findings should be confirmed at this later stage.

In this study, we used ACTA1-CreERT mice to conditionally knockout LSD1 in the skeletal muscle. The ACTA1 promoter is derived from human muscle actin gene, which is not expressed in the satellite cells, and has been widely used for the transgene expression in myofibers (Stantzou et al. Development 2017). Thus, the inactivation of LSD1 occurs in the existing myofibers, and alterations in satellite cell function, if any, would be indirect effects of the loss of LSD1 in mature myocytes or differentiating myoblasts.

To test whether postnatal muscle growth was affected in our LSD1-mKO mice, we administrated tamoxifen (4OHT) to pre-weaning mice (11 days old). LSD1 depletion did not affect the expression of muscle fiber genes, when muscle tissues were isolated from mice 11 days after the start of 4OHT (Additional Data).

These evidences exclude the contribution of satellite cells in the phenotypes observed in the LSD1-mKO mice. __Additional Data __will be added in the revised manuscript.

Major #2

LSD1 m-KO muscles seem to have more type I and IIA fibers than WT, even without DEX treatment. Is it possible to quantify the results in supplemental figure 4C?

As suggested, we quantitatively analyzed the fiber type compositions in Supplemental Fig. 4C using the data from WT (n=4) and LSD1-mKO (n=5) mice (Additional Data). We did not find a significant difference between these mice, confirming our finding that the loss of LSD1 accelerates the Dex-driven phenotypic changes. __Additional Data__will be added in the revised manuscript.

Major #3

The effect on fiber type is convincing, while variations in gene expression are of quite low amplitude. However, the atrophy should be induced by other means to ensure that the effects are specific to GC/nuclear receptors pathways; Denervation? Starvation? Not all the experiments need to be repeated, just key results such as, for example, exacerbation of atrophy in LSD1 m-KO, Foxk1 increase.

We agree that testing alternative atrophy models is important for generalizing our findings. For this, we employed a model for diabetes-related muscle atrophy. A pro-diabetic agent streptozotocin (STZ) disturbs the function of pancreatic islet leading to fast-fiber atrophy (O’Neill et al. Diabetes 2019). LSD1-mKO did not affect the muscle weight in STZ-treated mice (Additional Data). Consistently, there were no major difference in the expression of atrophy genes in STZ-treated WT and LSD1-mKO mice (Additional Data). These results suggest that the LSD1 function depends on the source of atrophy-inducing stress, and that the loss of LSD1 sensitized the muscle to GC-mediate signaling. Additional Data will be added in the revised manuscript.

Major #4

Autophagy data: the effect on the LC3I/LC3II ratio are modest. The autophagy part should be removed or completed with additional data to convincingly show that autophagy is affected. Links between LSD1 and mTOR have been published, so the mTOR pathway could be investigated in the model (S6k, S6 and 4EBP1 phosphorylation). Given AKT levels and phosphorylation are affected by the absence of LSD1 + DEX, it can be predicted that mTOR activity will change.

We have analyzed the expression of additional autophagy markers p62 and phosphorylated 4EBP1. Consistent with the upregulated expression of atrophy genes and increased LC3I/II ratio, LSD1-mKO mice had elevated levels of p62 and phosphorylated 4EBP1 (Additional Data). Altogether, the data suggest that Dex-induced muscle atrophy was exacerbated by the loss of LSD1. Additional Data will be added in the revised manuscript.

Major #5 The ability of LSD1 to retain FOXK1 in the nucleus is an important information that should be better supported experimentally. In the absence of such information, no mechanism can be proposed for the effect of LSD1 of FOXK1. The immunofluorescence images provided are not convincing and moreover they could be interpreted by a reduction in the level of FOXK1 protein (degradation?) rather than by a nuclear exclusion in the presence of DEX. This point should be addressed, authors could include western blot of nuclear and cytoplasmic fractions to better quantify the nuclear level of FOXK1 in absence of LSD1.

We agree that performing the suggested experiment would further enhance the quality of our study.

Major #6 The absence of centralized nuclei indicates that there is no fiber regeneration but it does not exclude the possibility that satellite cells were recruited to existing fibers and thus participated to hypertrophy. To eliminate this possibility, the average nuclei/cytoplasm volume should decrease if hypertrophy results from increased protein synthesis and not myonuclei accretion. This should be checked.

We histologically analyzed the sections of Gas muscles after Dex treatment and found that there is no evidence of central nuclei in either WT or KO mice (Supplemental Fig. 4D).

As mentioned above (Major #1), it is unlikely that the satellite cell function was responsible for the enhanced atrophic phenotype.

Major #7 The upregulation of ERR____g in the absence of LSD1 is convincing in the VWR conditions. ERR____g level should be evaluated in the sedentary LSD1 KO mice.

We have analyzed the expression of ERRg in sedentary mice, and found no significant difference between WT and KO mice (Additional Data). This suggests that the loss of LSD1 in combination with VWR training led to the increased expression of ERRg. Additional Data will be added in the revised manuscript.

Minor #1

There is a clear difference in the number of mouse replicates between treated (Dex or VWR) and non-treated mice, regardless the genotype. Experiments with non-treated mice lack adequate numbers to make a definitive conclusion. For example, there is a huge spread in the data in Figure 1 B and 4 D. If the number of animals would have been increased, would the absence of difference hold up?

We increased the number of non-treated animals in Figures 1B and 4B as suggested. Nonetheless, we did not find any significant differences in the muscle weight (Additional Data). These changes will be reflected on original Figures 1B and 4B.

Minor #2 The authors claim that: "Consistent with the results of the augmented endurance capacity, the Sol muscle in the KO mice showed enhanced succinate dehydrogenase (SDH) staining, indicating that the number of oxidative fibers increased (Figure 4F and Supplemental Figure 8F)". However, supplemental figure 8 D indicates that the number of type I fibers does not change compared to WT. Authors should clarify this statement.

Indeed, we found that the area of type I fiber but not the number was increased in the LSD1-KO Sol (Fig. 4D and Supplemental Fig. 8D). Because SDH staining reflects the OXPHOS capacity in all fiber types, it is possible that the OXPHOS capacity in the fibers other than type I had been augmented by LSD1-KO. Thus, for clarification, we will change the statement as follows: OXPHOS capacity of Sol was enhanced by the loss of LSD1.

Reviewer #2

__Methods

1__

The authors used the Cre-lox system with tamoxifen to generate skeletal muscle-specific LSD1 KO mice. It is clear that both the mRNA and protein levels of LSD1 in various muscles were dramatically reduced, but there is still some LSD1 expressed in skeletal muscle, especially in Sol muscle (Supplemental Figure 1C). The author needs to think about whether it is appropriate to use the term "LSD1 knockout" or "LSD1 deficiency".

We thank the reviewer for this comment. In this study, we crossed LSD1-floxed mice with ACTA1-creERT mice. This enables the deletion of critical exons of LSD1 in mature myocytes and myogenic precursors that have initiated the differentiation program. LSD1 is a ubiquitously expressed gene, and it is known that immature myogenic cells (e.g., satellite cells, Tosic et al. Nat Commun. 2018) and other non-myogenic cells such as hematopoietic and vascular cells abundantly express LSD1 (Kerenyi et al. Elife 2013, Yuan et al. Biochem Pharmacol. 2022). Thus, it is likely that LSD1 expression by these cell types were detected in our whole muscle western blots. We will add these statements in the text for clarification.

__Results

2__

To identify the transcriptional regulators that mediate the regulation of atrophy-associated genes by LSD1, the authors performed motif analyses on the promotor regions of upregulated genes in LSD1-mKO Gas. Based on the results and other reports, they focused on Foxk1 and proved LSD1 and Foxk1 cooperatively regulate the atrophy transcriptome in the presence of Dex. However, Figure 3C showed that a transcription factor Nfatc1 is also reduced in Sol muscle similar to Foxk1. Also, other studies demonstrated that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. More specifically, NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300 (Cell Rep. 2014 Sep 25; 8(6): 1639-1648). Furthermore, it has been reported that LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription (Cell Rep. 2017 Feb 21;18(8):1996-2006. doi: 10.1016/j.celrep.2017.01.078). It is unclear how the authors exclude Nfatc1 for the LSD1-mediated effects in different muscle fibers. Further experiments may be necessary to exclude Nfatc1.

We thank the reviewer for an insightful comment. In addition to Foxk1, we tested the involvement of NFATc1 in the gene regulation under LSD1-depleted state. We treated C2C12 with an LSD1 inhibitor S2101 in combination with a calcium ionophore that promotes the transcriptional function of NFATc1 by inducing its nuclear localization (Meissner et al. J Cell Physiol. 2007). While LSD1 inhibition promoted the expression of Pgc1a and Myh7, ionophore treatment had no additive effects (Additional Data). Because we found a physical association of Foxk1 with LSD1, we focused on the functional involvement of Foxk1 in LSD1-mediated repression of atrophy genes. We recently performed an ATAC-seq analysis in Dex-treated muscle, and found that the Foxk1 motif but not the NFATc1 motif was enriched in the LSD1-KO-specific open chromatin regions. This data further suggests the significant contribution of Foxk1 in the transcriptional regulation under LSD1 depletion.

#3

In figure 3D, only merged images were colored. It would be better to show colored images for Foxk1 and DAPI.

We will replace the images with the colored ones.

#4

Immunofluorescence analysis in C2C12 myotubes showed that Dex exposure reduced the nuclear retention of Foxk1, which was further promoted by the addition of T-3775440, an LSD1 inhibitor (Figure 3D). The author also used Foxk1-KO C2C12 myotubes to prove LSD1 and Foxk1 cooperation to regulate the expression of type I /IIA fiber and atrophy genes in Foxk1-KO cells. Are the effects of LSD1 dependent on Foxk1 or synergistically acting with Foxk1? The treatment of LSD1 inhibitor in Foxk1-KO C2C12 may be helpful to answer this question.

As suggested, we will examine the combination effect of LSD1 inhibition and Foxk1-KO. In addition, we will analyze chromatin association of LSD1 in Foxk1-KO cells by ChIP experiments, to test whether the function of LSD1 depends on Foxk1.

#5

In supplementary figure 2, body weight in the mKO+Dex group was reduced in comparison to that of WT+Dex. How about the body weight of mKO mice without Dex injection compared to that of WT? This data will be helpful to understand the effect of muscle-specific LSD1 deficiency on whole-body energy balance.

We measured the body weight of untreated mice, and found that there is no genotype effect (Additional Data). Thus, we think that LSD1-mKO alone does not influence the whole-body energy balance. We will include this data in the revised version.

#6

The authors analyzed the size distribution of myofibers and mentioned that large type I and type IIA fibers preferentially increased in the LSD1-mKO muscle, whereas large type IIB + IIX fibers decreased (Supplemental Figure 4, B, E, and F). It is better to show the results of statistics. If no significance were found, it should be mentioned in the result section.

We have performed statistical analyses on Supplemental Fig. 4E and F, and found that a fraction of large type I fibers was significantly larger in KO mice. This result will be added in the next version.

#7

Page 11, To reveal the genes regulated by LSD1 under the VWR condition, the authors performed additional RNA-seq analysis using Sol muscle. The non-hierarchical clustering analysis was informative and showed signaling pathways related to ‘mitochondrion’, ‘mitochondrion organization’, and ‘oxidative phosphorylation’ were altered in the Sol muscle deficient in LSD1 under the VWR condition (Figure 5B). However, it is unclear why they focus on Err-gamma to explain LSD1-KO phenotypes in Sol muscle. Is this gene also derived from RNA seq? It is better to show whether Err-gamma expression is also significantly altered based on RNA seq data.

Indeed, ERRg was upregulated by LSD1-KO+VWR and was included in the Cluster 6 genes together with the OXPHOS and mitochondria-related genes (Additional Data and Fig. 5A). These data prompted us to focus on ERRg as a potential factor that explains the LSD1-KO phenotype. Additional Data will be included in the revised version.

#8

The authors claim that LSD1 serves as an "epigenetic barrier" that optimizes fiber type-specific responses and muscle mass under stress conditions. This claim is derived from the loss of function studies. To generalize the functions of LSD1, the gain of function studies will be also necessary. Adding the characteristics of LSD1 overexpression in C2C12 cells will further improve the quality of the manuscript.

We agree that the gain of function studies will further strengthen the quality of our manuscript. As suggested, we will perform an LSD1 overexpression experiment using C2C12 cells and analyze the expression of atrophy and fast fiber related genes. Because Esrrg is completely silenced in C2C12 cells, it is difficult to monitor ERRg-mediated gene regulation in these cells. To overcome this, we will use a cardiomyocyte cell line, in which ERRg is functionally involved in differentiation (Sakamoto et al. Nat Commun 2022). We will overexpress LSD1 in these cells and examine whether the expression of ERRg and its downstream targets are altered.

__Discussion

9__

The authors mentioned supplementary figure 10 only at the end of the manuscript of the discussion section (page 15) without a specific explanation of the figures in the result section. The data are important in that LSD1 expression in human muscles declined with age and showed a negative correlation with the expression of the atrophy gene. It should be presented in the result section with a more detailed description.

We agree that these data are important and need further explanations. We will describe the details in the Results section and move the entire figure to the main figure.

#10

There are other studies to examine LSD1 and muscle regeneration or functions (e.g. Nat Commun 9, 366 (2018). ____https://doi.org/10.1038/s41467-017-02740-5____). More discussion to compare the current study and other studies will be necessary.

We thank the reviewer for this comment. We will add the discussion accordingly.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The authors investigated the functions of LSD1 in skeletal muscle in response to different stimulations. The study is quite interesting in that it revealed the dual roles of an epigenetic regulator LSD1 in the types of muscle fibers using a muscle-specific LSD1 KO mouse model and related cell lines with big data analysis. The LSD1 deficiency sensitized the skeletal muscle to Dex-induced atrophy and VWR-induced hypertrophy. LSD1 appears to suppress the catabolic pathways and the formation of type I fibers. Also, LSD1 may exert its effects together with Foxk1 to suppress genes related to protein catabolism, while repressing ERRg and its downstream oxidative genes, depending on the stimulus and muscle location. These findings will shed light on related fields in that LSD1 may be an epigenetic molecular switch that induces fiber-type-specific responses. Nevertheless, some questions need to be further answered to make up for the shortcomings of the study. The specific comments are as follows;

Methods

1. The authors used the Cre-lox system with tamoxifen to generate skeletal muscle-specific LSD1 KO mice. It is clear that both the mRNA and protein levels of LSD1 in various muscles were dramatically reduced, but there is still some LSD1 expressed in skeletal muscle, especially in Sol muscle (Supplemental Figure 1C). The author needs to think about whether it is appropriate to use the term "LSD1 knockout" or "LSD1 deficiency".

Results

1. To identify the transcriptional regulators that mediate the regulation of atrophy-associated genes by LSD1, the authors performed motif analyses on the promotor regions of upregulated genes in LSD1-mKO Gas. Based on the results and other reports, they focused on Foxk1 and proved LSD1 and Foxk1 cooperatively regulate the atrophy transcriptome in the presence of Dex. However, Figure 3C showed that a transcription factor Nfatc1 is also reduced in Sol muscle similar to Foxk1. Also, other studies demonstrated that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. More specifically, NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300 (Cell Rep. 2014 Sep 25; 8(6): 1639-1648). Furthermore, it has been reported that LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription (Cell Rep. 2017 Feb 21;18(8):1996-2006. doi: 10.1016/j.celrep.2017.01.078). It is unclear how the authors exclude Nfatc1 for the LSD1-mediated effects in different muscle fibers. Further experiments may be necessary to exclude Nfatc1.
2. In figure 3D, only merged images were colored. It would be better to show colored images for Foxk1 and DAPI.
3. Immunofluorescence analysis in C2C12 myotubes showed that Dex exposure reduced the nuclear retention of Foxk1, which was further promoted by the addition of T-3775440, an LSD1 inhibitor (Figure 3D). The author also used Foxk1-KO C2C12 myotubes to prove LSD1 and Foxk1 cooperation to regulate the expression of type I /IIA fiber and atrophy genes in Foxk1-KO cells. Are the effects of LSD1 dependent on Foxk1 or synergistically acting with Foxk1? The treatment of LSD1 inhibitor in Foxk1-KO C2C12 may be helpful to answer this question.
4. In supplementary figure 2, body weight in the mKO+Dex group was reduced in comparison to that of WT+Dex. How about the body weight of mKO mice without Dex injection compared to that of WT? This data will be helpful to understand the effect of muscle-specific LSD1 deficiency on whole-body energy balance.
5. The authors analyzed the size distribution of myofibers and mentioned that large type I and type IIA fibers preferentially increased in the LSD1-mKO muscle, whereas large type IIB + IIX fibers decreased (Supplemental Figure 4, B, E, and F). It is better to show the results of statistics. If no significance were found, it should be mentioned in the result section.
6. Page 11, To reveal the genes regulated by LSD1 under the VWR condition, the authors performed additional RNA-seq analysis using Sol muscle. The non-hierarchical clustering analysis was informative and showed signaling pathways related to 'mitochondrion', 'mitochondrion organization', and 'oxidative phosphorylation' were altered in the Sol muscle deficient in LSD1 under the VWR condition (Figure 5B). However, it is unclear why they focus on Err-gamma to explain LSD1-KO phenotypes in Sol muscle. Is this gene also derived from RNA seq? It is better to show whether Err-gamma expression is also significantly altered based on RNA seq data.
7. The authors claim that LSD1 serves as an "epigenetic barrier" that optimizes fiber type-specific responses and muscle mass under stress conditions. This claim is derived from the loss of function studies. To generalize the functions of LSD1, the gain of function studies will be also necessary. Adding the characteristics of LSD1 overexpression in C2C12 cells will further improve the quality of the manuscript.

Discussion

1. The authors mentioned supplementary figure 10 only at the end of the manuscript of the discussion section (page 15) without a specific explanation of the figures in the result section. The data are important in that LSD1 expression in human muscles declined with age and showed a negative correlation with the expression of the atrophy gene. It should be presented in the result section with a more detailed description.
2. There are other studies to examine LSD1 and muscle regeneration or functions (e.g. Nat Commun 9, 366 (2018). https://doi.org/10.1038/s41467-017-02740-5). More discussion to compare the current study and other studies will be necessary.

Significance

The study is quite interesting in that it revealed the dual roles of an epigenetic regulator LSD1 in the types of muscle fibers using a muscle-specific LSD1 KO mouse model and related cell lines with big data analysis.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

In this manuscript, Araki et. al show that LSD1 is required for muscle fiber maintenance upon chemical and environmental stress. By using a LSD1 conditional KO mouse model, along with C2C12 cell culture, the authors demonstrate that loss of LSD1 exacerbates glucocorticoid-induced muscle atrophy in fast fiber-dominant muscle. On the other hand, loss of LSD1 together with endurance exercise induces a hypertrophy in slow fiber-dominant muscle. Thus, these data suggest that LSD1 acts as a modulator of the muscle response to chemical and environmental stress, might be a target for therapeutic strategies against stress-induced myopathies. However, some concerns should be addressed in a revised version.

Major comments:

This study primarily uses the genetic mouse model in which LSD1 gene is inactivated after tamoxifen injection in 8 weeks old mice, as shown in supplemental figure 1 B and C. 8 weeks after birth post natal growth of muscle is not complete and the contribution of satellite cells to muscle growth is still significant. Therefore the timing of tamoxifen injection used cannot discriminate if the observed phenotype involves the function of LSD1 during the post-natal growth of the muscle or in the muscle fibers or both. One way to demonstrate the real contribution of LSD1 in the maintenance of muscle fibers plasticity under environmental stress would be to inject Tamoxifen later (around 10-12 weeks of age), in order to remove a possible bias caused by the contribution of satellite cells during the post-natal growth. At least key findings should be confirmed at this later stage.

• LSD1 m-KO muscles seem to have more type I and IIA fibers than WT, even without DEX treatment. Is it possible to quantify the results in supplemental figure 4C?
• The effect on fiber type is convincing, while variations in gene expression are of quite low amplitude. However, the atrophy should be induced by other means to ensure that the effects are specific to GC/nuclear receptors pathways; Denervation? Starvation? Not all the experiments need to be repeated, just key results such as, for example, exacerbation of atrophy in LSD1 m-KO, Foxk1 increase.
• Autophagy data: the effect on the LC3I/LC3II ratio are modest. The autophagy part should be removed or completed with additional data to convincingly show that autophagy is affected. Links between LSD1 and mTOR have been published, so the mTOR pathway could be investigated in the model (S6k, S6 and 4EBP1 phosphorylation). Given AKT levels and phosphorylation are affected by the absence of LSD1 + DEX, it can be predicted that mTOR activity will change.
• The ability of LSD1 to retain FOXK1 in the nucleus is an important information that should be better supported experimentally. In the absence of such information, no mechanism can be proposed for the effect of LSD1 of FOXK1. The immunofluorescence images provided are not convincing and moreover they could be interpreted by a reduction in the level of FOXK1 protein (degradation?) rather than by a nuclear exclusion in the presence of DEX. This point should be addressed, authors could include western blot of nuclear and cytoplasmic fractions to better quantify the nuclear level of FOXK1 in absence of LSD1.
• The absence of centralized nuclei indicates that there is no fiber regeneration but it does not exclude the possibility that satellite cells were recruited to existing fibers and thus participated to hypertrophy. To eliminate this possibility, the average nuclei/cytoplasm volume should decrease if hypertrophy results from increased protein synthesis and not myonuclei accretion. This should be checked
• The upregulation of ERR in the absence of LSD1 is convincing in the VWR conditions. ERR level should be evaluated in the sedentary LSD1 KO mice.

Minor comments:

• There is a clear difference in the number of mouse replicates between treated (Dex or VWR) and non-treated mice, regardless the genotype. Experiments with non-treated mice lack adequate numbers to make a definitive conclusion. For example, there is a huge spread in the data in Figure 1 B and 4 D. If the number of animals would have been increased, would the absence of difference hold up?
• The authors claim that: "Consistent with the results of the augmented endurance capacity, the Sol muscle in the KO mice showed enhanced succinate dehydrogenase (SDH) staining, indicating that the number of oxidative fibers increased (Figure 4F and Supplemental Figure 8F)". However, supplemental figure 8 D indicates that the number of type I fibers does not change compared to WT. Authors should clarify this statement.

Significance

Conceptually, Araki and colleagues have described the potential contribution of LSD1 in the maintenance of muscle fiber typing upon environmental stress. However, the technical approach used only partially allows to demonstrate the concept.

Field of expertise: Muscle atrophy and regeneration, Muscle stem cells, Epigenetic regulation in muscle, LSD1.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Thank you for giving us the opportunity to submit a revised draft of the manuscript “Tup1 is Required for Transcriptional Repression Necessary in Quiescence in S. cerevisiae” to Review Commons. We appreciate the time and effort that you and the other reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We believe that the experiments suggested in these comments would bring clarity to the manuscript, and wish that we had the ability to perform them all. Unfortunately our lab is closing, and the only remaining lab member is the PI, so we are only able to perform limited experiments to address some of the concerns raised during review. We have incorporated several changes in response to comments from the reviewers. Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript Word file with tracked changes.

Of particular note is the discussion of cellular morphology of the tup1∆ and sds3∆ strains. We realize that our findings are purely descriptive, and are not surprised that all three reviewers had comments on this data. This was the source of much discussion among the authors and consultation with other labs; we debated even including these observations in the manuscript, since we were unable to figure out the underlying mechanism. Ultimately we decided that it was worth reporting in case other labs may benefit from the knowledge, and we have altered the language in the manuscript (page 6) to better reflect this. However, if the reviewers feel that this observation would be better left out of the manuscript, we would be willing to remove Figure 6 and any discussion of these images.

Reviewer #1 Comments:1. The authors chose to examine 3-day SP cells to interrogate quiescence because tup1∆ cells are highly flocculant, interfering with the isolation of purified quiescent cells. These cells are a mixture of both nonquiescent and quiescent cells, so it is not correct to state that they represent a quiescent cell population. The addition of EDTA to the gradients used to isolate quiescent cells could eliminate flocculation and permit the isolation of quiescent cells. EDTA is also often added to media in low amounts to reduce flocculation. The authors need to indicate the proportion of quiescent cells in their SP cultures by applying these tools.

We appreciate the suggestion, but the phenotype of this strain is not typical flocculation (see photo below, also added to the paper as Supplementary Figure 1). We did add EDTA (pH 8.0) to a final concentration of 10 mM to two separate tup1∆ and it did not visibly affect the clumping of cells. Furthermore, changes to the cell wall are a distinct feature of quiescent S. cerevisiae and contribute to the ability to separate different cell types by density-gradient centrifugation, so it is difficult to anticipate how EDTA would affect our ability to isolate Q cells. We have provided more explanation in the manuscript to better explain this (page 3).

1. The authors reported that while Xpb1 and Tup1 share many overlapping binding sites, but that Xbp1 does not regulate Tup1's binding. What other factors might be responsible for their shared binding? Could histone deacetylation play a role? This could be addressed by a Tup1 ChIP in an sds3∆ mutant.

This is a good thought; histone acetylation levels may have a role in regulating Tup1 localization and we would have liked to address this if we had more time. Unfortunately, we had some difficulty performing ChIP of Tup1, because initially we used a FLAG tag which caused a phenotype similar to deletion of Tup1, and had to switch to making myc-tagged strains. This delay meant we did not have time to pursue creating myc-tagged Tup1 in an sds3∆ strain, and now we do not have the ability to follow up on this for revisions.

1. Has PolII occupancy been examined in Log vs SP cells of tup1∆ to determine if Tup1 inhibits PolII association with its genes that are repressed ?

We did not look at PolII occupancy in our Tup1 deletion, and could not find any existing datasets with this information. It is our hope that another lab is able to carry out this experiment, because it could be very enlightening, but it is beyond the scope of this work.

1. The observation that tup1∆ cells have several nuclear puncta is intriguing, although the cytological images need to be improved.

The nuclear puncta we see in the tup1 deletion are definitely a puzzle. We had limited time to investigate this phenomena, and in discussing the matter with some other labs it seemed doubtful that more advanced imaging would yield anything of use to us. We realized that we accidentally omitted important details for this figure and have updated the manuscript to add them. We imaged 2 biological replicates for each strain and imaged many yeast samples for each strain (which has been added to the caption for Figure 6) and found that our findings were statistically significant (p

Reviewer #2 Comments1. The authors acknowledge that it would be better to work with purified quiescent cells but couldn't isolate pure populations. As a result, a mixture of quiescent and nonquiescent cells are analyzed in stationary phase. They say this is because Tup1 deletion strains are flocculent. But they performed ChIP-Seq on Myc-tagged Tup1 strain. Don't these cells express Tup1? If not, could this be performed in wild-type yeast with Myc-tagged Tup1? It seems important to separate quiescent from nonquiescent yeast for the authors' conclusions.

It is true that we could have done ChIP-seq for Tup1 in purified Q cells. We considered it, but decided to look at the mixed population so that we could directly compare our RNA-seq results from the tup1∆ strain. It’s a balance between having some results that are specific to quiescence, versus being able to directly compare the effects of deletion of Tup1 at the sites where it binds. We are now unable to perform this experiment, but we have updated the language in the manuscript (page 3) to better reflect this choice.

1. The Chipseq data in Fig 1B do not have a y axis and it is consequently not clear whether these data are normalized and shown with the same axis.

Thank you for pointing this out - these data are normalized to RPKM during processing, and we have updated the caption for figure 1 and the methods on page 10 to reflect this information. Normalizing the data in IGB itself, however, causes an adjustment in the y-axis that makes the tracks appear to be inconsistent. In any case, we are not making claims about the relative amount of signal, and as it is common in the field to not include y-axes on IGB tracks, we have opted to keep the y-axis for Figure 1B as-is.

1. In Fig 2, it seems important to determine how many genes are different between WT and Tup1 deletion strains in log phase. Are just as many genes different? Or is Tup1 more important in diauxic shift and stationary phase than log phase?

We did intend to focus only on diauxic shift and stationary phase data for this paper, since there has already been so much work on the role of Tup1 in log phase. As mentioned above, comparisons of RNA between log and DS/Q is difficult. We attempted to find a publicly available dataset to perform some analysis for revisions, but unfortunately most previous work on the effect of Tup1 on transcription was performed via tiling arrays, which is not comparable.

1. Are the genes that are regulated by Tup1 normally regulated during diauxic shift or stationary phase compared with log growth?

Because there is a massive global decrease in the level of total RNA in diauxic shift and quiescence (McKnight, Boerma, et al., 2015) it is impossible to directly compare transcript levels between these states in our experiments. If there was time, we could have attempted to repeat these experiments with an external spike-in control; this is potentially something another lab could do to follow up on our findings.

1. What fraction of the genes that are differentially expressed in Tup1 knockout yeast have Tup1 binding at the promoter? Enhancer? What fraction can be explained by Tup1, Hap1, Nrg1, Mig1 individually and together?

We have added the number of genes that are differentially expressed in Tup1 knockout yeast during DS to the manuscript (page 3). Regarding enhancers, the genome of S. cerevisiae is very compact, and there is not evidence of long-distance activation of genes as seen in metazoans (Dujon, 1996; Dobi & Winston, 2007; Spiegel and Arnone, 2021). Upstream activating sequences (UASs) are generally considered the closest equivalent to enhancers in cerevisiae, and they tend to function within a few hundred base pairs of the promoter. Our analysis only identifies the nearest gene; it would be difficult to parse out locations in the promoter versus a UAS without a more advanced analysis that is beyond our capabilities now.

As for the effect of Hap1, Nrg1, and Mig1, we were able to look for their motifs in the genes that are differentially expressed in the Tup1 knockout but we do not have binding data for these factors in quiescence or stationary phase so it is impossible to conclusively state what role those TFs play. This would be a very interesting followup to our work, but is outside the scope of this manuscript.

References:

Dujon, B. 1996. The Yeast Genome Project: What did we learn? Trends Genet. 12, 263-270.

Dobi, K.C.; Winston, F. 2007. Analysis of Transcriptional Activation at a Distance in Saccharomyces cerevisiae. Mol Cell Biol. 27(15), 5575-5586. https://doi.org/10.1128/MCB.00459-007

Spiegel, JA; Arnone, J.T. 2021.Transcription at a Distance in the Budding Yeast Saccharomyces cerevisiae. Appl. Microbiol. 1(1), 142-149. https://doi.org/10.3390/applmicrobiol1010011

1. The methodology used to generate the gene ontology enrichments should be described in the methods.

Thank you for noticing this omission; we have added the relevant information to the manuscript (page 10) and have also added the related citation (page 11).

1. The authors should provide genomewide data to support the statement that Tup1 and Rpd3 ChIP datasets have substantial overlap. They should also provide genomewide data to support the statement that there is substantial overlap between Rpd3 and Tup1. How much overlap is observed and how much is expected by chance?

We have compared the existing ChIP data for Rpd3 binding in quiescent cells to our ChIP data for Tup1 in 3-day cultures and included this in the manuscript (page 4, Supplementary Figure 2B), along with a p-value.

1. For Sds3, similar to Tup1 inactivation, it would be helpful to know how many genes change in with Sds3 inactivation in log phase in addition to diauxic shift and stationary phase.

As with our response to comment #3, we focused only on diauxic shift and stationary phase data for this paper, and analysis of this data would be difficult without a spike-in control. While there are some existing datasets for RNA-seq of Rpd3 knockouts, this would include both Rpd3L and Rpd3S activity, rather than just Rpd3L, which is our focus with the Sds3 deletion strains. As such, we did not perform RNA-seq of sds3∆ in log phase.

1. If the argument is that Sds3 and Xbp1 cooperate with Tup1 to affect gene expression, testing the gene expression changes that are associated with Tup1 in Sds3 or Xbp1 knockout strains would help the authors make this point.

We do not have tup1∆/sds3∆ or tup1∆/xbp1∆ double knockout strains. We attempted to make these strains but could not, which may indicate that these double deletions are synthetic lethal. Deletion of sds3 alone causes a significant reduction in growth rate, so it is perhaps not surprising that we could not create the double knockouts.

1. The final phenotype of extra DAPI positive blobs in the nucleus is not very specific or clear.

We agree, please see our comments at the top of this letter.

Reviewer #3 (Major comments):Did tup1∆/sds3∆ double mutant show the same phenotype with tup1∆ (or sds3∆) single mutant in G0? If Tup1 actually plays role in tandem with Sds3 in the gene regulation during G0, the epistatic relationship might be estimated.

We do not have tup1∆/sds3∆ or tup1∆/xbp1∆ double knockout strains. We attempted to make these strains but could not, which may indicate that these double deletions are synthetic lethal. Deletion of sds3 alone causes a significant reduction in growth rate, so it is perhaps not surprising that we could not create the double knockouts.

The histone acetylation was not synergistically augmented in the above double mutant?

Please see the response above.

The authors showed that tup1∆ but not sds3∆ cells contain multiple DAPI signals but sds3∆ cells show abnormal cell shape in G0 phase. These phenotypic abnormalities in these mutants suggest a potential mitotic defect. Both mutants showed very similar abnormalities in H3K23 acetylation and gene expressions in quiescent state. Why these showed distinctly different abnormality in cell morphology during G0?

Unfortunately we were unable to investigate this further.

Did iswi2∆ cells also show abnormality in G0 phase?

No, they did not; thank you for asking, this is a good question. We have added this information to the manuscript (page 6).

(Minor comments)Supplementary figure1. This data seems to be very important. I recommend to use this data in the main figure with statistical analysis (p-values) to show the significant overlap of Tup1 and Rdp3 distribution.

We have compared the existing ChIP data for Rpd3 binding in quiescent cells to our ChIP data for Tup1 in 3-day cultures and included this in the manuscript (page 4, Supplementary Figure 2B) . We do feel that this data belong in the supplement, however, because the data is not exactly equivalent to our studies: quiescent cells and 3-day cultures are not the same, and knockout of Rpd3 eliminates function of both Rpd3L and Rpd3S complexes, while knocking out Sds3 targets only the Rpd3L complex.

Figure 4. Histone acetylation level data in Figure 4A and the data for gene repressions by Tup1 and Sds3 in Figure 4C seem to be very important. However, statistical analysis data (p-values) was not presented. Please show the statistical analysis data (p-values) as in figure 3 to show that the Tup1 and Sds3 contribute similarly in histone deacetylation and repression. The author did not find the significant changes of histone deacetylation in xbp1∆ cells but said that when filtered in Xbp1 binding motif Xbp1 depletion has similar effect on the acetylation level. Please show this data.

We have added language in the manuscript comparing genes with altered acetylation levels to those that are differentially expressed in our RNA-seq datasets, along with a p-value, to page 5.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In this study, the authors found that Tup1 corepressor coordinates with Rpd3L HDAC complex in the deacetylation of histone H3K23 during quiescence entry. They also found that Tup1 coordinates with ISWI2 to regulate +1 nucleosome at HXT family genes for the gene repression during G0. Finally, they showed that loss of Tup1 results in abnormal shape of nuclei during G0. These data suggest a critical role of Tup1 corepressor in the proper gene regulations in tandem with Rpd3 HDAC and ISWI2 remodeler upon quiescence entry. This study seems to be a critical progress from their previous study on the roles of Rpd3 complex in the quiescence entry (McKnight et al. 2015 Mol. Cell). My comments were listed bellow.

Major comments

Did tup1∆/sds3∆ double mutant show the same phenotype with tup1∆ (or sds3∆) single mutant in G0? If Tup1 actually plays role in tandem with Sds3 in the gene regulation during G0, the epistatic relationship might be estimated.

The histone acetylation was not synergistically augmented in the above double mutant?

The authors showed that tup1∆ but not sds3∆ cells contain multiple DAPI signals but sds3∆ cells show abnormal cell shape in G0 phase. These phenotypic abnormalities in these mutants suggest a potential mitotic defect. Both mutants showed very similar abnormalities in H3K23 acetylation and gene expressions in quiescent state. Why these showed distinctly different abnormality in cell morphology during G0?

Did iswi2∆ cells also show abnormality in G0 phase?

Minor comments

Supplementary figure1. This data seems to be very important. I recommend to use this data in the main figure with statistical analysis (p-values) to show the significant overlap of Tup1 and Rdp3 distribution.

Figure 4. Histone acetylation level data in Figure 4A and the data for gene repressions by Tup1 and Sds3 in Figure 4C seem to be very important. However, statistical analysis data (p-values) was not presented. Please show the statistical analysis data (p-values) as in figure 3 to show that the Tup1 and Sds3 contribute similarly in histone deacetylation and repression. The author did not find the significant changes of histone deacetylation in xbp1∆ cells but said that when filtered in Xbp1 binding motif Xbp1 depletion has similar effect on the acetylation level. Please show this data.

Significance

The current work by the authors significantly progressed their work (McKnight et al. 2015 Mol. Cell) showing important role of Rpd3 deacetylase complex in the quiescence entry. This study will significantly contribute to understanding the gene regulation mechanisms in the G0 entry.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In this paper, the authors investigate the role of the Tup1 transcriptional co-repressor in transcriptional repression after glucose depletion in quiescent S. cerevisiae. Tup1 N terminal helix facilitates oligomerization and interacts with histone tails and a beta propeller domain interacts with histone deacetylases and DNA binding factors. Tup1 interacts with nucleosomes by binding histone H3 and H4 tails, binds to hypoacetylated tails and stabilizes +1 nucleosomes repositioned by Isw2. Tup1 had been identified as a gene essential for viability of cells in G0 and implicated in glucose repression in yeast. The authors found that Tup1-Ssn6 binds new targets when S cerevisiae are glucose deprived and as they enter G0. Tup1 was found to repress the expression of genes involved in glucose metabolism and glucose transport. Tup1 was discovered to coordinate with the Rpd3L complex to deacetylate H3K23. Tup1 was also found to coordinate with Isw2 to affect nucleosome positions at hexose transporter family genes. Some cells with Tup1 deletion had multiple DAPI puncta in stationary phase, suggesting a possible role for Tup1 in mitosis.

Overall Comments

In Fig 1, the shift in Tup1 binding with diauxic shift and stationary phase is clear. The data clearly show an effect of Tup1 on histone acetylation and nucleosome positioning. However, whether Tup1 has an important functional role in quiescence is not clear.

Comments

1. The authors acknowledge that it would be better to work with purified quiescent cells but couldn't isolate pure populations. As a result, a mixture of quiescent and nonquiescent cells are analyzed in stationary phase. They say this is because Tup1 deletion strains are flocculent. But they performed ChIP-Seq on Myc-tagged Tup1 strain. Don't these cells express Tup1? If not, could this be performed in wild-type yeast with Myc-tagged Tup1? It seems important to separate quiescent from nonquiescent yeast for the authors' conclusions.

2. The Chipseq data in Fig 1B do not have a y axis and it is consequently not clear whether these data are normalized and shown with the same axis.

3. In Fig 2, it seems important to determine how many genes are different between WT and Tup1 deletion strains in log phase. Are just as many genes different? Or is Tup1 more important in diauxic shift and stationary phase than log phase?

4. Are the genes that are regulated by Tup1 normally regulated during diauxic shift or stationary phase compared with log growth?

5. What fraction of the genes that are differentially expressed in Tup1 knockout yeast have Tup1 binding at the promoter? Enhancer? What fraction can be explained by Tup1, Hap1, Nrg1, Mig1 individually and together?

6. The methodology used to generate the gene ontology enrichments should be described in the methods.

7. The authors should provide genomewide data to support the statement that Tup1 and Rpd3 ChIP datasets have substantial overlap. They should also provide genomewide data to support the statement that there is substantial overlap between Rpd3 and Tup1. How much overlap is observed and how much is expected by chance?

8. For Sds3, similar to Tup1 inactivation, it would be helpful to know how many genes change in with Sds3 inactivation in log phase in addition to diauxic shift and stationary phase.

9. If the argument is that Sds3 and Xbp1 cooperate with Tup1 to affect gene expression, testing the gene expression changes that are associated with Tup1 in Sds3 or Xbp1 knockout strains would help the authors make this point.

10. The final phenotype of extra DAPI positive blobs in the nucleus is not very specific or clear.

Significance

Discovering transcription factors that are critical for the entry into and maintenance of quiescence is an important area of discovery as relatively little is known about gene regulation during quiescence and understanding this process is fundamentally important for our understanding of cell biology. Previous studies had implicated Tup1 in stationary phase in yeast. These studies provide additional insight into the mechanism.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In this manuscript, the authors have investigated the role of the Tup1 corepressor in transcriptional repression that occurs during the establishment of quiescence in S. cerevisiae. Using ChIP-seq, they found that Tup1 is present at many sites during log phase growth, and that it localizes to new targets upon glucose exhaustion at a point called diauxie; post-diuaxie, Tup 1 remains associated with these sites in stationary phase (SP), which they define as growth of yeast cells for 3 days after inoculation in glucose-rich medium. To understand the significance of Tup1 relocalization during quiescence establishment, they performed differential expression (DE) analysis following RNA-seq in WT and tup1∆ cells at the diauxic shift and 3-day SP. The large number of DE genes in both cell states were consistent with a role for Tup1 in the regulation of key targets associated with the initiation of quiescence, although there was not a strict correlation between Tup1 occupancy at these DE genes. Next, they investigated the relationship of Tup1 to Xbp1 and Rpd3, factors that play a role in transcriptional repression during quiescence. They noted significant overlap between the binding sites for the three factors in either isolated quiescent cells or 3-day SP cells using published and newly acquired ChIP-seq data. However, the absence of Xbp1, a transcription factor that recruits the Rpd3 histone deacetylase in quiescent cells, did not alter Tup1 binding to its targets in these cells. RNA-seq analysis of xbp1∆ and tup1∆ SP cells found a significant overlap between the genes that were repressed in the absence of the two transcription factors. Moreover, Tup1, Xbp1, and Rpd3 were noted to be required for H3K23 deacetylation at repressed genes in SP. Finally, the authors asked if the Tup1 affects the position of nucleosomes at TSSs in SP cells by performing MNase-seq. They found that Tup1 and the chromatin remodeler Isw2, which interacts with Tup1, are required to position nucleosomes at the promoters of a family of glucose transporter genes, targets of Tup1 during the initiation of quiescence.

Comments:

1. The authors chose to examine 3-day SP cells to interrogate quiescence because tup1∆ cells are highly flocculant, interfering with the isolation of purified quiescent cells. These cells are a mixture of both nonquiescent and quiescent cells, so it is not correct to state that they represent a quiescent cell population. The addition of EDTA to the gradients used to isolate quiescent cells could eliminate flocculation and permit the isolation of quiescent cells. EDTA is also often added to media in low amounts to reduce flocculation. The authors need to indicate the proportion of quiescent cells in their SP cultures by applying these tools.

2. The authors reported that while Xpb1 and Tup1 share many overlapping binding sites, but that Xbp1 does not regulate Tup1's binding. What other factors might be responsible for their shared binding? Could histone deacetylation play a role? This could be addressed by a Tup1 ChIP in an sds3∆ mutant.

3. Has PolII occupancy been examined in Log vs SP cells of tup1∆ to determine if Tup1 inhibits PolII association with its genes that are repressed ?

4. The observation that tup1∆ cells have several nuclear puncta is intriguing, although the cytological images need to be improved.

Significance

This manuscript presents some new information that links Tup1, a multifacteted transcriptional co-repressor, to the repression of transcription that occurs during the establishment of yeast quiescence. The McKnight lab has previously shown that the Rpd3 HDAC plays an important role in this process, in part through its recruitment by the TF Xbp1. Tup1 also binds to Rpd3, and the overlap between the sites where Tup1 and Xpb1 bind and the targets that they repress, suggests a shared function in establishing transcriptional reprogramming. Whether this shared function is based only on the recruitment of the Rpd3 histone deacetylation is unclear, and, more importantly, there is also some question if deacetylation is in fact the main factor driving transcriptional repression during the initiation of quiescence. The finding that Tup1 is responsible for repositioning nucleosomes at a class of glucose transporters to repress the transcription of these genes suggests a specific function for Tup1 during the establishment of quiescence; however, this is apparently not a broad function. Thus, we are left with a lot of nice descriptive information as a result of well-done experiments that has not revealed much about the mechanism by which Tup1 regulates the global transcriptional reprogramming that occurs during quiescence.

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Manuscript number: RC-2022-01536

Corresponding author(s): Michael Glotzer

[Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

If you wish to submit a preliminary revision with a revision plan, please use our "Revision Plan" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

1. General Statements We thank the reviewers for their thoughtful and helpful comments. In general, the reviews were highly positive, although their reviews indicated parts of the manuscript that needed further clarification. We have made extensive changes that improve the clarity and rigor of this submission. We have performed several additional experiments which have extended our analysis in several ways detailed below. None of the conclusions have changed.

The following is a list of eight major changes implemented during the revisions. Point-by-point responses to the reviewers comments follow on subsequent pages.

1. The reviews made clear that we needed to more explicitly discuss the AIR-1 depletion phenotype. This phenotype is complex, it does not result in a complete loss of asymmetry, unlike, for example, depletion of the centrosome component SPD-5. This is because, in AIR-1 depleted embryos, a PAR-2 and cortical flow-dependent pathway induces PAR-2 accumulation at both anterior and posterior poles that induces flows from each pole to the lateral region (Reich 2019, Kapoor 2019, Zhao 2019, Klinkert 2019; PMIDs 31155349, 31636075, 30861375, 30801250). These flows also modulate ECT-2 localization. To clarify this point which came up in multiple reviews, we now include an explanation of the complexity of the AIR-1 phenotype and we present an analysis of ECT-2 localization in embryos depleted of both AIR-1 and PAR-2.

In addition to the 95% confidence intervals that were present on our graphs, we now include indications of the results of statistical tests of significance to the results of different treatments.

We have revised the analysis ECT-2 accumulation in two ways. First, in the previous draft, we assessed the anterior accumulation over the anterior 40% and the posterior 15% of the embryo. We have revised this analysis comparing the anterior and posterior 20% of the cortex, respectively. This is simpler and more logical in contexts where embryos are symmetric. In addition, we altered the measurements of the length of the posterior boundary. Previously we used a common threshold value, below which we counted pixels to assess boundary length. During the revisions, we noticed that this value was not appropriate for our mutant transgenes which accumulated to higher levels. Therefore, we revised our analysis pipeline such that, for each embryo, we measure the average intensity of the cortex in the anterior 60% of the embryo. We set a threshold of 0.85* this average anterior intensity value. As before, cortical positions below this threshold contribute to the boundary length. This is a more robust and simpler means of evaluating the size of the posterior domain. Neither of these changes affect any of our conclusions, but they are simpler and more rigorous.

Most of our figures include quantification of the degree of ECT-2 asymmetry as well as the average anterior and posterior accumulation of ECT-2 as a function of time. While the images show the intensity profiles across the embryo, previously, we did not explicitly show a quantification of the average intensity of ECT-2 as a function of position along the embryo. A new graph, Figure 2Bv, shows this for control embryos and embryos in which tubulin is depleted and depolymerized. This shows that the MT depolymerization results in lower accumulation at the posterior of the embryo and higher accumulation at the anterior.

We provide documentary and quantitative evidence that ZYG-9 depletion induces potent cortical flows (Figure 3c and Figure 3, supplement 3), further bolstering the central role of cortical flows in inducing ECT-2 asymmetry.

As requested by reviewer 2 (R2b), we have included the analysis of ECT-2 distribution in Gα depleted embryos. As expected due to the lack of spindle elongation, the displacement of ECT-2 from the posterior cortex is greatly attenuated.

As requested by reviewer 2 (R2d), we now show that ECT-2C fragments accumulate on the cortex in embryos depleted of ECT-2.

One other important point raised by several reviewers concerns the behavior of the ECT-2 T634E allele. This allele, due to the substitution of a phosphomimetic residue, accumulates on the cortex at about 50% the level of the wild-type version. To investigate the possibility that this quantitative difference was the cause of the phenotype, we depleted both the wild-type and mutant ECT-2 constructs by RNAi (these are the sole sources of ECT-2 in the animals). First, we find that wild-type ECT-2 can be depleted to 20% of wild type levels with only a 13% rate of cytokinesis failure (when T634E is depleted to 20%, embryos fail more than 50% of the time). Thus the two-fold reduction in cortical ECT-2 seen in T634E not likely highly significant (ECT-2 is not haploinsufficient). In addition, embryos with ECT-2 T634E initiate ingression in a timely manner, but the furrows ingress more slowly than wild-type. In contrast, depletion of ECT-2 to 20% results in a delay in furrow initiation, but once these furrows form, they ingress at rates similar rates to wild-type. Thus, the T634E variant exhibits a behavior that is quite distinct from that resulting from a (strong) reduction in the levels of wild-type ECT-2.

Point-by-point description of the revisions

This section is mandatory. Please insert a point-by-point reply describing the revisions that were already carried out and included* in the transferred manuscript. *

(Reviewer comments: italicized 9 pt font, author response: plain text 10 pt font. Numbers have been added to the reviewer comments e.g. R2c=Reviewer 2, third comment)

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary

R1a* In this study the authors addressed how Ect2 localization is controlled during polarization and cytokinesis in the one-cell C. elegans embryo. Ect2 is a central regulator of cortical contractility and its spatial and temporal regulation is of uttermost importance. After fertilization, the centrosome induces removal of Ect2 from the posterior plasma membrane. During cytokinesis Ect2 activity is expected to be high at the cell equator and low at the cell poles. Similarly to polarization, the centrosome provides an inhibitory signal during cytokinesis that clears contractile ring components from the cell poles. Whether and how the centrosomes regulate Ect2 localization is not know and investigated in the study. *

This is an accurate summary of the goals of this study.

R1b *The authors start by filming endogenously-tagged Ect2 and find that Ect2 localizes asymmetrically, with high anterior and low posterior membrane levels during polarization and cytokinesis. They reveal that the centrosome together with myosin-dependent flows results in asymmetric Ect2 localization. Previous studies had suggested that Air1, clears Ect2 from the posterior during polarization and the authors expand those finding by showing that Air1 function is also required to displace Ect2 from the posterior membrane during cytokinesis. *

*To elucidate if Ect2 displacement is induced by phosphorylation of Ect2 by Air1, the authors investigate the localization of a C-terminal Ect2 fragment containing the membrane binding PH domain. When the predicted Air1 phosphorylation sites are mutated to alanine, the Ect2 fragment still localizes asymmetrically but exhibits increased membrane accumulation. *

*Finally, they investigate the functional role of Air-1 during furrow ingression. They demonstrate that embryos deficient of Air1 and NOP1 have impaired furrow ingression. Lastly, the authors sought to confirm that there is a direct effect of Air1 on Ect2 function by generating a phosphomimetic point mutation of Ect2 using Crispr. They find that the membrane localization of phosphomimetic Ect2 is reduced and consequently furrow ingression is impaired. *

This is an accurate summary of our results.

Major comments

R1c *It is not convincing that the six putative phosphorylation sites are targeted by the Air1. If Air1 phosphorylation displaces Ect2 from the membrane, a reduction in Ant/Post Ect2 ratio is expected in the phosphodeficient mutants, like after air1 RNAi. However this is not observed for cytokinesis or polarization (Fig. 5D(i); E). This suggests that phosphorylation of those sites is not essential for the asymmetric Ect2 localization. *

In otherwise wild-type embryos, phosphorylation of these sites is not required for asymmetric ECT-2 localization. Non-phosphorylatable ECT-2 variants exhibit asymmetric localization because these proteins relocalize due to myosin-directed flows. To test the role of phosphorylation, we examine the distribution of ECT-2 and ECT-2C fragments in myosin-depleted embryos in which the flows are blocked, under these conditions, transient local depletion is observed with the phosphorylatable variants, Fig 5E.

While AIR-1 promotes normal polarity establishment, as shown in several recent papers, cortical changes nevertheless occur in the absence of AIR-1. Specifically, a parallel PAR-2 dependent pathway induces weaker flows from both poles toward the equator. To further substantiate the effect of PAR-2 accumulation on ECT-2 accumulation in AIR-1 depleted embryos, we assayed ECT-2 accumulation in air-1(RNAi); par-2(RNAi) embryos (Figure 4, supplement 2). These results show that ECT-2 is nearly symmetric in these double depleted embryos. In addition we have edited the text to describe the unusual bi-polar PAR-2 accumulation that occurs in AIR-1 depleted embryos.

R1d *The authors aim to demonstrate that phosphorylation of the identified sites is important for cytokinesis. For this they investigate contractile ring ingression in the phosphomimetic point mutation. Since ring ingression is slower and fails in nop1 mutant they authors conclude that this demonstrates a functional importance of this site. I am not surprised that embryos ingress slower in this mutant since Ect2 localization to the membrane is reduced. This however does not show that this phosphorylation site is the target of the centrosome signal. Importantly, authors would need to demonstrate that Rho signaling and thus Ect2 activity, is increased at the poles, when phosphodeficient Ect2 is the only Ect2 in the embryo. *

The fact that a phosphomimetic residue at this site leads to reduced membrane localization is highly relevant, as we suggest that phosphorylation of this site contributes to the mechanism by which AIR-1 generates asymmetric ECT-2. Given the role of AIR-1 in regulating polarity, a version of ECT-2 that can not be phosphorylated would be predicted to be dominant lethal, necessitating a conditional expression strategy which does not currently exist in the early C. elegans embryo system (indeed we were unable to recover a T-> A allele at this site, despite extensive efforts). To avoid this issue, we used a viable, fertile, hypomorphic allele that is predicted to be less responsive to AIR-1 activity. The goal of this experiment was to evaluate whether the putative AIR-1 sites affect not only the NOP-1 pathway for furrow ingression, but also impact furrowing that is centralspindlin-dependent.

To complement this finding have performed experiments in which ECT-2 was partially depleted We used RNAi to partially deplete ECT-2 and ECT-2 T634E and measured the total embryo fluorescence of each ECT-2 variant and the kinetics of furrow ingression. Partial depletion of wt ECT-2, to ~ 20% of control levels leads to delay in furrow formation and all but 2/18 (11%) of embryos complete cell division. In contrast, a similar depletion of ECT-2T634E depletion results in a failure of furrow ingression in ~52 % of embryos. Furthermore, while ECT-2T634E embryos initiate furrowing with normal kinetics, they exhibit a slower rate of furrow ingression, in contrast, partial depletion of WT ECT-2 results in a delay in furrow initiation, but once initiated, the rate of furrow ingression is not significantly affected. These results demonstrate that ECT-2T634E behavior can not simply be explained by a modest reduction in membrane binding.

R1e *The authors use the Aurora A inhibitor MLN8237: It was shown prior (De Groot et al., 2015) that this inhibitor is not highly specific for Aurora A, and that it also inhibits Aurora B. Thus experiments need to be repeated with MK5108 or MK8745. They should also be conducted during polarization. Why does Aurora A inhibition not abolish asymmetry? That would be expected? *

The role of AIR-1 in symmetry breaking during polarization is previously published, including with chemical inhibitors (Reich 2019, Kapoor 2019, Zhao 2019, Klinkert 2019, PMID 31155349, 31636075, 30861375, 30801250). ECT-2 localization depends on both the spatial regulation of AIR-1 activity and the distribution of cortical factors that contribute to ECT-2 cortical association, as a result of cortical flows. During acute, chemical perturbation of AIR-1 it is likely that these factors, which were polarized prior to drug treatment, remain polarized, allowing the residual cortical ECT-2 to remain asymmetric. The reviewer is correct about the specificity of MLN8237 and we do not rely on it alone to demonstrate the role of AIR-1. Rather this experiment is a complement to our AIR-1 depletion studies, which are sufficient to establish specificity. We present this experiment merely to show that AIR-1 acutely regulates ECT-2 during cytokinesis in embryos that were entirely unperturbed during polarization.

R1f *There is no statistical analysis of the results in the entire study. For all claims stating a change in Ant/Post Ect2 ratio or Ect2 membrane localization selected time points should be statistically compared: for example the main point of Fig.1 is that Ect2 becomes more asymmetric during anaphase. Thus a statistical analysis of the Ect2 ratio at anaphase onset (t=0s) and eg. t=90 s after anaphase onset should be performed; or Fig. 3A nop-1 mutant Ant/Post Ect2 ratio during polarization: again statistical analysis of control and nop-1 mutant embryos is needed at a particular time point. *

All of the graphs were presented with the mean of ~10 embryos per condition and included the 95% confidence intervals. In the revised manuscript, we have included tests of statistical significance, at each time point. While non-overlapping confidence intervals generally suggest statistical significance, we include these analyses on the graphs as it can be difficult to assess statistical significance when the confidence intervals overlap.

R1g *The aim of Fig. 2B is to demonstrate that Ect2 localization is independent of microtubules, however they still observe some microtubules with the Cherry-tubulin marker and those are even very close to the membrane and therefore could very well influence Ect2 on the membrane. Therefore I am not convinced that this experiment rules out that microtubules have no role in regulating Ect2 localization. *

We do not exclude that microtubules play a contributing role in ECT-2 phosphoregulation, but rather we conclude that the primary cue is the centrosome. Indeed, microtubules can play an important role in controlling spindle positioning which affects the proximity of the centrosome to the cortex.

The manuscript states, “Despite significant depletion of tubulin and near complete depolymerization of microtubules (Figure 2B, insets), we observed strong displacement of ECT-2 from a broad region of the posterior cortex during anaphase (Figure 2B).” Thus, despite dramatic reductions in microtubules, not only does ECT-2 become polarized, it becomes hyperpolarized. In contrast, were microtubules directly involved in ECT-2 displacement, one would expect a reduction in polarization as a result microtubule depolymerization. Conversely, though SPD-5 depleted embryos contain far more microtubules than embryos in which microtubule assembly is suppressed, ECT-2 is not polarized in SPD-5 depleted embryos. Thus in the manuscript, we conclude, “Collectively, these studies suggest that ECT-2 asymmetry during anaphase is centrosome-directed.” This conclusion is well supported by the results shown.

R1h *Throughout the paper the authors should tone down their statement that Air1 breaks symmetry by phosphorylating Ect2, since phosphorylation of Ect2 by Air2 is not shown. *

We agree with this comment and will make the necessary edits to the text. Indeed, this is the reason why we had included the final section in our original draft, “Limitations of this study” which makes this point explicitly.

R1i *I understand that the establishment of Ect2 asymmetry is important for polarization. However, how does asymmetric Ect2 localization result in more active Ect2 at the cell equator, which is required for the formation of the active RhoA zone? Would we not expect an accumulation of Ect2 at the cell equator, or if that is not the case more active Ect2 at the equator versus the poles? *

The pseudocleavage furrow forms as a result of the anterior enrichment of active RHO-1 and its downstream effectors. There is no evidence for a local accumulation of active RHO-1 specifically at the site of the pseudocleavage furrow. Rather, this furrow forms at the boundary between the portion of the embryo where RHO-1 is active and the posterior of the embryo where RHO-1 is far less active (Figure 1 Supplement 2). We suggest that aster-directed furrowing during cytokinesis likewise results from asymmetric accumulation of the same components, without them necessarily being specifically enriched solely at the furrow.

While cytokinesis generally involves an equatorial contractile ring, furrow formation can be driven by an asymmetric - i.e. non-equatorial - accumulation of actomyosin. This behavior is exemplified during pseudocleavage during which the entire anterior cortex is enriched for actomyosin and the posterior is depleted of myosin (Figure 1 Supplement 2). Several published studies provide evidence that the asymmetric pattern of myosin accumulation contributes to cytokinesis (PMID 22918944, 17669650).

Minor comments

R1j *Can the authors explain why the quantification of Ant/Post Ect2 ratio in control embryos differs in different figures? For example: in Fig. 1D i) a slight increase of Ect2 asymmetry ratio is seen at around 80 s after anaphase onset. In comparison, in Fig. 2C (i) this increase is not obvious. Are those different genetic backgrounds? *

In figure 1 D, time 0 begins at anaphase onset, whereas in 2C, time 0 is specified at the time of nuclear envelope breakdown (NEBD). The duration between NEBD and anaphase onset is ~130 sec and an increase in ECT-2 polarization is observed at 220 s post NEBD, ie 90 sec post anaphase onset comparable to that seen in Fig 1D.

R1k *One key point of the paper is that myosin-dependent cortical flows amplify Ect2 asymmetry during polarization and cytokinesis. During polarization the data is convincing, however during cytokinesis Ect2 ratio is only slightly decreased after nmy-2 depletion, again is this decrease even significant? *

Figure 3 supplement 1 shows a significant difference in ECT-2 asymmetry between control and myosin-depleted embryos.

R1l *In the introduction: "Centralspindlin both induces relief of ECT-2 auto-inhibition and promotes Ect2 recruitment to the plasma membrane" it should be added 'Equatorial' membrane, since Ect2 membrane binding is, to my knowledge, not compromised in centralspindlin mutants or in Ect2 mutants that cannot bind centralspindlin. *

Generally speaking, the reviewer is correct that cortical accumulation of ECT-2 globally is centralspindlin independent. However, as seen in e.g. ZYG-9 depleted embryos, ECT-2 is recruited to the posterior cortex in a centralspindlin-dependent manner. Thus centralspindlin can promote ECT-2 accumulation to the cortex and the site of that accumulation will be dictated by the position of the spindle midzone.

R1m *Labels in the figures are often very small eg Fig. 1 ii-v) and difficult to read. In addition it is easier for the reader if the proteins shown in the fluorescent images is also labeled in the figure (eg Fig. 2B add NG-Ect2). *

These useful suggestions have been incorporated.

R1n *Material and methods it should be mentioned which IPTG concentration was used. *

The IPTG concentration (1 mM) has been added to the revised text.

R1o *The authors speculate that the Air1 phosphorylation sites in Ect2 PH domain prevent binding to phospholipid due the negative charge. At the same time, the authors propose that the PH domain binds to a more stable protein on the membrane, which is swept along with the cortical flows and they propose anillin could be that additional binding partner. I might miss something, but do the authors suggest Ect2 has two binding partners: anillin and the phospholipids? It would be necessary to explain this better. *

*The authors should test if anillin represents the suggested myosin II dependent Ect2 anchor. For this they should check if Ect2 localization to the membrane is altered upon on anillin RNAi. *

This summary of our model is largely correct, though we do not know the identity of the more stable cortical anchor(s). While we suspect the PH domain binds to a phospholipid, ECT-2 cortical localization also requires ~100 residues C-terminal to the PH domain. It is likely that this domain interacts with a cortical component.

In preliminary experiments, ECT-2 accumulation is not strictly anillin-dependent. However, functional redundancy may obscure a contribution of anillin. Anillin was mentioned simply because of the evidence for a physical interaction between ECT-2 and anillin (Frenete PMID 22514687). In the revised manuscript we also include the possibility that ECT-2 accumulations involves one or more anterior PAR proteins. The identity of the cortical anchor(s) is an interesting question for future studies. We consider this question beyond the scope of the current manuscript.

R1p *The title of fig. 3 does not fit the statement the authors want to make, since the key point is how Ect2 polarization is affected and not membrane localization in general. *

Thank you for this suggestion. The title has been changed to “Cortical flows contribute to asymmetric cortical accumulation of ECT-2”

R1q *In Fig 4A/C. After air1 depletion the authors observe a reduction in Ect2 asymmetry. Why are the centrosomes not marked in the figures? Because they cannot be detected? The authors would also need to show that the mitotic spindle and centrosomes are no altered by air1 RNAi in the zyg9 mutant. Otherwise the observed effect might be indirect. *

Centrosomes are perturbed by depletion of AIR-1 (Hannak, PMID 11748251), but they are still detectable and their positions will be added to figure 4. As has been extensively demonstrated, AIR-1 depletion does lead to attenuated spindles and defects in spindle assembly, some of which are also seen TPXL-1 depleted embryos. These consequences of AIR-1 depletion does complicate the analysis, but this is typical of factors that regulate many processes. This is one of the key reasons why we used ZYG-9 depletion in combination with AIR-1 depletion to overcome these indirect effects.

R1r *The authors state that tpxl-1 depletion attenuates Ect2 asymmetry, this is not seen in the quantification ((Fig. 4B(i)). The main phenotype they observe is that Ect2 levels on the membrane increase (Fig. 4 (ii) and (iii). They go on testing the function of tpxl1 by depleting tpxl1 in the zyg9 mutant, where the centrosomes are close to the posterior cortex. Here they see no effect on Ect2 asymmetry. Based on that they conclude that tpxl1 has no role in this process. To me this finding is not surprising since the centrosome is close the cortex in zyg9 mutant embryos. Therefore sufficient amounts of active Air1 could reach the membrane and displace Ect2. Thus an amplification of the inhibitory signal by tpxl1 on astral microtubules might not be required. The authors need to mention this possibility and tone down their statment (also in the discussion) that tpxl1 is not required for this process. *

In the text, we state, “Cortical ECT-2 accumulation is enhanced by TPXL-1 depletion, though the degree of ECT-2 asymmetry is unaffected (Figure 4B).… we observed robust depletion of ECT-2 at the posterior pole in zyg-9 embryos depleted of TPXL-1, but not AIR-1 (Figure 4C). We conclude that while AIR-1 is a major regulator of the asymmetric accumulation of ECT-2, the TPXL-1/AIR-1 complex does not play a central role in this process.” We consider this to be an accurate description of the results. In sum, we have found no evidence that TPXL-1 contributes to generating ECT-2 asymmetry, beyond its well established role in regulating spindle length and position. The are several other processes that are known to be AIR-1 dependent and TPXL-1 independent; these primarily involve the centrosome (Ozlu, PMID 16054030). Given that TPXL-1 associates with astral microtubules, the fact that microtubule depletion can enhance ECT-2 asymmetry also argues against a requirement for TPXL-1.

R1s *It was shown that the C-terminus of Ect2 is sufficient and the PH domain is required for Ect2 membrane localization in C. elegans (Chan and Nance, 2013; Gomez-Cavazos et al., 2020). Papers should be cited. *

Thank you for this helpful comment. Chan and Nance 2013 indeed shows that the ECT-2 C-term is sufficient to localize to the cell cortex. In contrast, the Gomez-Cavasos paper (PMID 32619481) shows in figure S2 that the PH domain is required for cortical localization of ECT-2; this paper does not focus extensively on cortical accumulation of ECT-2. We have cited Chan and Nance in the revised manuscript.

R1t *The authors find that nmy-2 depletion results in loss of asymmetry for the Ect2 C-term and Ect2 3A fragment during polarization. Why is the same experiment not shown for cytokinesis? *

Strong depletion of NMY-2 prevents polarity establishment, resulting in symmetric spindles, which in turn results in symmetric ECT-2 accumulation. Thus, the requested experiment would not provide significant additional information.

R1u *Air1 is targeted to GFP-C-term Ect2 fragment via GFP-binding to determine the influence on GFP-C-term Ect2 localization (Fig. 5F). They state that they see a reduction of Ect2 C-term but not of C-term 3A after targeting. The reader has to compare Fig. 5D with F. Since the differences are not big, they need to compare the Ect2 C-term and Ect2 C-term 3A with and without Air1 targeting in the same graph (plus statistics). Otherwise this statement is not convincing. *

It is not straightforward to directly compare ECT-2C in the presence and absence of GBP-mCherry-AIR-1, because the GBP:AIR-1 fusion protein recruits a large fraction of ECT-2C to the centrosome. For this reason we think it is best to compare the behavior over time of ECT-2C and ECT-2C3A in the presence of GBP-mCherry-AIR-1. At the onset of anaphase, these two fragments localize similarly, but they then diverge over time.

R1v *In Fig. 6A the authors determine the contribution of air1 to furrowing. For this they deplete air1 in the nop1 mutant. According to previous studies, air1 mutants have a monopolar spindle. How can the authors analyze the function of air1 in cytokinesis when the spindle is monopolar? Did the authors do partial air1 depletion? They authors need to show that there is not major effect on the spindle and centrosome for their conditions. For comparison air1(RNAi) alone has to be included, otherwise the experiment is not conclusive. *

AIR-1 depletion does not result in a monopolar spindle in C. elegans embryos, though the spindle is attenuated and disorganized (PMID 9778499). TPXL-1 depletion also results in short, well organized spindles (PMID 19889842). The concerns are the reason we performed the ZYG-9 depletion experiments in Figure 4C to ensure the centrosomes are proximal to the cortex.

R1w *Upon air1(RNAi) in the nop1 mutant NMY2 intensity seems decreased and not increased. Can the authors comment on that, since that is opposite of what is expected. *

This is expected as previous studies have shown that NOP-1 contributes to RHO-1 activation during polarization and cytokinesis (Tse, PMID 22918944). (NOP stands for No Pseudocleavage).

R1x *In Fig 6B they introduce a phosphomimetic point mutation in S634 [sic, T634] in the endogenous Ect2 locus. It not clear why the authors chose this site out of the six putative sites and why they only chose one and not 3 or 6 sites? This needs some explanation. *

In our early work with ECT-2 transgenes, we found that a T634E mutation strongly affected cortical ECT-2C, so we decided to assess its affect on the function and localization of endogenous ECT-2. While we were able to recover a T634E variant, we were not able to recover a T634A variant, despite considerable effort. Based on these experiences, we anticipated that we would be unable to recover a mutant version of ECT-2 in which all sites were changed to phosphomimetic.

R1y *In the model (fig. 7) no astral microtubules are shown during pronuclear meeting and metaphase. Astral microtubules are present at this stage and should be added to the schematic. *

MTs will be added to the figure.

Reviewer #1 (Significance (Required)):

R1z *The centrosomes inhibit cortical contractility during polarization and cytokinesis in the one-cell C. elegans embryo. Centrosome localized Air1 was proposed to be part of this inhibitory signal, however the phosphorylation target of Air1 is not known. The identification of Ect2 as a phosphorylation target of Air1 would be a great advancement in the field. However, the presented manuscript lacks convincing data that Ect2 is the phosphorylation target of Air1 during polarization and cytokinesis. *

We explicitly acknowledge that we have not directly shown that AIR-1 phosphorylates ECT-2. However, we have shown that (i) AIR-1 inhibits cortical ECT-2 localization, (ii) the negative regulator of AIR-1, SAPS-1, promotes AIR-1 cortical accumulation, (iii) that the cortical localization domain of ECT-2 has putative AIR-1 sites, which, when mutated to non-phosphorylatable residues leads to increased cortical accumulation of ECT-2 (and (iv) phosphomimetic residues reduce its cortical accumulation), and (v) that these AIR-1 sites are required to render GFP-ECT-2C responsive to GBP-AIR-1. For these reasons we feel that our data makes a strong, albeit indirect, case that AIR-1 regulates ECT-2, even though we clearly acknowledge that we do not directly show that AIR-1 directly phosphorylates ECT-2.

Direct proof would require the demonstration that AIR-1 phosphorylates ECT-2 in vivo. This would be difficult to show as ECT-2 phosphorylation is likely transient, it likely affects only a subset of the total ECT-2 pool, and it likely results in loss of membrane association of ECT-2. As it it not possible to synchronize C. elegans embryos, biochemical analysis would be very difficult. Even a phosphospecific antibody for the putative ECT-2 phosphosites might not be particularly informative, as it would be predicted to give a diffuse cytoplasmic signal.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

R2a* In this work, Longhini and Glotzer investigate the localization of an essential regulator of polarity and cytokinesis, RhoGEF ECT-2, in the one-cell C. elegans embryo. The authors show that centrosome localized Aurora A kinase (AIR-1 in C. elegans) and myosin-dependent cortical flows are critical in asymmetric ECT-2 accumulation at the membrane. Since membrane interaction of ECT-2 is dependent on the Pleckstrin homology domain present at the C-terminus of ECT-2, they further analyzed the importance of putative AIR-1 consensus sites present in this domain. The authors linked the relevance of these sites in controlling ECT-2 localization and its significance on cytokinesis. The manuscript is well written, the work is interesting, and the data quality is high. *

We thank the reviewer for their critique.

Major comments:

R2b - In Fig. 2, the authors claim that the centrosomes and the position of the mitotic spindle are critical in regulating the asymmetric enrichment of ECT-2 at the membrane. To test the relevance of spindle positioning on ECT-2 localization, the authors depleted PAR-3 and PAR-2. The authors observed that the ECT-2 asymmetry is affected in these settings. However, PAR-3 or PAR-2 depletion impacts polarity, which is critical for many cellular processes, including spindle positioning. Can the authors try to specifically misposition the spindle without affecting polarity? For instance, by depleting Galpha/GPR-1/2 and assessing the impact of such depletion on ECT-2 localization.

Thank reviewer for good suggestion. We have performed the suggested experiment (presented in Figure 2, supplement 2). As one might predict, ECT-2 starts out polarized as Gα is not required for polarity establishment. During anaphase, ECT-2 becomes more symmetric in Gα depleted embryos as compared to wild-type.

R2c *-I wonder why the intensity of ECT-2 at the anterior and posterior membrane decreases in air-1(RNAi) post anaphase onset (Fig. 4A)? Moreover, I fail to observe a significant asymmetric distribution of ECT-2 in embryos depleted for PERM-1. Therefore it appears that the difference between DMSO and MLN8237-treated embryos is not substantial (at least in the images)? *

We do not have a complete or rigorous explanation for all the changes in cortical ECT-2, but they are highly reproducible. We speculate that there are cell cycle regulated changes in ECT-2 accumulation, in addition to its regulation by AIR-1. For example, in figure 1, a strong reduction in both anterior and posterior cortical ECT-2 is evident beginning at approximately -350 sec, which may reflect the initial stages of Cdk1 activation. This may result from cell cycle regulated modulation of ECT-2, as there is evidence that mammalian ECT-2 is subject to a very potent inhibition membrane association by Cdk1 (PMID 22172673). Alternatively, there could be cell cycle modulation of the cortical factor that serves as the “co-anchor” of ECT-2. The ability of GBP-AIR-1 to induce GFP-ECT-2C dissociation also appears cell cycle regulated.

Consistent with a cell cycle regulated component, note that NEBD is delayed in AIR-1 depleted embryos (PMID 17669650, 17419991, 30861375). This delay results in a shorter interval between NEBD and e.g. the peak in Cdk1 activation, explaining the earlier decrease in AIR-1(RNAi) embryos vs. control, relative to NEBD.

Our quantitative analysis indicates a significant increase of cortical ECT-2 upon treatment with MLN8237. In addition, the quantitation in the previous version did show a significant polarization of ECT-2 in PERM-1-depleted embryos prior to treatment. We have revised this figure to simply show an acute increase in cortical ECT-2 upon drug treatment, as the focus of this experiment was solely to show that ECT-2 cortical accumulation is acutely responsive to chemical inhibition during cytokinesis in otherwise normal embryos.

*-The data in Fig. 5 and 6 are exciting but raise a few concerns: *

R2d *a). The authors show that ECT-2C localization mimics the localization of endogenous tagged ECT-2. However, all these analyses with ECT-2C and various mutants are performed in the presence of endogenous ECT-2. Can the author check the localization of these mutant strains in conditions where the endogenous proteins are depleted? I understand that the cortical flow would be perturbed in conditions where endogenous ECT-2 is depleted. However, I suspect that one can analyze the anaphase-specific distribution. *

We have examined ECT-2C localization in embryos depleted of ECT-2. Cortical localization of ECT-2C is not dependent upon endogenous ECT-2. This result is now shown in figure 5 supplement 1. However, as the reviewer suggested, embryos depleted of ECT-2 do not show a high degree of ECT-2C asymmetry as ECT-2 is required for the cortical flows that amplify the symmetry breaking during polarization. During cytokinesis, ECT-2C does show a modest change in localization at the poles; the extent of the polar reduction is limited and the changes are symmetric as ECT-2 displacement causes spindles to be symmetrically positioned and limits their elongation during anaphase.

R2e *b). Can the author comment on why ECT-2C does not accumulate at a similar level as ECT-2C(3A or 6A) at the cell membrane when AIR-1 is depleted (compare Fig. 5D with Supplemental Fig. 5)? *

When ECT-2C(3A or 6A) are expressed in otherwise wild-type embryos, embryo polarization occurs, resulting in anterior-directed flows that concentrate the factor(s) that enables the anterior enrichment of ECT-2 (and ECT-2C 3A/6A). By contrast, when AIR-1 is depleted, most embryos exhibit a “bipolar” phenotype in which PAR-2 is recruited to both anterior and posterior poles, and the actomyosin network becomes somewhat concentrated laterally (PMID 30801250, 30861375, 31636075). The differential positioning of the actomyosin network in AIR-1 depleted embryos is likely responsible for the interesting difference that the reviewer points out. This section of the results states. “Nevertheless, these variants accumulated in an asymmetric manner. ECT-2C asymmetry temporally correlated with anteriorly-directed cortical flows (Figure 5 D,E), raising the possibility that asymmetric accumulation of endogenous ECT-2 drives flows that cause asymmetry of the transgene, irrespective of its phosphorylation status.”

R2f *c). Does the cortical localization of the ECT-2C(6A) mutant become symmetric upon further depletion of AIR-1? Of course, if the asymmetric distribution of ECT-2C(6A) is dependent on the presence of endogenous protein in the cellular milieu, the point raised earlier will help address this concern. *

We have not performed this exact experiment with ECT-2C-3A though we have performed it with a longer ECT-2 C-terminal fragment (aa 559-924). As expected, due to the considerations described above, the asymmetry of ECT-2C-3A is reduced when AIR-1 is depleted. Likewise, ECT-2C-6A is becomes symmetric when endogenous ECT-2 is depleted due to the dependence of its asymmetry on cortical flows, as discussed above.

In the revised manuscript, we provide additional explanation of the AIR-1 depletion phenotype which will explain the origin of the asymmetric distribution of ECT-2.

R2g *d). The authors predict that the AIR-1 mediated phosphorylation delocalizes ECT-2 from the polar region of the cell cortex. Since the posterior spindle pole is much closer to the posterior cortical region, the delocalization is much more robust at the posterior cell membrane. I wonder why targetting AIR-1 at the membrane (GBP-mCherry-AIR-1) does not entirely abolish GFP-ECT-2C membrane localization? Can the author include the localization of GBP-mCherry-AIR-1 in the data? Also, do we know for sure if GBP-mCherry-AIR-1 is kinase active? *

The GBP-mCherry-AIR-1 transgene was obtained from the Gönczy lab which demonstrated that it has some activity (PMID 30801250). Given that centrosomal AIR-1 (as compared to astral AIR-1) is the primary pool of AIR-1 responsible for modulating cortical ECT-2 levels, it is a not clear that the GBP-fused form of AIR-1 is as active as the centrosomal pool of AIR-1; indeed we suspect it is significantly less active, similar to the manner in which TPXL-1/AIR-1 appears less active towards ECT-2 than centrosomal AIR-1. Indeed as the reviewer suggests, were this pool of AIR-1 highly active, we would expect that its cortical recruitment would preclude embryo polarization, and this transgene would cause lethality when expressed with a GFP-tagged cortical protein. These concerns notwithstanding, we do observe a specific reduction in the anterior accumulation of ECT-2C as compared to ECT-2C3A, suggesting that this form of the kinase has some ability to modulate ECT-2C.

Co-expression of GFP-ECT-2C with GBP-mCherry-AIR-1 induces the centrosomal/astral accumulation of GFP-ECT-2C, which is highly visible in the figure and not seen in the absence of GBP-mCherry-AIR-1. Not surprisingly, the co-expression also induces a cortical pool of GBP-mCherry-AIR-1 that is not seen in the absence of GFP-ECT-2C. These redistributions indicate formation of the complex between GFP-ECT-2C and GBP-mCherry-AIR-1. The mCherry-AIR-1 images could be added as insets to the figure, but in our opinion, they would not make a substantive contribution, given the dramatic accumulation of centrosomal GFP-ECT-2C.

R2h *e). The authors show that centrosomal enriched AIR-1 [spd-5(RNAi)], but not the astral microtubules localized AIR-1 [tpxl-1(RNAi)], is vital for ECT-2 membrane localization. Interestingly, the authors showed that AIR-1 acts in the centralspindlin-directed furrowing pathway (Fig. 6A). I wonder if the authors can combine NOP-1 depletion with TPXL-1 depletion? I guess this will further help to exclude the function of TPXL-1 in the centralspindlin-directed furrowing pathway. *

We would like to clarify that our data indicates that AIR-1 acts on both the centralspindlin-independent furrowing (e.g. the anterior furrow in 4C), as well as centralspindlin-dependent furrowing (Figure 6).

While the experiment the reviewer proposes appears simple in theory, the interpretation is potentially a bit more complex, due to the role of TPXL-1 in spindle elongation, which can affect centralspindlin-directed furrowing. That said, there are two published experiments and one experiment in the manuscript that indicate that centralspindlin dependent furrowing can occur in TPXL-1 depleted embryos. First, Lewellyn et. al. showed that while tpxl-1(RNAi) embryos furrow, tpxl-1(RNAi); zen-4(RNAi) embryos do not, suggesting centralspindlin can function in the absence of TPXL-1. Second, the same paper shows that embryos doubly depleted of TPXL-1 and GPR-1/2 exhibit multiple furrows. Our previous work has shown that furrowing in Galpha-depleted embryos is centralspindlin dependent (Dechant and Glotzer). Furthermore, in the current manuscript we found that embryos depleted of both TPXL-1 and ZYG-9 form posterior furrows (8/8 embryos, 6/8 furrows were strong furrows) although the appearance of these furrows is delayed, presumably due to the reduction in spindle elongation due to TPXL-1-depletion. As described in the manuscript, these posterior furrows have been previously shown to be centralspindlin dependent and NOP-1 independent.

In accordance with these results, and in direct response to the reviewer’s specific suggestion, we do observe furrowing in nop-1(it142); TPXL-1(RNAi) embryos (10/10 embryos furrow, 9/10 complete cytokinesis) . Thus, all of the available results indicate that TPXL-1 is largely dispensable for centralspindlin dependent furrowing. However, the role of TPXL-1 in centralspindlin-dependent furrowing is not a focus of the manuscript, thus we do not favor including this result, as it distracts from the primary focus of the study.

R2i *f). Why do NMY-2-GFP cortical levels appear lower in 30% of the embryos that show various degrees of cytokinesis defects (Fig. 6A)? *

There are a number of possible origins of the variability. As shown in (Reich 2019, Kapoor 2019, Zhao 2019, Klinkert 2019, PMID 31155349, 31636075, 30861375, 30801250), AIR-1 depletion results in variable polarization (unpolarized PAR-2, bipolarized PAR-2, anterior PAR-2, posterior PAR-2). Furthermore, spindles in AIR-1 depleted embryos exhibit somewhat variable positioning. While we were unable to correlate these sources of variability with furrow formation, these results demonstrate that AIR-1 depletion impairs furrowing directed by centralspindlin, which was not entirely expected, given that (i) AIR-1 depletion potently suppresses NOP-1 dependent flows of cortical myosin, as evidenced by the loss of an anterior furrow in AIR-1(RNAi); nop-1(it142) embryos and (ii) centralspindlin directed furrowing can occur in the posterior in ZYG-9 depleted embryos both in the presence or absence of AIR-1 (Figure 4C).

R2j *g). The authors report that phosphomimetic mutation at the phospho-acceptor residue in ECT-2 impacts its cortical accumulation. This strain, together with NOP-1 depletion, affects furrow ingression. One explanation for this phenotype is that phosphomimetic mutant weakly accumulates at the membrane. However, one interesting observation is that ECT-2T634E enriches at the central spindle (Fig. 6B, panel 120 sec), which somehow I could not find in the text. Could this additional localization of ECT2 at the central spindle contribute to the cytokinesis defects that the authors have observed? The microscopy images the authors have included show that ECT-2T634E significantly localizes at the equator at the time of furrow initiation. Can the authors add the localization of ECT2 wild-type and ECT-2T634E in NOP-1 depleted conditions where they see an apparent impact on the cytokinesis? Similarly, if the authors include the localization of NMY-2 in these conditions-it will further add more weightage to the data. *

We regularly detect trace amounts of ECT-2 on the central spindle and this is slightly enhanced at in the ECT-2T634E mutant. However, given the large cytoplasmic pool of ECT-2, it seems unlikely that the slight enrichment of ECT-2 on the central spindle significantly affects the cortical pool of ECT-2, though the reduction in cortical ECT-2 may facilitate its enrichment on the central spindle.

As shown in figure 3B, depletion of NOP-1 does not dramatically affect cortical ECT-2 levels in wild-type embryos. Likewise, we did not observe a significant effect of NOP-1 depletion in ECT-2 T634E, thus we decided not to include this negative result.

As discussed in general point 8, we suggest the modest reduction in the membrane pool of ECT-2 is unlikely to be the primary cause of the T634E, but rather the ability of AIR-1 to modulate induce its relocalization. Consistent with this interpretation, the embryos that failed ingression tended to have more symmetric spindles, which could limit the residual cortical flows that facilitate furrow ingression.

Minor comments:

R2k -An explanation of how the timing of NEBD was analyzed in multiple settings would be helpful.

Depending on the experiment, we used either ECT-2:mNG fluorescence (it is excluded from the nucleus until NEBD) and/or the Nomarski images to score NEBD.

R2l ____-*The authors mentioned on p. 6-'Despite significant depletion of tubulin.....during anaphase'. These experiments are performed in the near complete depolymerization of microtubules; thus, regular anaphase will not establish. I understand that the authors are monitoring localization wrt the timing similar to anaphase in the non-perturbed condition, and thus a bit of change in the sentence is required. *

Thank you for highlighting this point. We have substituted “following mitotic exit” for “anaphase”. In these images, mitotic exit can be scored by the emergence of contractility.

R2m*-After testing the relevance of SPD-5 (that primarily acts on PCM and not on centrioles)-the authors write on p. 6 that 'two classes of explanation...early embryo'. I did not understand the importance of this sentence here. *

To clarify, we deleted the words “classes of” from the sentence in question and following that sentence we added the word, “first” indicating that we were explaining the first of the two possible explanations

R2n*-The observed impact of spd-5 (RNAi) on ECT-2 localization could be because of the effects of SPD-5 depletion on centrosomal AIR-1? The authors can link the impact of SPD-5 depletion not only with the centrosome but also with AIR-1 in the discussion. *

Indeed, it is well established that SPD-5 is required for centrosomal AIR-1 (Hamill DR, et. Al Dev Cell (2002). The revised discussion now states, “Specifically, during both processes, ECT-2 displacement requires the core centrosomal component SPD-5, which is required to recruit AIR-1 to centrosomes{Hamill et al., 2002, #1201}, but ECT-2 displacement is not inhibited by depolymerization of microtubules and it does not require the AIR-1 activator TPXL-1 (see below).”

R2o-In the various Figure legends, sometimes the authors mention time '0' as anaphase, and other time as anaphase onset.

In all cases, anaphase onset was intended and the legends will be corrected.

Reviewer #2 (Significance (Required)):

R2p *The manuscript is well written, the work is interesting, and the data quality is of good quality. *

We thank the reviewer for their encouragement as well as for their thoughtful critique!

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

R3a* Symmetry breaking is the process by which uniformity of the system is broken. Many biological systems, such as the body axes establishment and cell divisions in embryos, undergo symmetry breaking to pattern cellular interior design. C. elegans zygote has been a classic model system to study the molecular mechanism of symmetry breaking. Previous studies demonstrated critical roles of centrosomes and microtubules in breaking symmetry in the actin cytoskeleton during anterior-posterior polarization and cytokinesis. It, however, remains elusive how centrosomes and/or microtubules regulate the assembly and contractility of the actin cytoskeleton. Recent reports identified Aurora-A AIR-1 as the key centrosomal kinase that suppresses the function of the actin cytoskeleton, but little is known about a substrate of the kinase during symmetry breaking events. *

Longhini and Glotzer proposed in this manuscript that RhoGEF ECT-2 plays a critical role in symmetry breaking of the actin cytoskeleton under the control of AIR-1 kinase. Kapoor and Kotak (2019) previously proposed the same GEF as a downstream effector of centrosomes, but this work did not provide direct evidence for ECT-2 as the AIR-1 effector. This manuscript identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. Although this manuscript lacks direct in vivo and in vitro evidence for phosphorylation of ECT-2 by AIR-1 kinase, the above findings reasonably support a model where in AIR-1 promotes the local inhibition of ECT-2 on the cortex. Design of the experiments, the quality of images, and data analysis are reasonable, and the main text was written very well. The main conclusion of this work will attract many readers in cell and developmental biology fields. I basically support its publication in the journals supported by Review Commons with minor revisions (see below).

We thank the reviewer for their encouraging remarks and helpful comments.

Minor comments

R3b 1) In Figures 2A and 2B, the authors claimed apparent correlation between spindle rocking and ECT-2 displacement. However, because both MTs and ECT-2 in Fig2AB images are blur, I cannot convince myself whether ECT-2 intensities on the cortex showed negative correlation with the distance between the posterior centrosome and the cortex. The authors may want to provide quantitative data set and use a statistical test to support this conclusion.

Only figure 2A focuses on the rocking. The important structure to assess is the position of the centrosome, as the astral arrays of microtubules are largely radially symmetric (except towards the spindle midzone). As this point in the manuscript were were not discriminating between the astral microtubules and the centrosomes, rather focusing on the overall position of the aster as a whole. Figures 2B, 2D, Fig 2 Supplements 1 and 2, Fig 3C, and Fig 4B, summarized in figure 7A provide quantitive evidence that the centrosome-cortex distance is an important determinant of ECT-2 cortical accumulation.

R3c *2) Figure 2D would [sic; presumably should] show a ratio between the anterior/posterior pole and the lateral cortex. *

The reviewer is presumably noticing that the lateral cortex is brighter than the poles when PAR-3 is depleted. While we agree with this assessment, the point of this experiment was to evaluate whether both centrosomes are equally capable of regulating cortical ECT-2 at the respective poles. It appears to us that comparing the anterior and posterior poles is the appropriate measurement to make to address this point and comparison of the poles to the lateral cortex in par-3(RNAi) vs control would be confusing to readers.

R3d *3) In Figure 3D, the authors need to clarify why they measured ECT-2 dynamics only within the "anterior pole". It would be reasonable to measure ECT-2 dynamics by FRAP and cortical high-speed live imaging on the posterior and the lateral cortex during symmetry breaking. *

We measured ECT-2 recovery at a variety of sites with similar recovery kinetics. The comparison of ECT-2 dynamics on anterior and posterior furrows were shown in order to compare ECT-2 dynamics on centralspindlin-dependent and -independent furrows.

We now provide additional supplemental data on ECT-2 dynamics during symmetry breaking. When ECT-2 is polarized, the residual signal is too low to obtain a measure of its recovery.

R3e 4) In Figure 4 supplement, a difference between with or without ML8237 seems marginal. The authors need to show a statistical test to claim "rapid enhancement of cortical ECT-2 after ML8237 treatment".

We will provide a statistical analysis. As the inhibitor affects ECT-2 globally, the anterior/posterior ratio doesn’t change significantly. To avoid confusion, we now present total cortical ECT-2 levels upon anaphase onset in this experiment as this is the most relevant parameter.

R3f *5) I would strongly suggest the authors to clearly state in the first paragraph of discussion that "this working hypothesis is not supported by direct evidence for phosphorylation of ECT-2 by AIR-1 kinase in vitro and in vivo." It should be reasonable to weaken the statement "by Aurora A-dependent phosphorylation of the ECT-2 PH domain" in p13. *

We agree with the underlying sentiment (as indicated by the “limitations” section that was present in the original version) and we have revised these sentences accordingly: “Our studies suggest that asymmetric, posteriorly-shifted, spindle triggers an initial focal displacement of ECT-2 from the posterior cortex by Aurora A-dependent phosphorylation of the ECT-2 PH domain, though the evidence for this phosphorylation event is indirect.”

Reviewer #3 (Significance (Required)):

*See the second paragraph of the Evidence, Reproducibility, and Clarity section. *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Symmetry breaking is the process by which uniformity of the system is broken. Many biological systems, such as the body axes establishment and cell divisions in embryos, undergo symmetry breaking to pattern cellular interior design. C. elegans zygote has been a classic model system to study the molecular mechanism of symmetry breaking. Previous studies demonstrated critical roles of centrosomes and microtubules in breaking symmetry in the actin cytoskeleton during anterior-posterior polarization and cytokinesis. It, however, remains elusive how centrosomes and/or microtubules regulate the assembly and contractility of the actin cytoskeleton. Recent reports identified Aurora-A AIR-1 as the key centrosomal kinase that suppresses the function of the actin cytoskeleton, but little is known about a substrate of the kinase during symmetry breaking events.

Longhini and Glotzer proposed in this manuscript that RhoGEF ECT-2 plays a critical role in symmetry breaking of the actin cytoskeleton under the control of AIR-1 kinase. Kapoor and Kotak (2019) previously proposed the same GEF as a downstream effector of centrosomes, but this work did not provide direct evidence for ECT-2 as the AIR-1 effector. This manuscript identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. Although this manuscript lacks direct in vivo and in vitro evidence for phosphorylation of ECT-2 by AIR-1 kinase, the above findings reasonably support a model where in AIR-1 promotes the local inhibition of ECT-2 on the cortex. Design of the experiments, the quality of images, and data analysis are reasonable, and the main text was written very well. The main conclusion of this work will attract many readers in cell and developmental biology fields. I basically support its publication in the journals supported by Review Commons with minor revisions (see below).

Minor comments

1. In Figures 2A and 2B, the authors claimed apparent correlation between spindle rocking and ECT-2 displacement. However, because both MTs and ECT-2 in Fig2AB images are blur, I cannot convince myself whether ECT-2 intensities on the cortex showed negative correlation with the distance between the posterior centrosome and the cortex. The authors may want to provide quantitative data set and use a statistical test to support this conclusion.
2. Figure 2D would show a ratio between the anterior/posterior pole and the lateral cortex.
3. In Figure 3D, the authors need to clarify why they measured ECT-2 dynamics only within the "anterior pole". It would be reasonable to measure ECT-2 dynamics by FRAP and cortical high-speed live imaging on the posterior and the lateral cortex during symmetry breaking.
4. In Figure 4 supplement, a difference between with or without ML8237 seems marginal. The authors need to show a statistical test to claim "rapid enhancement of cortical ECT-2 after ML8237 treatment".
5. I would strongly suggest the authors to clearly state in the first paragraph of discussion that "this working hypothesis is not supported by direct evidence for phosphorylation of ECT-2 by AIR-1 kinase in vitro and in vivo." It should be reasonable to weaken the statement "by Aurora A-dependent phosphorylation of the ECT-2 PH domain" in p13.

Significance

See the second paragraph of the Evidence, Reproducibility, and Clarity section.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In this work, Longhini and Glotzer investigate the localization of an essential regulator of polarity and cytokinesis, RhoGEF ECT-2, in the one-cell C. elegans embryo. The authors show that centrosome localized Aurora A kinase (AIR-1 in C. elegans) and myosin-dependent cortical flows are critical in asymmetric ECT-2 accumulation at the membrane. Since membrane interaction of ECT-2 is dependent on the Pleckstrin homology domain present at the C-terminus of ECT-2, they further analyzed the importance of putative AIR-1 consensus sites present in this domain. The authors linked the relevance of these sites in controlling ECT-2 localization and its significance on cytokinesis. The manuscript is well written, the work is interesting, and the data quality is high.

Major comments:

• In Fig. 2, the authors claim that the centrosomes and the position of the mitotic spindle are critical in regulating the asymmetric enrichment of ECT-2 at the membrane. To test the relevance of spindle positioning on ECT-2 localization, the authors depleted PAR-3 and PAR-2. The authors observed that the ECT-2 asymmetry is affected in these settings. However, PAR-3 or PAR-2 depletion impacts polarity, which is critical for many cellular processes, including spindle positioning. Can the authors try to specifically misposition the spindle without affecting polarity? For instance, by depleting Galpha/GPR-1/2 and assessing the impact of such depletion on ECT-2 localization.
• I wonder why the intensity of ECT-2 at the anterior and posterior membrane decreases in air-1 (RNAi) post anaphase onset (Fig. 4A)? Moreover, I fail to observe a significant asymmetric distribution of ECT-2 in embryos depleted for PERM-1. Therefore it appears that the difference between DMSO and MLN8237-treated embryos is not substantial (at least in the images)?
• The data in Fig. 5 and 6 are exciting but raise a few concerns:
  • a). The authors show that ECT-2C localization mimics the localization of endogenous tagged ECT-2. However, all these analyses with ECT-2C and various mutants are performed in the presence of endogenous ECT-2. Can the author check the localization of these mutant strains in conditions where the endogenous proteins are depleted? I understand that the cortical flow would be perturbed in conditions where endogenous ECT-2 is depleted. However, I suspect that one can analyze the anaphase-specific distribution.
  • b). Can the author comment on why ECT-2C does not accumulate at a similar level as ECT-2C(3A or 6A) at the cell membrane when AIR-1 is depleted (compare Fig. 5D with Supplemental Fig. 5)?
  • c). Does the cortical localization of the ECT-2C(6A) mutant become symmetric upon further depletion of AIR-1? Of course, if the asymmetric distribution of ECT-2C(6A) is dependent on the presence of endogenous protein in the cellular milieu, the point raised earlier will help address this concern.
  • d). The authors predict that the AIR-1 mediated phosphorylation delocalizes ECT-2 from the polar region of the cell cortex. Since the posterior spindle pole is much closer to the posterior cortical region, the delocalization is much more robust at the posterior cell membrane. I wonder why targetting AIR-1 at the membrane (GBP-mCherry-AIR-1) does not entirely abolish GFP-ECT-2C membrane localization? Can the author include the localization of GBP-mCherry-AIR-1 in the data? Also, do we know for sure if GBP-mCherry-AIR-1 is kinase active?
  • e). The authors show that centrosomal enriched AIR-1 [spd-5(RNAi)], but not the astral microtubules localized AIR-1 [tpxl-1(RNAi)], is vital for ECT-2 membrane localization. Interestingly, the authors showed that AIR-1 acts in the centralspindlin-directed furrowing pathway (Fig. 6A). I wonder if the authors can combine NOP-1 depletion with TPXL-1 depletion? I guess this will further help to exclude the function of TPXL-1 in the centralspindlin-directed furrowing pathway.
  • f). Why do NMY-2-GFP cortical levels appear lower in 30% of the embryos that show various degrees of cytokinesis defects (Fig. 6A)?
  • g). The authors report that phosphomimetic mutation at the phospho-acceptor residue in ECT-2 impacts its cortical accumulation. This strain, together with NOP-1 depletion, affects furrow ingression. One explanation for this phenotype is that phosphomimetic mutant weakly accumulates at the membrane. However, one interesting observation is that ECT-2T634E enriches at the central spindle (Fig. 6B, panel 120 sec), which somehow I could not find in the text. Could this additional localization of ECT2 at the central spindle contribute to the cytokinesis defects that the authors have observed? The microscopy images the authors have included show that ECT-2T634E significantly localizes at the equator at the time of furrow initiation. Can the authors add the localization of ECT2 wild-type and ECT-2T634E in NOP-1 depleted conditions where they see an apparent impact on the cytokinesis? Similarly, if the authors include the localization of NMY-2 in these conditions-it will further add more weightage to the data.

Minor comments:

• An explanation of how the timing of NEBD was analyzed in multiple settings would be helpful.
• The authors mentioned on p. 6-'Despite significant depletion of tubulin.....during anaphase'. These experiments are performed in the near complete depolymerization of microtubules; thus, regular anaphase will not establish. I understand that the authors are monitoring localization wrt the timing similar to anaphase in the non-perturbed condition, and thus a bit of change in the sentence is required.
• After testing the relevance of SPD-5 (that primarily acts on PCM and not on centrioles)-the authors write on p. 6 that 'two classes of explanation...early embryo'. I did not understand the importance of this sentence here.
• The observed impact of spd-5 (RNAi) on ECT-2 localization could be because of the effects of SPD-5 depletion on centrosomal AIR-1? The authors can link the impact of SPD-5 depletion not only with the centrosome but also with AIR-1 in the discussion.
• In the various Figure legends, sometimes the authors mention time '0' as anaphase, and other time as anaphase onset.

Significance

The manuscript is well written, the work is interesting, and the data quality is of good quality.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary

In this study the authors addressed how Ect2 localization is controlled during polarization and cytokinesis in the one-cell C. elegans embryo. Ect2 is a central regulator of cortical contractility and its spatial and temporal regulation is of uttermost importance. After fertilization, the centrosome induces removal of Ect2 from the posterior plasma membrane. During cytokinesis Ect2 activity is expected to be high at the cell equator and low at the cell poles. Similarly to polarization, the centrosome provides an inhibitory signal during cytokinesis that clears contractile ring components from the cell poles. Whether and how the centrosomes regulate Ect2 localization is not know and investigated in the study.

The authors start by filming endogenously-tagged Ect2 and find that Ect2 localizes asymmetrically, with high anterior and low posterior membrane levels during polarization and cytokinesis. They reveal that the centrosome together with myosin-dependent flows results in asymmetric Ect2 localization. Previous studies had suggested that Air1, clears Ect2 from the posterior during polarization and the authors expand those finding by showing that Air1 function is also required to displace Ect2 from the posterior membrane during cytokinesis. To elucidate if Ect2 displacement is induced by phosphorylation of Ect2 by Air1, the authors investigate the localization of a C-terminal Ect2 fragment containing the membrane binding PH domain. When the predicted Air1 phosphorylation sites are mutated to alanine, the Ect2 fragment still localizes asymmetrically but exhibits increased membrane accumulation.

Finally, they investigate the functional role of Air-1 during furrow ingression. They demonstrate that embryos deficient of Air1 and NOP1 have impaired furrow ingression. Lastly, the authors sought to confirm that there is a direct effect of Air1 on Ect2 function by generating a phosphomimetic point mutation of Ect2 using Crispr. They find that the membrane localization of phosphomimetic Ect2 is reduced and consequently furrow ingression is impaired.

Major comments

It is not convincing that the six putative phosphorylation sites are targeted by the Air1. If Air1 phosphorylation displaces Ect2 from the membrane, a reduction in Ant/Post Ect2 ratio is expected in the phosphodeficient mutants, like after air1 RNAi. However this is not observed for cytokinesis or polarization (Fig. 5D(i); E). This suggests that phosphorylation of those sites is not essential for the asymmetric Ect2 localization.

The authors aim to demonstrate that phosphorylation of the identified sites is important for cytokinesis. For this they investigate contractile ring ingression in the phosphomimetic point mutation. Since ring ingression is slower and fails in nop1 mutant they authors conclude that this demonstrates a functional importance of this site. I am not surprised that embryos ingress slower in this mutant since Ect2 localization to the membrane is reduced. This however does not show that this phosphorylation site is the target of the centrosome signal. Importantly, authors would need to demonstrate that Rho signaling and thus Ect2 activity, is increased at the poles, when phosphodeficient Ect2 is the only Ect2 in the embryo.

The authors use the Aurora A inhibitor MLN8237: It was shown prior (De Groot et al., 2015) that this inhibitor is not highly specific for Aurora A, and that it also inhibits Aurora B. Thus experiments need to be repeated with MK5108 or MK8745. They should also be conducted during polarization. Why does Aurora A inhibition not abolish asymmetry? That would be expected?

There is no statistical analysis of the results in the entire study. For all claims stating a change in Ant/Post Ect2 ratio or Ect2 membrane localization selected time points should be statistically compared: for example the main point of Fig.1 is that Ect2 becomes more asymmetric during anaphase. Thus a statistical analysis of the Ect2 ratio at anaphase onset (t=0s) and eg. t=90 s after anaphase onset should be performed; or Fig. 3A nop-1 mutant Ant/Post Ect2 ratio during polarization: again statistical analysis of control and nop-1 mutant embryos is needed at a particular time point.

The aim of Fig. 2B is to demonstrate that Ect2 localization is independent of microtubules, however they still observe some microtubules with the Cherry-tubulin marker and those are even very close to the membrane and therefore could very well influence Ect2 on the membrane. Therefore I am not convinced that this experiment rules out that microtubules have no role in regulating Ect2 localization.

Throughout the paper the authors should tone down their statement that Air1 breaks symmetry by phosphorylating Ect2, since phosphorylation of Ect2 by Air2 is not shown.

I understand that the establishment of Ect2 asymmetry is important for polarization. However, how does asymmetric Ect2 localization result in more active Ect2 at the cell equator, which is required for the formation of the active RhoA zone? Would we not expect an accumulation of Ect2 at the cell equator, or if that is not the case more active Ect2 at the equator versus the poles?

Minor comments

Can the authors explain why the quantification of Ant/Post Ect2 ratio in control embryos differs in different figures? For example: in Fig. 1D i) a slight increase of Ect2 asymmetry ratio is seen at around 80 s after anaphase onset. In comparison, in Fig. 2C (i) this increase is not obvious. Are those different genetic backgrounds?

One key point of the paper is that myosin-dependent cortical flows amplify Ect2 asymmetry during polarization and cytokinesis. During polarization the data is convincing, however during cytokinesis Ect2 ratio is only slightly decreased after nmy-2 depletion, again is this decrease even significant?

In the introduction: "Centralspindlin both induces relief of ECT-2 auto-inhibition and promotes Ect2 recruitment to the plasma membrane" it should be added 'Equatorial' membrane, since Ect2 membrane binding is, to my knowledge, not compromised in centralspindlin mutants or in Ect2 mutants that cannot bind centralspindlin.

Labels in the figures are often very small eg Fig. 1 ii-v) and difficult to read. In addition it is easier for the reader if the proteins shown in the fluorescent images is also labeled in the figure (eg Fig. 2B add NG-Ect2).

Material and methods it should be mentioned which IPTG concentration was used.

The authors speculate that the Air1 phosphorylation sites in Ect2 PH domain prevent binding to phospholipid due the negative charge. At the same time, the authors propose that the PH domain binds to a more stable protein on the membrane, which is swept along with the cortical flows and they propose anillin could be that additional binding partner. I might miss something, but do the authors suggest Ect2 has two binding partners: anillin and the phospholipids? It would be necessary to explain this better. The authors should test if anillin represents the suggested myosin II dependent Ect2 anchor. For this they should check if Ect2 localization to the membrane is altered upon on anillin RNAi.

The title of fig. 3 does not fit the statement the authors want to make, since the key point is how Ect2 polarization is affected and not membrane localization in general.

In Fig 4A/C. After air1 depletion the authors observe a reduction in Ect2 asymmetry. Why are the centrosomes not marked in the figures? Because they cannot be detected? The authors would also need to show that the mitotic spindle and centrosomes are no altered by air1 RNAi in the zyg9 mutant. Otherwise the observed effect might be indirect.

The authors state that tpxl-1 depletion attenuates Ect2 asymmetry, this is not seen in the quantification ((Fig. 4B(i)). The main phenotype they observe is that Ect2 levels on the membrane increase (Fig. 4 (ii) and (iii). They go on testing the function of tpxl1 by depleting tpxl1 in the zyg9 mutant, where the centrosomes are close to the posterior cortex. Here they see no effect on Ect2 asymmetry. Based on that they conclude that tpxl1 has no role in this process. To me this finding is not surprising since the centrosome is close the cortex in zyg9 mutant embryos. Therefore sufficient amounts of active Air1 could reach the membrane and displace Ect2. Thus an amplification of the inhibitory signal by tpxl1 on astral microtubules might not be required. The authors need to mention this possibility and tone down their statment (also in the discussion) that tpxl1 is not required for this process.

It was shown that the C-terminus of Ect2 is sufficient and the PH domain is required for Ect2 membrane localization in C. elegans (Chan and Nance, 2013; Gomez-Cavazos et al., 2020). Papers should be cited.

The authors find that nmy-2 depletion results in loss of asymmetry for the Ect2 C-term and Ect2 3A fragment during polarization. Why is the same experiment not shown for cytokinesis?

Air1 is targeted to GFP-C-term Ect2 fragment via GFP-binding to determine the influence on GFP-C-term Ect2 localization (Fig. 5F). They state that they see a reduction of Ect2 C-term but not of C-term 3A after targeting. The reader has to compare Fig. 5D with F. Since the differences are not big, they need to compare the Ect2 C-term and Ect2 C-term 3A with and without Air1 targeting in the same graph (plus statistics). Otherwise this statement is not convincing.

In Fig. 6A the authors determine the contribution of air1 to furrowing. For this they deplete air1 in the nop1 mutant. According to previous studies, air1 mutants have a monopolar spindle. How can the authors analyze the function of air1 in cytokinesis when the spindle is monopolar? Did the authors do partial air1 depletion? They authors need to show that there is not major effect on the spindle and centrosome for their conditions. For comparison air1(RNAi) alone has to be included, otherwise the experiment is not conclusive.

Upon air1(RNAi) in the nop1 mutant NMY2 intensity seems decreased and not increased. Can the authors comment on that, since that is opposite of what is expected.

In Fig 6B they introduce a phosphomimetic point mutation in S634 in the endogenous Ect2 locus. It not clear why the authors chose this site out of the six putative sites and why they only chose one and not 3 or 6 sites? This needs some explanation.

In the model (fig. 7) no astral microtubules are shown during pronuclear meeting and metaphase. Astral microtubules are present at this stage and should be added to the schematic.

Significance

The centrosomes inhibit cortical contractility during polarization and cytokinesis in the one-cell C. elegans embryo. Centrosome localized Air1 was proposed to be part of this inhibitory signal, however the phosphorylation target of Air1 is not known. The identification of Ect2 as a phosphorylation target of Air1 would be a great advancement in the field. However, the presented manuscript lacks convincing data that Ect2 is the phosphorylation target of Air1 during polarization and cytokinesis.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

The authors do not wish to provide a response at this time.

We will provide the point-by-point response when we submit the full revision of our manuscript

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

This paper presents an investigation of the mechanisms of how chitin is synthesized in Drosophila by investigating the chitin synthetase Kkv and two proteins related/redundant proteins that are required for chitin production Exp and Reb.

The authors show that synthesis of nascent chitin polymers is separable from the secretion of chitin and that Ex/Reb is specifically required for chitin translocation/secretion. To understand the functions of Exp/Reb, the authors perform structure/function analyses and examine the localization of the proteins. They find that Na-MH2 domain in Exp/Reb is required for chitin translocation, and that a motif the authors name CM2 is required for Exp localization. For Kkv, they show the WGTRE domain is required for ER exit and that a coiled-coiled domain is required for KKV localization and full Kkv activity. By using live imaging and mutations that disrupt membrane trafficking, the authors show that Kkv, which is a transmembrane protein, cycles to the membrane, and like most membrane proteins, is endocytosed and transits through the endocytic system and is returned to the apical surface. Interestingly, despite being dynamically moved around the cell, chitin synthesis produces highly organized extracellular matrixes. Considering that constitutive production of chitin by Kkv everywhere in the cell would create a mess, these results underscore that regulated organized secretion/translocation of chitin is central to generating patterned extracellular matrixes (as the saying goes, "location, location, location"). Consistent with Exp/Reb being important regulators in extracellular matrix patterning, Exp/Reb not only are required for export of chitin, in the absence of Exp/Reb, the pattern of Kkv localization at the apical surface is altered. Unexpectedly however, by using super resolution microscopy the authors show that Kkv and Exp/Reb have complementary rather than matching localizations. Thus, while it is not clear exactly how Exp/Reb are regulating Kkv, they are doing something very interesting.<br /> Overall, this paper will be of broad interest to the cell biology and developmental biology communities, and to the translational community working to develop chitin as a commercial biopolymer. It is also generally clearly written, although I think there are some inaccuracies in the how some points are phrased. The experiments are well done, and subject to the revisions out lined below.

Major concerns:

• A major conclusion of the paper is that Exp/Reb are not required for chitin synthesis. On the most basic level this statement is well supported, because chitin grains are made in the cytoplasm in the Exp/Reb mutants. However, I think the field would be better served with a more nuanced consideration or the role of Reb/Exp. From the data presented, it seems that in the absence of Reb/Exp, the total amount of chitin produced is greatly reduced. I think it would be worth considering Exp/Reb, or the synthesis process in general, as having processivity or duty cycle or quality control such that in the absence of Exp/Reb while Kkv may make short chitin polymers, or occasional long polymers, the major production of chitin doesn't get going without Exp/Reb. Thinking of Reb/Exp as processivity factors in addition to export factors dramatically changes how one thinks of the proteins and the process of chitin synthesis. While these considerations can be handed with some discussion, it would be very interesting to look at the length of the chitin polymers in the Reb/Exp mutants and see if the average chain length is much reduced. This would help distinguish between Exp/Reb reving up the total number of Kkv molecules that produce chitin and Exp/Reb allowing the same number of Kkv molecules to stay active and produce much longer chitin chains. A caveat here is that I have no idea how hard this is to do, so I won't put this at the level of a required revision, but this result would significantly deepen the analysis in the paper.
• In looking that the subcellular localization of the Kkv and Reb in regular and super resolution, the authors I think the authors missed an important, but straight forward way to gain insight into the apparent complementary distribution of Kkv and Exp/Reb. In stage 16 WT embryos, Kkv has a distinct ringed pattern that corresponds to the tanedial ridges (e.g. clearly visible in Fig. 6A and 6G). How those ridges are set up is unclear, although there are some interesting Turing-pattern models out there. One prediction might be that Exp/Reb should be in between the Kkv rings. If so, maybe Exp/Reb are key components of patterning chitin secretion to make this 3D patterned matrix? Alternatively, maybe Exp/Reb act on a smaller length scale and will match the Kkv ring pattern, just not overlapping with Kkv at the very fine scale. These are straightforward experiments and again could provide key insights into the function of Exp/Reb.
• In general, most of the figures do not include WT or a control for comparison. This makes it hard for non-experts to assess what the effect of a mutation or condition is. For example, there are no examples of WT or Df(exp reb) in Figures 1-4. I realize this would increase the number of panels, but the paper would be more accessible if comparisons were within figures instead of comparing between main and supplementary figures and other papers.
• To bolster the case the Exp/Reb directly regulate Kkv distribution, the authors should examine the distribution of Kkv in a catalytically null Kkv mutant, or drugs that block Kkv, or mutations in other genes required for Kkv activity to show that the altered distribution of Kkv in Exp/Reb mutants is a direct consequence of the lack of Exp/Reb rather than in indirect consequence of lack of extracellular chitin, which causes gross perturbations in the trachea. Also, are there differences in the distributions of Kkv in salivary glands with or without the presence of Exp/Reb? If Exp/Reb change the distribution of Kkv in the salivary glands, which normally do not express Kkv and presumably many other components of the chitin ECM system, this would be a powerful argument that there is a direct effect.

Minor concerns.

• Page 5 "These intracellular chitin punctae disappeared from stage 14, when chitin is then deposited extracellularly (Fig 1B')." Fig. 1B' is stage 15 embryos.
• Page 5 "lead to tracheal morphogenetic defects". It would be helpful to the reader if the text or legend told the reader what they were looking for? Broken tubes? Inflated tubes? Variable tubes?
• Fig. 1H. Main text says "co-expression of Kkv and expMH2/rebMH2 did not lead to tracheal morphogenetic defects (Fig 1H, ...". The tracheal dorsal trunk in Fig. 1H does not look WT. The legend does not state the stage, but the DT looks to have an enlarged diameter and it might be too long. Please present measurements on stage 16 trachea to confirm that there is no effect on tracheal morphology.
• Fig. 3E there is a lot of GFP-Kkv that is not in co-localized with the KDEL marker. Can the authors clarify what compartment all the other staining is? ER?
• Section 3.1. The authors imply that the WGTRE domain is specifically required for ER exit. However, an alternative is that absent the WGTRE domain, the protein just does not fold correctly, which would also preclude ER exit, but would be a different problem for the protein to make chitin if it isn't folded.
• Page 15. I disagree with statement "At stage 16, control embryos showed a highly homogeneous apical distribution of Kkv in stripes, corresponding to the taenidial folds, and Kkv vesicles were largely absent (Fig 6G)." In Fig. 6G, the tandeal ring pattern is clearly visible, as are the fusion cells. If Kkv distribution were "highly homogeneous" these structures/pattern would not be visible.
• Page 15. I also disagree with the characterization of the apical Kkv distribution in st 15 embryos. "In control embryos we detected a very uniform and homogenous pattern of apical Kkv (Fig 6I).". To my eye, the pattern is punctate and random for the clumps of stain, with the underlying beginnings of the tanidial pattern starting to be visible. The pattern appears neither uniform nor homogenous.
• P16. The degree of order in the distribution of Kkv is overstated. The authors state that "The results of this analysis, showed that Kkv on the apical membrane, is evenly distributed following a regular pattern (Fig. 6L,L',L',M)." However, given that there is barely a visibly perceptible difference between the actual distribution of Kkv in 6L' and a calculated random distribution in 6L", and that the pattern is neither visibly even or regular, it would be more representative to say something to the effect that the analysis shows there is "underlying order" or "some degree of order" or a "non-random pattern". Visually, the key difference between 6L ' and L" is that there are fewer closely clustered Kkv dots. You could still have an uneven distribution of Kkv that maintains minimum spacing, which is a kind of ordered organization, but not one that would be assumed from the description. It would be helpful if the authors instead of just saying a "regular pattern" also stated the nature of the pattern they observe, i.e. Grid? Stripes? Minimum spacing?
• Discussion. Another model for the role of Exp/Reb could be to bind and neutralize an inhibitor of Kkv activity. This would account complementary distribution of Kkv and Exp/Reb.
• Fig. 6L. what tissue is being analyzed? Presumably trachea, but this should be specified as salivary glands are also mentioned in the legend.
• Fig. 7 C models. I believe that the super resolution data is not accurately accounted for in the models. In both model 1 and model 2, Kkv and Exp/Reb are shown to be in close proximity, but the super resolution data suggests that most Kkv and Exp/Reb are separated hundreds of nanometers. Further, showing Kkv and Exp/Reb as touching was not supported by the coIP experiments, which failed to detect an interaction. It is possible that only a small fraction of Exp/Reb that is in close proximity to Kkv is active, but if so, this should be explicitly mentioned in the models to reconcile the data showing that Kkv and Exp/Reb are mostly not anywhere near each other.
• -Image analysis. Please detail the criteria for "apical" and "basal" regions were the basis for freehand segmentation. What was counted as apical and what was basal?
• Abstract and Introduction: The authors state that "We find that Kkv activity in chitin translocation, but not in polymerization, requires the activity of Exp/Reb, and in particular of its conserved Na-MH2 domain.", but then follow that with the statement that "Furthermore, we find that Kkv and Exp/Reb display a largely complementary pattern at the apical domain, and that Exp/Reb activity regulates the topological distribution of Kkv at the apical membrane." Many readers, will find the use of "furthermore" confusing because they will take furthermore as the about to be described data logically following the previous data, but then run headlong into the fact the Kkv and Exp/Reb show a complementary distribution, which does not obviously follow from Kkv activity requiring Exp/Reb. The authors could clarify this and highlight the interesting, unexpected and exciting nature of their results by replacing "Furthermore" with "Unexpectedly" or "Surprisingly", and emphasizing the important role of Exp/Reb in Kkv organization. Maybe something like: Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane.

Significance

The topic is interesting from the aspect of cell biology in terms of how a long polymer is created intracellularly, secreted and spatially organized to create a sophisticated extracellular matrix. The topic is also of general interest because chitin is central to the body plan of all insects, crustaceans and many other species, and chitin is of increasing interest as a biopolymer that could have extensive commercial uses.

In addition to an informative structure/function analysis of the Kvv and Exp/Reb, the results identify what is, to my knowledge, the first regulator of the spatial organization of chitin sythase in insects and it unexpectedly shows a complementary pattern to the the synthase. This highlights just how little we understand about how complex extracellular matrixes are synthesized.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

This paper deciphers very nicely the genetics and cellular events where and how chitin polymers become synthesized and translocated towards the apical cell membrane for further release into the extracellular space. Altogether this is fundamental work of high significance explaining how chitin is produced and released.

The authors initially detected unusual intracellular chitin by overexpression of the chitin synthase kkv in tracheal cells before regular chitin deposition occurs. In addition, they recognized that the kkv gain of function mediated unusual intracellular chitin vesicles and in later stages in exp/reb mutants. These findings were the starting point of further experiments, suggesting that Kkv synthesizes chitin and that Kkv-mediated chitin deposition requires Exp/Reb activity to translocate and release chitin. Their genetic studies further show that chitin polymerization and translocation are uncoupled.

All primary studies were tested in embryonic tracheal cells and as a proof of principle control in salivary glands which do not express chitin. Elegant rescue experiments in the mutant background showed that the Exp/Reb Nα-MH2 domains are required but not sufficient for chitin translocation and deposition and are dispensable for protein localization. Additionally, they identified another conserved domain (CM2) which is required Exp/Reb localization but not for chitin translocation. Similarly, they investigated Kkv domains by rescue experiments in kkv mutant embryos. They identified the WGTRE domain as essential for ER exit and the coiled-coil region for proper apical Kkv localization. Altogether they provide evidence that Kkv requires proper localization at the apical membrane, which is likely coordinated by Exp/Reb. This precise Kkv localization is linked with Kkv activity and chitin deposition. Therefore, this work is new and fundamental to many disciplines such as insect biology, chitin biology, cell & developmental biology, and others. Thus, the work is worth publishing but requires some changes from my point of view.

Major Comments

1. The authors nicely show that Kkv is able to synthesize chitin in a constitutive manner and that it can accumulate intracellularly. However, this needs some more input to understand the underlying biological sense. For example, what are the chitin vesicles' nature of early vesicles (st 13) and the unusual late (st15) chitin vesicles? Exocytosis, endocytosis or recycling? This can be clarified to understand chitin translocation and that synthesis and translocation are uncoupled. The authors tested rab5DN mutant salivary glands to exclude endocytosis. However, the chitin-positive vesicle size and amount in the rab5 mutant appear different from the control experiment, where much more intracellular chitin accumulates. Thus, it may suggest that some chitin vesicles are independent of Rab5-mediated endocytosis, others probably not. Indeed, the authors identified some KKv and some other chitin vesicles in all discussed intracellular processes; however, additionally, chitin appears to accumulate also in the cytoplasm. The authors conclude that Kkv protein might be able to polymerize chitin at all different intercellular stages, including endocytosis and degradation pathway. First, I wonder why chitin was found within membranous vesicles and, at the same time, within the cytoplasm. Second, does it make sense in the biological context when tracheal cells or other chitin-producing organs want to secrete chitin at the apical membrane while Kkv has the ability to produce chitin in all cellular areas, even in endosomes? In this context, another fundamental question concerning chitin secretion and subsequent organization could be investigated with the author's tools. Are the chitin vesicles loaded with chitin binding proteins or deacetylases that organize the formation of the nano and makro fibrillar chitin matrix in tracheal tubes? For example, previous research showed a reduced luminal accumulation of the 2A12 antigen in kkv mutants and expRNAi knockdown embryos.
2. Observation of Extracellular kkv-GFP: does extracellular anti-GFP staining co-localize with the anti-Kkv antibody?
3. Putative Kkv microvesicles: the authors state that extracellular GFP staining could be Kkv located in microvesicles. I wonder whether the observed extracellular GFP puncta contain a membrane or other membranous proteins.
4. Fig.1:
5. general remarks: some images of this figure could be improved by showing the single channels of CBP to judge whether chitin is secreted and/or vesicles appear. -In addition, some images show higher magnifications, others overview only. It would be beneficial to visualize the small vesicles additionally with higher magnifications.
6. Fig.1M: This image is problematic due to the epidermal background staining. The tracheal system is hard to recognize. A single channel of Cbp ist not indicated.
7. Fig. 1P: Apical membrane marker or any cytoplasmic marker would be extremely useful to judge subcellular Exp localization in this experiment - this image is hard to compare with Exp localization in Fig S2D.

Fig. 2O: Apical/Basal accumulation, what are the numbers at the Y-axis?

Fig. 3F: The authors state that the WGTRE domain is required for ER exit of Kkv based on colocalization studies with KDEL and FK2. However, the study with FK2 is not convincing as immunostainings are of poor quality. The GFP construct appears not to be expressed in all tracheal cells, and moreover, the FK2 staining is faint. Thus, judging whether the protein is not ubiquitinated from the presented image is challenging. However, it does not change the key message, Kkv does not exit ER. By the way, there is a new paper showing Serca to be essential for ER exit of Kkv, which would fit the discussion of the kkv domains.

Fig. 7 - model: First, Since the authors do not show that Kkv is part of a membranous microvesicle, I'm skeptical whether this should be part of a model that explains the shown data. Therefore, I'm asking the authors to delete it or to show it more clearly. Second, the meaning of the yellow arrowheads is not indicated. Third, the explanation in the legend is sound, but showing the two options could be improved.

Minor comments:

1. Missing reference: Chirin has been recognized importance in physiology (Zhao et al., 2019; Zhu et al., 2016) but also as a biomaterial (? Reference). Suggestion: DOI: 10.3390/ma15031041 (Improving Polysaccharide-Based Chitin/Chitosan-Aerogel Materials by Learning from Genetics and Molecular Biology) This paper discusses the current usage and potential of chitin as a biomaterial in many disciplines.)
2. 3.1, second paragraph final sentence: double point

Referees cross-commenting

Rev#1 asks the same questions as I do. Technical questions and the idea to compare endogenous Kkv.The same is true with Rev#3. Overlapping questions concerning technical things about figure illustration and clarity of presented stainings. Altogether, the criticism will improve the manuscript

Significance

This paper deciphers very nicely the genetics and cellular events where and how chitin polymers become synthesized and translocated towards the apical cell membrane for further release into the extracellular space. Altogether this is fundamental work of high significance explaining how chitin is produced and released.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In this manuscript, the authors provide data on the function of exp/reb and Kkv in chitin deposition. They show chitin polymerization and deposition are uncoupled and exp/reb are required for the deposition of the chitin by regulating the distribution of kkv at the apical membrane. However, there is no direct interaction between Kkv and Exp/reb. The functional analysis of Kkv and Exp/reb is interesting.

The overexpression lines are used throughout the manuscript to analyze protein functions. Since ectopic expression of kkv and exp leads to chitin synthesis and deposition. Authors use this overexpression system to analyze the functional domain of kkv and Exp/reb. It is reasonable. However overexpression line might not represent the endogenous protein perfectly, it might cause some issues to answer certain questions.

Major comments

1. Fig. 4 Does ectopic overexpression of Kkv-GFP have the same expression pattern as the endogenous Kkv? The overexpression line may lead to ectopic expression. the colocalization of endogenous Kkv and intracellular vesicles would be more accurate.
2. Are Kkv and Exp/reb expressed at the same time endogenously? If kkv is expressed earlier than Exp, can intracellular chitin be detected in wild-type embryos at early stages? Fig. 1b shows overexpression of Kkv at S13 has intracellular chitin (exp is not expressed at this stage).
3. Fig. 1B no intracellular chitin is detected. Fig. 1H intracellular chitin is detected. Does Overexpression of exp-MH2 interfere with the endogenous Exp function?
4. For measurement, some detailed info is needed, for example, what is your area of interest?

Minor comments:

1. Fig. 2 and Fig. 3. how do you define the region of apical and basal? An apical marker is needed here. N is the total number of embryos or the number of sections in the same embryo?
2. Fig. 5H What is your area of interest to measure vesicles? Which tracheal segment do you measure? Some details need to be provided here.
3. Fig. 5 what is your area of interest when you measure Kkv?

Significance

This work further advance the knowledge about chitin synthesis and deposition

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

1. General Statements

All three reviewers demonstrated similar concerns and provided clear guidance on potential revisions. All three reviewers recognized the importance of our study to the mitosis, genome instability and DNA damage fields, and identify limitations regarding potential therapeutic implications of the study. To mitigate these limitations and extend the breadth of our study, the revised manuscript now includes colony formation assays to more directly evaluate the impact of mitotic DNA damage therapies in cancer cell proliferation. It also includes several of the experimental suggestions/clarifications proposed by the reviewers. Lastly, we include here a clear plan of additional experiments that we agree to conduct.

2. Description of the planned revisions

Reviewer #1

Major comments:

1) In the opinion of the reviewer, the study is somewhat unbalanced as it starts with a high throughput analysis of a large number of compounds but only etoposide treatment is investigated in detail in the key experiments shown in Figures 5 and 6. The effect of topoisomerase II inhibitor on kinetochore-MT stability has already been demonstrated by Bakhoum et al, 2014. If authors wish to generalize that similar phenotypes are observed after various types of DNA damage, they should test additional compounds (such as Lomustine, Mitomycin C, and Carboplatin). In addition, measuring of the kinetochore-MT half-life in figure 5 should be performed with better time resolution within the first 5 minutes. This would allow better comparison of the measured half lives that are much shorter than 5 minutes.

R: We agree with the reviewer and we will perform additional measurements of kinetochore microtubule half-life with these different compounds and include shorter time points to increase the temporal resolution within the first 5 min.

3) Involvement of Kid and Kif4A in arm ejection of the polar chromosomes is an interesting observation in context of mitotic DNA damage. However, it is unclear how the distribution of the chromokinesines was evaluated in Figure 6A-D. Was the signal quantified at the metaphase plate or at polar chromosomes? It seems that Kif4A localizes to the polar chromosome caused by etoposide treatment whereas no signal is visible in DMSO control (Fig. 6C).

R: Since whole cells were exposed to DNA damage, we had previously quantified total fluorescence intensity of Kid and Kif4a on all chromosomes (aligned and unaligned). We nevertheless concede that some chromosomes might have been more exposed (or are more susceptible) to DNA damage and we will therefore provide a quantification of the fluorescence intensity ratio of Kid and Kif4A levels on polar chromosomes relative to the metaphase plate, with and without mitotic DNA damage.

4) Authors convincingly showed that SAC is activated by mitotic damage and this is also consistent with previous reports. However they did not address if DDR pathways contribute to the activation of SAC. This would be interesting especially in context of a recent report that showed Bub3 as a direct substrate of ATM (Xiao et al. 2022, JBC). I wonder if the polar chromosomes are formed and missegregate also in the absence of ATM activity.

R: This is an interesting suggestion and we have already obtained data from one experiment regarding the formation of polar chromosomes upon ATM inhibition. We will perform additional independent validation of these data and include our findings in the fully revised manuscript.

Reviewer #2

Major comments:

…They need to present the % of cells with polar chromosomes, and it would also be informative to understand the rate of cells with lagging chromosomes, or that underwent anaphase with polar chromosomes with and without chromokinesin depletion.

R: We will quantify the frequency of the different segregation errors upon DNA damage, with and without Chromokinesin depletion.

It would be very helpful for them to provide a schematic model between DNA damage, overstable microtubules, satisfied SAC, monotelic attached chromosomes, and the role of chromokinesins. At present these connections are very unclear.

R: We thank the reviewer for drawing attention to this point. We will provide a step-by-step model in the fully revised manuscript.

Reviewer #3

Major comments:

According to Figure 2C, the ratio of "Exit with micronuclei (from misaligned chromosome(s))" is relatively low compared to other phenotypes such as "Mitotic arrest" or "Cell death." I wonder if polar chromosome phenotype is also correlated with these other cell fates. Please clarify which fate is correlated with polar chromosome formation after DNA damage.

R: We will provide these correlations. We already know that those cells that arrest in mitosis is due to misaligned chromosomes. We will also perform the correlation between cells that died and the presence of misaligned chromosomes.

In Figure 3, the authors used Nocodazole-treated background to assess the involvement of SAC in DNA-damaging compound-induced mitotic delay. However, as shown in Figure 2B, DNA-damaging compounds cause a minor delay in mitosis, which might be challenging to analyze in the presence of Nocodazole. There is also a possibility that DNA damage response (DDR) works independently and adjunctly to delay mitosis. Because one of the major claims of the authors is that "the SAC is the only mechanism that is required to delay mitosis in the presence of long-term mitotic DNA damage (page 10, line278)", I recommend Nocodazole wash-out (as in Figure 2B) to examine the effect of MPS1-IN-1 (and ideally an inhibitor of the DDR pathway, such as ATMi) on mitotic delay induced by DNA-damaging compounds.

R: We now clarify that the observed mitotic delay in the presence of DNA damaging compounds occurred after nocodazole washout. As so, nocodazole was no longer present in the system. We also draw the attention that DNA damage in the presence of nocodazole, a condition that promotes maximal SAC activity, was fully dependent on MPS1 activity (Figure 4A). We have also obtained data from one experiment regarding the formation of polar chromosomes after nocodazole wash-out and ATM inhibition. We will perform additional independent validation of these data and include our findings in the fully revised manuscript.

Figure 6E-G: I wonder whether siKid+siKif4a affected %polar chromosomes or not.

R: We will perform this experiment and include the results addressing this point in the fully revised manuscript.

Page 10, line 287: the authors claim that "we show that long-term mitotic DNA damage..., causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesisns,...." However, only Mad1 localization data is provided in Figure 6E-G, and whether siKid + siKif4a rescues the missegregation of polar chromosomes is not clear. The authors should either provide supporting evidence or revise this sentence for clarity.

R: We will determine whether Kid+Kif4a depletion rescues the missegregation of polar chromosomes (i.e. reduces the frequency of cells that exit with polar chromosomes in the presence of mitotic DNA damage).

3. Description of the revisions that have already been incorporated in the transferred manuscript

Reviewer #1

Major comments:

2) Authors claim that the observed phenotype of the chromosomal missegregation following the mitotic DNA damage occurs specifically in cancer cells but the data supporting this statement is poor. They need to show more data in non-transformed cells or remove the statement.

R: Data from non-transformed RPE-1 cells are now included in the revised manuscript.

Minor comments:

1) Specificity of Aurora-B pT232 antibody should be validated to exclude cross-reactivity with Aurora-A at spindle pole (Fig S8C)

R: Antibody specificity against active Aurora A and B was validated with specific kinase inhibitors. These data are now included in the revised manuscript.

Reviewer #2

Major comments:

1. The term 'long-term DNA damage during mitosis' is confusing. DNA damage occurs when? (before, or during mitosis?). Their live cell data shows they are following cells that underwent mitosis within a certain time window after damage occurred, but it is not clear if it occurs only before, or sometimes during mitosis.

R: We realize that this was indeed confusing. The only experiment where mitotic DNA damage might have been introduced before mitosis is in Figure 1A-C. All other experiments were directly or indirectly based on live-cell data in which only cells that were already in mitosis when they were exposed to DNA damage were analysed. Because these live-cell experiments revealed that DNA damage before mitosis prevented mitotic entry at the used concentration of the DNA-damaging agents, mitotic cells scored upon DNA damage in fixed cell experiments must have been already in mitosis when DNA damage was applied. We also now include a scheme of each experimental set up in the respective figures to clearly indicate how the data was obtained and to facilitate the respective interpretations. This is now clarified in the main text.

All damage occurred in cells treated with nocodazole - could this have impacted the results? Was similar DNA damage induced if cells were arrested in monastrol/STLC, or MG132 for example? They show the distal Mad2 data in STLC but not the damage. Also Figure S4A/B appears to be from one experiment only which makes it difficult to interpret.

R: We have additionally induced DNA damage when the cells were arrested in STLC. yH2AX levels as inferred by western blot analysis were indistinguishable from cells treated with nocodazole. These data are now included in the revised manuscript. We also clarify that data presented in previous Figure S4 are from 2 independent experiments.

Why not show the RPE1 data for polar chromosomes?

R: Data from non-transformed RPE-1 cells are now included in the revised manuscript.

Confusing interpretation - monotelic attachments, yet also stable attachments,..? Please can they clarify what is meant by these terms.

R: Monotelic attachments in which a single chromatid is attached to microtubules are intrinsically unstable due to the lack of centromeric tension. However, mitotic DNA damage alters this scenario and stabilizes monotelic attachments. We have now clarified this point in the main text.

They focus most of the study on understanding why polar chromosomes arise after DNA damage. However, this phenotype seems to be a relatively minor effect. Eg. In Figure 2b only a few cells exhibit very extended mitosis, and in Figure 2c only a small percentage exit mitosis with misaligned chromosomes. Furthermore, in Figure 4B the percentage of polar chromosomes with only distal Mad2 is low. In Figure 4A the images are not clear whether a 'both' or 'distal' is being shown. It does not seem as if any are distal only, and an example of this would be helpful.

R: We thank the reviewer for alluding to this important point. The frequency of cells with polar chromosomes with/without DNA damage is indicated in Figure 2C. The respective duration of mitosis due to polar chromosomes is clearly and significantly increased as shown in Figure 2B. In fixed material we chose 70 min after nocodazole washout in these experiments because there is no difference in the frequency of cells with polar chromosomes between DMSO and DNA-damage-treated cells, allowing a direct comparison of the types of kinetochore-microtubule attachments on polar chromosomes only (please see new Figure 5). We now clarify that beyond this time frame, only DNA-damage-treated cells show polar chromosomes. We also draw the reviewer’s attention to the B&W panels where the different types of attachments are clearly highlighted.

It is also not clear what 'other' means in Figure 2C.

R: The phenotypes classified as ‘other’ in Figure 2C are detailed in Figure S1C. This was indicated in the figure 2 legend, but we have now clarified it also in the main text.

They conclude that chromokinesin depletion rescues the polar chromosomes phenotype. However they do not directly assess the rate of polar chromosome formation, only the % of polar chromosomes that are only distal Mad2 as far as I can see?

R: Similar to reviewer 1, we thank this reviewer for alluding to this important point. The frequency of cells with polar chromosomes with/without DNA damage is indicated in figure 2C. The respective duration of mitosis due to polar chromosomes is clearly and significantly increased as shown in Figure 2B. In fixed material we chose 70 min after nocodazole washout in these experiments because there is no difference in the frequency of cells with polar chromosomes between DMSO and DNA-damage-treated cells, allowing a direct comparison of the types of kinetochore-microtubule attachments (please see new Figure 5). We now clarify that beyond this time frame, only DNA-damage-treated cells show polar chromosomes.

It is not clear from how many experiments data are shown for the Mad1 distal experiments in Figures 4 and S4 (there are no error bars, so is this one experiment only?). This should be indicated in figure legends, and repeated if performed only once.

R: Data presented in Figure 4 is a pool from 3 independent experiments and data presented in Figure S4 is a pool from 2 independent experiments. For these reasons, there are no error bars to include. This is now clarified in the figure legends.

Reviewer #3

Major comments:

1. Page 6, line 155: the authors claim that "In contrast, among other defects, treatment with any of the DNA-damaging compounds caused a significant mitotic delay due to the presence of misaligned chromosomes near the spindle poles." Although Figure 2A shows a representative image of polar chromosomes, I do not find quantitative data that analyze %polar chromosomes in mitosis treated with DNA-damaging compounds. I also do not find the data supporting the claim that polar chromosomes caused a mitotic delay. Because most subsequent analyses were performed based on this result, the quantitative data should be provided here. For the latter, I suggest showing "time in mitosis (Fig 2B)" separately with or without polar chromosomes.

R: The frequency of cells with polar chromosomes with/without DNA damage is indicated in figure 2C. The respective duration of mitosis due to polar chromosomes is clearly and significantly increased as shown in Figure 2B. In fixed material we chose 70 min after nocodazole washout in these experiments because there is no difference in the frequency of cells with polar chromosomes between DMSO and DNA-damage-treated cells, allowing a direct comparison of the types of kinetochore-microtubule attachments (please see new Figure 5). We now clarify that beyond this time frame, only DNA-damage-treated cells show polar chromosomes. We now highlight in figure 2C what fraction of cells underwent mitotic arrest due to polar chromosomes, as well as those that exited mitosis with polar chromosomes.

In Figure 3, the authors used Nocodazole-treated background to assess the involvement of SAC in DNA-damaging compound-induced mitotic delay. However, as shown in Figure 2B, DNA-damaging compounds cause a minor delay in mitosis, which might be challenging to analyze in the presence of Nocodazole. There is also a possibility that DNA damage response (DDR) works independently and adjunctly to delay mitosis. Because one of the major claims of the authors is that "the SAC is the only mechanism that is required to delay mitosis in the presence of long-term mitotic DNA damage (page 10, line278)", I recommend Nocodazole wash-out (as in Figure 2B) to examine the effect of MPS1-IN-1 (and ideally an inhibitor of the DDR pathway, such as ATMi) on mitotic delay induced by DNA-damaging compounds.

R: We now clarify that the observed mitotic delay in the presence of DNA damaging compounds occurred after nocodazole washout. As so, nocodazole was no longer present in the system. We also draw the attention that DNA damage in the presence of nocodazole, a condition that promotes maximal SAC activity, was fully dependent on MPS1 activity (Figure 4A).

Line 226, (our unpublished observations): because the authors claim that "the formation of polar chromosomes due to the stabilization of kinetochore-microtubule attachments upon long-term mitotic DNA damage is likely exclusive to cancer cells," the authors should present data on RPE-1 cells at least for %polar chromosome formation (as suggested in comment 1) and Mad1 localization. Plus, even though the data is provided, the statement "exclusive to cancer cells (page 8, line 230)" is speculative and should be toned down. Mad1 localization data is also important because the authors claim that "long-term mitotic NA damage specifically stabilized kinetochore-microtubule attachments in cancer cells (page 10, line 288)" in the discussion.

R: Data from non-transformed RPE-1 cells are now included in the revised manuscript.

For the Mad1 assay, such as in Fig. 4A, the authors analyzed the CENP-C pair with two or one Mad1 foci formation. However, in some representative pictures, for example, Fig S4A-Etoposide, I found pairs of CENP-C signals on the polar chromosome without any Mad1 foci (the one next to the pairs shown in the square). As the authors argue, these kinetochores may represent polar chromosomes that eventually satisfy SAC and may be important. I, therefore, wonder why those kinetochores are omitted from the assay. Please explain this point in the manuscript if there is any reason.

R: We have now provided a clearer example and clarified in the main text that only chromosomes outside the spindle area were considered polar chromosomes.

Minor comments:

Page 7, line 168: the authors claim that "regardless of the type of DNA lesion, long-term mitotic DNA damage persists throughout mitosis and promotes micronuclei formation from polar chromosomes." However, the former claim is not fully supported by Figure S3, which addressed the effect of Etoposide only; the latter claim is not fully supported by Figure 2C, which lacks clarity (as pointed out in comment 2) and statistical analysis. Please revise this sentence.

R: We now present the levels of yH2AX after treatment with Lomustine, Mitomycin C, and Carboplatin and compared it with DMSO-treated controls and Etoposide. We also include statistics for the cell fate and respective chromosome segregations errors after treatment with the different DNA damaging agents.

Line 182: it would be helpful for readers to explain why MG132 was used.

R: This is now explained in the main text.

Line 210: it would be helpful for readers to explain briefly what PA-GFP means and how the assay works.

R: This is now explained in the main text.

Figure 1E: some color codes for each compound are difficult to distinguish. I also found it challenging to locate some lines on the graph. I recommend separating this graph, for example, by types of DNA lesions caused by compounds, and color codes that are easy to distinguish should be used.

R: We have now changed the most confusing colors and provide a higher temporal resolution chart in the low yH2AX region to facilitate visualization.

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

R: None

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In the manuscript entitled "Long-term mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells," the authors propose that DNA damage on mitotic chromosomes causes chromokinesin-mediated polar chromosomes, which eventually results in missegregation and micronuclei formation. They first performed screening of compounds that cause DNA damage on mitotic chromosomes and found that DNA damage delayed mitosis in the nocodazole wash-out experiment. The authors found that several DNA damage-inducing compounds all caused an increase of asymmetric Mad1 localization on polar chromosomes. Using photoactivatable GFP-a-tubulin, the authors showed that a-tubulin stabilizes after Etoposide treatment. They finally showed that chromokinesin Kid and Kif4a knockdown rescues the asymmetric Mad1 localization.

Major comments:

1. Page 6, line 155: the authors claim that "In contrast, among other defects, treatment with any of the DNA-damaging compounds caused a significant mitotic delay due to the presence of misaligned chromosomes near the spindle poles." Although Figure 2A shows a representative image of polar chromosomes, I do not find quantitative data that analyze %polar chromosomes in mitosis treated with DNA-damaging compounds. I also do not find the data supporting the claim that polar chromosomes caused a mitotic delay. Because most subsequent analyses were performed based on this result, the quantitative data should be provided here. For the latter, I suggest showing "time in mitosis (Fig 2B)" separately with or without polar chromosomes.
2. According to Figure 2C, the ratio of "Exit with micronuclei (from misaligned chromosome(s))" is relatively low compared to other phenotypes such as "Mitotic arrest" or "Cell death." I wonder if polar chromosome phenotype is also correlated with these other cell fates. Please clarify which fate is correlated with polar chromosome formation after DNA damage.
3. In Figure 3, the authors used Nocodazole-treated background to assess the involvement of SAC in DNA-damaging compound-induced mitotic delay. However, as shown in Figure 2B, DNA-damaging compounds cause a minor delay in mitosis, which might be challenging to analyze in the presence of Nocodazole. There is also a possibility that DNA damage response (DDR) works independently and adjunctly to delay mitosis. Because one of the major claims of the authors is that "the SAC is the only mechanism that is required to delay mitosis in the presence of long-term mitotic DNA damage (page 10, line278)", I recommend Nocodazole wash-out (as in Figure 2B) to examine the effect of MPS1-IN-1 (and ideally an inhibitor of the DDR pathway, such as ATMi) on mitotic delay induced by DNA-damaging compounds.
4. Line 226, (our unpublished observations): because the authors claim that "the formation of polar chromosomes due to the stabilization of kinetochore-microtubule attachments upon long-term mitotic DNA damage is likely exclusive to cancer cells," the authors should present data on RPE-1 cells at least for %polar chromosome formation (as suggested in comment 1) and Mad1 localization. Plus, even though the data is provided, the statement "exclusive to cancer cells (page 8, line 230)" is speculative and should be toned down. Mad1 localization data is also important because the authors claim that "long-term mitotic NA damage specifically stabilized kinetochore-microtubule attachments in cancer cells (page 10, line 288)" in the discussion.
5. For the Mad1 assay, such as in Fig. 4A, the authors analyzed the CENP-C pair with two or one Mad1 foci formation. However, in some representative pictures, for example, Fig S4A-Etoposide, I found pairs of CENP-C signals on the polar chromosome without any Mad1 foci (the one next to the pairs shown in the square). As the authors argue, these kinetochores may represent polar chromosomes that eventually satisfy SAC and may be important. I, therefore, wonder why those kinetochores are omitted from the assay. Please explain this point in the manuscript if there is any reason.

Minor comments:

1. Page 7, line 168: the authors claim that "regardless of the type of DNA lesion, long-term mitotic DNA damage persists throughout mitosis and promotes micronuclei formation from polar chromosomes." However, the former claim is not fully supported by Figure S3, which addressed the effect of Etoposide only; the latter claim is not fully supported by Figure 2C, which lacks clarity (as pointed out in comment 2) and statistical analysis. Please revise this sentence.
2. Line 182: it would be helpful for readers to explain why MG132 was used.
3. Line 210: it would be helpful for readers to explain briefly what PA-GFP means and how the assay works.
4. Figure 6E-G: I wonder whether siKid+siKif4a affected %polar chromosomes or not.
5. Page 10, line 287: the authors claim that "we show that long-term mitotic DNA damage..., causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesisns,...." However, only Mad1 localization data is provided in Figure 6E-G, and whether siKid + siKif4a rescues the missegregation of polar chromosomes is not clear. The authors should either provide supporting evidence or revise this sentence for clarity.
6. Figure 1E: some color codes for each compound are difficult to distinguish. I also found it challenging to locate some lines on the graph. I recommend separating this graph, for example, by types of DNA lesions caused by compounds, and color codes that are easy to distinguish should be used.

Referees cross-commenting

I generally agree with other reviewers' comments and confirmed that they raised similar concerns.

Significance

It has been described previously that mitotic arrest induces DNA damage and that the DDR pathway during mitosis is attenuated. The data presented in this manuscript provide a potentially novel cellular response against DNA damage during mitosis. The manuscript will be of interest to those in the field of the cell cycle (especially mitosis), the DDR, and tumor chemotherapies. While the finding that DNA damage during mitosis causes polar chromosomes is potentially interesting, the manuscript is still rather descriptive, and data that address the molecular mechanism is insufficient for the level that the authors conclude. Although the data quality is high, I think some essential data supporting their conclusion and clarity of the description are missing from the manuscript, which can be addressed before publication.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Novais-Cruz et al present an interesting and generally well-performed study on the impact of DNA damage just prior to mitosis on chromosome segregation fidelity. Overall, the experiments are performed and presented to a high standard, and the key findings are potentially of interest. However, in its present form the manuscript is overall quite confusing and it is difficult to assess the robustness of their conclusions. Their observations and mechanistic model connecting the observations is not clear at all in the current form. Several key pieces of data are missing that would help craft the story, and more explanation is needed to connect their overarching hypothesis to the data better. The below points serve as an illustration of the missing information that would be needed in order to make a proper judgement of whether the data support their main conclusions.

1. The term 'long-term DNA damage during mitosis' is confusing. DNA damage occurs when? (before, or during mitosis?). Their live cell data shows they are following cells that underwent mitosis within a certain time window after damage occurred, but it is not clear if it occurs only before, or sometimes during mitosis.
2. All damage occurred in cells treated with nocodazole - could this have impacted the results? Was similar DNA damage induced if cells were arrested in monastrol/STLC, or MG132 for example? They show the distal Mad2 data in STLC but not the damage. Also Figure S4A/B appears to be from one experiment only which makes it difficult to interpret.
3. Why not show the RPE1 data for polar chromosomes?
4. Confusing interpretation - monotelic attachments, yet also stable attachments,..? Please can they clarify what is meant by these terms.
5. They focus most of the study on understanding why polar chromosomes arise after DNA damage. However, this phenotype seems to be a relatively minor effect. Eg. In Figure 2b only a few cells exhibit very extended mitosis, and in Figure 2c only a small percentage exit mitosis with misaligned chromosomes. Furthermore, in Figure 4B the percentage of polar chromosomes with only distal Mad2 is low. In Figure 4A the images are not clear whether a 'both' or 'distal' is being shown. It does not seem as if any are distal only, and an example of this would be helpful.
6. It is also not clear what 'other' means in Figure 2C.
7. They conclude that chromokinesin depletion rescues the polar chromosomes phenotype. However they do not directly assess the rate of polar chromosome formation, only the % of polar chromosomes that are only distal Mad2 as far as I can see? They need to present the % of cells with polar chromosomes, and it would also be informative to understand the rate of cells with lagging chromosomes, or that underwent anaphase with polar chromosomes with and without chromokinesin depletion.
8. It would be very helpful for them to provide a schematic model between DNA damage, overstable microtubules, satisfied SAC, monotelic attached chromosomes, and the role of chromokinesins. At present these connections are very unclear.
9. It is not clear from how many experiments data are shown for the Mad1 distal experiments in Figures 4 and S4 (there are no error bars, so is this one experiment only?). This should be indicated in figure legends, and repeated if performed only once.

Significance

Novais-Cruz et al present an interesting and generally well-performed study on the impact of DNA damage just prior to mitosis on chromosome segregation fidelity. Overall, the experiments are performed and presented to a high standard, and the key findings are potentially of interest to the mitosis, and genomic instability fields.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

In the presented manuscript, authors investigated consequences of DNA damage on progression through mitosis. In agreement with previous reports, they observed a mitotic delay that was dependent on activation of the spindle assembly checkpoint (SAC). Starting with a high throughput screening authors concluded that the SAC-dependent mitotic delay is a common feature and is not limited to a specific type of DNA damage. They followed with a more detailed analysis using selected compounds and showed that mitotic DNA damage promotes formation of polar chromosomes with stable kinetochore-microtubule attachments. The live-cell imaging revealed that the cells carrying DNA damage eventually exited mitosis with the misaligned chromosomes forming micronuclei in the daughter cells. Most of the conclusions are supported by the experimental data with some exceptions detailed below.

Major comments:

1. In the opinion of the reviewer, the study is somewhat unbalanced as it starts with a high throughput analysis of a large number of compounds but only etoposide treatment is investigated in detail in the key experiments shown in Figures 5 and 6. The effect of topoisomerase II inhibitor on kinetochore-MT stability has already been demonstrated by Bakhoum et al, 2014. If authors wish to generalize that similar phenotypes are observed after various types of DNA damage, they should test additional compounds (such as Lomustine, Mitomycin C, and Carboplatin). In addition, measuring of the kinetochore-MT half-life in figure 5 should be performed with better time resolution within the first 5 minutes. This would allow better comparison of the measured half lives that are much shorter than 5 minutes.
2. Authors claim that the observed phenotype of the chromosomal missegregation following the mitotic DNA damage occurs specifically in cancer cells but the data supporting this statement is poor. They need to show more data in non-transformed cells or remove the statement.
3. Involvement of Kid and Kif4A in arm ejection of the polar chromosomes is an interesting observation in context of mitotic DNA damage. However, it is unclear how the distribution of the chromokinesines was evaluated in Figure 6A-D. Was the signal quantified at the metaphase plate or at polar chromosomes? It seems that Kif4A localizes to the polar chromosome caused by etoposide treatment whereas no signal is visible in DMSO control (Fig. 6C).
4. Authors convincingly showed that SAC is activated by mitotic damage and this is also consistent with previous reports. However they did not address if DDR pathways contribute to the activation of SAC. This would be interesting especially in context of a recent report that showed Bub3 as a direct substrate of ATM (Xiao et al. 2022, JBC). I wonder if the polar chromosomes are formed and missegregate also in the absence of ATM activity.

Minor comments:

1. Specificity of Aurora-B pT232 antibody should be validated to exclude cross-reactivity with Aurora-A at spindle pole (Fig S8C)

Significance

This study addresses the impact of mitotic DNA damage on chromosome segregation which is an important but largely unexplored topic. Authors extend earlier observations by Bakhoum et al, 2014 and demonstrate that also the misaligned polar chromosomes result in formation of micronuclei and may promote chromosomal instability. The study will be of interest mainly for the mitosis filed. The possibility that the described phenotype may have implications for cancer therapies is interesting but will surely require more detailed studies.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Manuscript number: RC-2022-01406

Corresponding author(s): Harris, Reuben

1. General Statements [optional]

We would like to thank all three reviewers for their time and thorough assessment of our manuscript. We appreciate their constructive feedback and believe our work has been considerably strengthened by addressing the comments, suggestions, and concerns raised during peer review. In the following responses, we address the reviewer’s critiques point-by-point.

2. Point-by-point description of the revisions

Reviewer 1

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Moraes et al. build upon their recent studies of APOBEC3 antagonism by EBV BORF2 by showing that additional RNR subunits encoded by other herpesviruses share this activity, suggesting that the host-virus arms races involving APOBEC3 proteins is more widespread than previously thought. Furthermore, the authors show that herpesviruses infecting primates that lack A3B (New World Monkeys) do not apparently exhibit a capacity to antagonize human A3B, suggesting that this function was not required during the evolution of those viruses (while it seemingly was important for viruses infecting hosts that encode A3B). Overall, this is a technically sound submission that combines confocal immunofluorescence, co-immunoprecipitation, and enzymatic assays to comprehensively test the sensitivity of different A3s to counteraction by viral RNRs. The enzymatic (deamination) assays performed prove to be the most insightful, since co-IP and colocalization microscopy was not entirely sufficient to reveal which domains of A3 are important for targeting by RNRs. It is well-written, well-organized, and well-referenced, and will be of interest to readers who study APOBEC3s, herpesviruses, and host-virus arms races more generally.

Response: Thank you for appreciating the technical aspects and broader impact of our studies.

Major points:

1. Figure 4B: the fluorescence microscopy data does not match well with the Co-IP (in Figure 4A). For example, L1B in A3A enhances the A3A-BORF2 co-IP but no clear differences are observed in colocalization. Or are the authors claiming the presence of L1B results in greater colocalization between A3A and BORF2, because there is slightly less diffuse BORF2 in the cytoplasm under these conditions? If that is the case, then quantitative colocalization analysis will need to be performed. In general, virtually none of the colocalization analysis in Figure 4B matches well with the co-IP results of Figure 4A. The authors take this to suggest that L7, and not L1, is most important determinant for BORF2 binding to A3s, but in that case, then the colocalization data is disconnected functionally from co-IP results. This is not necessarily a large problem, since the authors ultimately test the enzymatic activity of A3s in the presence of different RNRs. These latter functional experiments more objectively define what regions of A3s are important for antagonism by RNRs.

Response: Thank you for giving us an opportunity to clarify these important points. We understand how the fluorescence microscopy data in Figure 4B may at first appear to disagree with the co-IP results in Figure 4A. However, we would like to point out that WT A3A—which has a shorter L1 region and binds less strongly to BORF2 compared to A3B—is nevertheless efficiently relocalized by BORF2 (PMID 35476445, 31534038, 31493648). We believe that this observation can be explained by compensatory avidity interactions during cytoplasmic aggregate formation in living cells, a process mediated by the formation of a non-canonical BORF2-BORF2 dimer as detailed in our recent cryo-EM studies (PMID 35476445). These avidity interactions explain how a weaker interaction (as indicated by weaker co-IP levels) can still result in the formation of large cytoplasmic aggregates. We have therefore revised our text to explain this apparent incongruity (page 11, lines 10-13).

Can the authors discuss/cite more about the actual subcellular compartments that the A3s are being relocated towards by the RNPs? In general, the authors' comments are limited to whether the A3 is predominantly in the nucleus, or not.

Response: Previous imaging studies with markers for cytoplasmic organelles by our lab suggested that BORF2-A3B aggregates accumulate within the endoplasmic reticulum (ER) (PMID 30420783). However, in our recent cryo-EM studies of the BORF2-A3B complex (PMID 35476445), we discovered that disrupting BORF2-BORF2 dimerization prevents aggregate formation but does not affect EBV BORF2’s ability to bind to A3B and relocalize the complex to the cytoplasmic compartment. In other words, dimerization-deficient mutants of BORF2 clearly cause A3B-BORF2 heterodimers to appear diffusely cytoplasmic. Therefore, we no longer have a reason to implicate the ER and we aim to clarify this in future studies aiming to define the full molecular composition of the large cytoplasmic aggregates.

Since the authors draw a connection between the absence of A3B in New World Monkeys and the fact that New World Monkey-specific viruses don't seem to counteract A3s, can the authors discuss what could be learned by studying human individuals who lack A3B and the evolution of herpesviruses in those individuals?

Response: This is a very interesting point, but we would prefer not to speculate on this in our manuscript. Although there is indeed an A3B deletion allele in the human population (predominantly southeast Asia), its worldwide allele frequency is quite low and most people still have 1 or 2 copies of this antiviral gene. Thus, the deletion allele frequency is not high enough to remove the selective pressure on the virus to maintain A3B counteraction activity through its RNR.

        However, we did discover one Old World monkey species that completely lacks *A3B* (*Colobus angolensis*). We showed that the RNR from the gamma-herpesvirus that infect these monkeys (ColHV-1) lacks the ability to antagonize human A3B, ancestral A3B, human A3A, or the endogenous A3A of its natural host (__Figure 8__ and __Figure 8—figure supplement 3__). Thus, as you predicted, relieving the selective pressure within a species over an evolutionary period of time likely resulted in loss of A3B-antagonism activity by the viral RNR (page 19, lines 8-16).

Minor points:

1. I'm not sure it makes sense to call out Figures 1A-D in the Introduction section, rather than the Results section.

Response: We have changed the Introduction and removed the original Figure 1 completely. We have however added a new Figure 1, which provides a structural rationale for our overall experimental approach.

Reviewer #1 (Significance (Required)):

This work represents a step-wise advance from the authors' previous work on herpesvirus RNPs and counteraction of host APOBEC3s. I study host-virus molecular arms race on evolutionary scales and this article is of interest and significance to me, and I assume to others in the field as well. The findings found within the submission are interesting but not necessarily informative about human health and disease. However, the subsequent work that this manuscript inspires is likely to tell us more about herpesvirus evolution in human patients and the mechanisms by which APOBEC3s promote cancer.

Response: We thank you again for appreciating the broader significance of our work and how the results present here may inspire important future studies.

Reviewer 2

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: Building off the groups prior work on A3B and EBV BORF2 interactions, here they have expanded their studies to examine additional herpesvirus RNRs, demonstrating which features are conserved. Using a combination of IP experiments and IF, they have included KSHV ORF61 and HSV-1 ICP6 RNRs, and demonstrated that the A3 loop structures, L1, L3, and L7 from A3A, A3B, and A3G play varying roles in determining the ability to interact with the different RNRs. They then go on to demonstrate that the ability of BORF2 to block the deaminase activity of A3B is dependent on the tyrosine at position 481. Lastly, and most interestingly, they show that RNRs from Old World monkeys, but not New World monkeys, can bind to A3A and A3B, lead to their re-localization, and block deaminase activity.

Response: We thank you for appreciating the molecular details and broader impact of our studies. Please note that we have revised the paper to focus on gamma-herpesviruses by removing the less informative results with HSV-1 and adding new studies on Old/New World viral RNRs including comparisons with ancestral A3B.

Major comments: The vast majority of this work is very convincing. The authors claims are clearly reflected in the data presented for the most part. However, the work done with HSV-1 ICP6 co-IP is not very convincing. The authors claim that L7 and L3 swaps from A3Bctd to A3Gctd decreases pulldown (lines 5-12, p.7; lines 18-21, p.8; line 17, p.16). The figures (2A, 3A, 4A) however show only A3A being pulled down with ICP6. The re-localization data however does seem more consistent with the above claims. The authors note this in line 9, p.8. However, they come to a different conclusion in line 2, p.8, regarding the discrepancy between IP and IF data.

Response: As mentioned above, we have removed the less convincing results with HSV-1 ICP6. We believe uncovering the mechanistic details of HSV-1 ICP6 interaction with A3B will require significant additional work and, therefore, would prefer to address this question in future studies.

The data and methods are clearly presented, with the exception of the supplemental figures, where it is unclear how the predicted modeling was conducted.

Response: We apologize for the brief description in our earlier submission. We have revised our Methods section and included a more detailed description regarding the generation of protein structural models (page 29, lines 20-23; page 30, lines 1-6).

Experiments all seem to be sufficiently replicated.

Response: Thank you.

Minor comments:

The references to prior studies seem comprehensive. Text and figures were all very clear. Introducing the supplemental figure 1 earlier, may provide clarity to the argument about degree of relatedness (line 2, p.7).

Response: We agree with this suggestion and have made changes to introduce the structural model of KSHV ORF61 in our new Figure 1.

The suggestion of ORF61 interaction with L3 as an anchor region (line 10-12, p.9) was not very clear/could benefit from a bit more elaboration.

Response: We agree with this comment and have placed the predicted structural model of KSHV ORF61 bound to A3Bctd in our new Figure 1 and we have changed the text to clarify the role of A3B L3 in binding to KSHV ORF61 (page 10, lines 4-8).

Reviewer #2 (Significance (Required)):

This work builds on the conceptual framework of host-pathogen interactions and co-evolution, adding new examples of co-divergence of primate herpesviruses with their respective host restriction factors. Following up on past findings (Cheng et al., 2019; Shaban et al., 2021), and reports from others (Stewart et al., 2019), they outline the degree to which their initial findings (BORF2 and A3B interactions) are conserved across other herpesvirus RNRs, and place them in the context of the evolution of the A3 gene locus and expansion.

This work will be of great interest to virologists. Especially those that work in the field of host pathogen evolution and the molecular arms race.

My background is in host-pathogen interactions and herpesvirus evolution. I lack the sufficient expertise to evaluate the predicted modeling.

Response: We thank you again for appreciating the novelty and significance of our work. We are also hopeful that it will be of great interest to virologists.

---

Reviewer 3

---

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript, Moraes and colleagues build upon previous publications from this group to 1) characterize the variation in the ability of orthologs of BORF2 from six different herpesviruses to bind and/or relocalize and/or inhibit the deaminase activity of A3A and A3B; 2) use swaps and other mutagenesis to measure whether various regions and amino acids in A3A, A3B, and A3G contribute to the observed variation in the ability of RNR subunits from different viruses to bind these A3s.

The data convincingly show that different regions of different A3s contribute differently to binding of RNRs from different viruses. These same regions also have variable effects on RNR-mediated relocalization and inhibition of the deamination activity of A3A, A3B, and A3G. In the last set of experiments presented in the manuscript, the authors show that the RNR from the four viruses isolated from humans and rhesus macaques are able to bind human A3B, while the RNRs from two New World monkey viruses are unable to bind human A3B. Finally, the authors suggest a correlation between the timing of the birth of A3B in the branch leading to the last common ancestor of hominoids/Old World monkeys and the gain of A3 binding/antagonism by herpesvirus RNRs. However, these evolutionary implications are not convincingly supported by the current datasets and would require a significant burden of initial experiments to test.

Response: We thank the reviewer for the nice summary of our work and for appreciating the loop swaps experiments showing differential RNR binding to APOBEC3s. In our original submission, we compared the RNRs of 4 viruses infecting Catarrhini primates and 2 viruses infecting New World primate species. We found that only the RNRs from viruses that infect Catarrhini primates bind, relocalize, and inhibit human A3B. We have now performed additional experiments to further investigate this remarkable association (Figures 7, 8, and associated supplementary material) which are detailed in our revised manuscript and summarized below:

First, we have expanded the scope of our experiments to include all publicly available RNR sequences from primate gamma-herpesviruses (i.e., 11 RNRs in contrast to our initial 6 RNRs). Second, we tested this whole panel against human A3B and found that only the RNRs from viruses that infect Old World primates that encode A3B are able to bind, relocalize, and inhibit human A3B (Figures 6 & 8). In comparison, binding to human A3A in co-IP experiments is invariably weaker and/or not detectable, relocalization phenotypes are less pronounced, and DNA deaminase activity is not inhibited (Figure 6 and Figure 8—figure supplement 2B). __Third, a subset of this RNR panel was tested against the A3A enzymes of their natural host species (__Figure 7) and, again, only the RNRs form viruses that infect Old World primates bind and relocalize the A3A enzymes tested. Fourth, as an addition test of this idea, we grafted a short helical loop structure (HLS) from EBV BORF2 into the marmoset CalHV-3 RNR and showed that this small change enabled the chimeric protein to bind to both human A3B and A3A (likely through L7), though not to the natural marmoset A3A protein. Fifth, we used all available present-day primate A3B sequences to reconstruct the most likely ancestral A3B sequence and showed that this enzyme is nuclear and as active (if not more active) than human A3B (Figure 8). This ancestral A3B protein is also bound, relocalized, and inhibited by most present day RNRs from gamma-herpesviruses that infect species with A3B, but not by the RNRs of any of the NWM-infecting viruses tested. The only exception to this association between A3B and Catarrhini-infecting gamma-herpesviruses is the RNR of the African Colobus virus ColHV-1, which we found can likely be explained by the loss of A3B in its host species due to a deletion that occurred approximately 10-14 mya after the split of the Colobinae subfamily into African and Asian tribes, which further supports the idea that A3-antagonism by gamma-herpesvirus RNRs is maintained by the selective pressure imposed by the antiviral activity of A3B.

Major Comments

1) The use of only the human orthologs of A3A and A3B limit the inferences that can be made regarding the ability of RNRs from various viruses to bind the A3s from the host species of that virus. For example, human A3A (and other hominoid A3As) have a rather distinct Loop 1 sequence, where that same loop in rhesus A3A is a much more similar to A3B. It follows that the RNRs from rhesus-tropic viruses could very well bind and inhibit A3A from rhesus. Likewise, the A3B-RNR interactions within and between species could differ markedly. Indeed, we know that the loops of A3s are some of the most rapidly evolving regions of these genes.

Response: We agree fully with these points and have addressed them through several new experiments. We have now tested the RNRs from rhesus macaque and NWM gamma-herpesviruses against the A3A enzymes of their natural host species (Figure 7) and found that only RNRs form viruses that infect Old World primates bind and relocalize the A3A enzymes tested. In addition, we used all available present-day primate A3B sequences to reconstruct the most likely ancestral sequence and showed that this ancestral A3B enzyme is antagonized exclusively by the RNRs of present-day gamma-herpesviruses that infect A3B-encoding primates.

2) If RNR's ability to bind A3s correlated or was driven by the birth of A3B in catarrhine primates, the evolution of the binding/antagonism trait would be highly unparsimonious. The most parsimonious scenario would be emergence of A3 antagonism in the LCA of alpha and gammaherpesviruses (since the authors show A3 binding in HSV-1 and several gammaherpesviruses) with a loss of the trait in NWM-infecting viruses; alternatively, the trait could have been horizontally transferred gained 3 independent times, but this is certainly unlikely and not supported by any data. However, it is also possible that the RNRs from NWM infecting viruses do, in fact, bind/antagonize the A3 orthologs from NWMs. This needs to be tested before addressing the complexities of the birth of the antagonism trait.

Response: Please see our responses above. All of our results support a model in which the birth of A3B in an ancestral primate selected for a gamma-herpesvirus with A3B binding and neutralization activity and that this activity has been maintained through evolution and still manifests today in all of the tested present-day RNRs of gamma-herpesviruses that infect species with A3B.

Minor Comments

1) The authors should state more discreetly what is new to this paper and what was shown in previous paper and in some cases repeated here. For example, figure 1 is all repeated experiments from previous papers which is unusual for a manuscript.

Response: This is a fair point and we have removed the original Figure 1 and replaced it with a structural model that provides a strong rationale for the rest of our studies. For the sake of clarity, we have also revised our text and made the necessary changes to ensure a clear distinction between new and repeated results.

2) The authors conclude that RNRs bind to A3s via partially distinct surfaces, but they don't actually test binding. Swaps and mutations do not show that the site of mutation is a site of interaction. but they do test the requirement of these AAs or regions for binding. Formally, these mutations could be exerting an allosteric effect on the binding interface of RNR and A3. In combination with the CryoEM data, these new data do support the model that these are different surfaces of interaction, but the wording should be more precise to present this.

Response: In our revised manuscript we use a combination of in silico protein structure prediction and docking to model the binding interface between human A3Bctd and KSHV ORF61 (new Figure 1). This approach predicts an interaction with the L3 region of A3B, which we validate through co-IP and co-localization experiments (Figure 3). In contrast, EBV BORF2 requires the L7 region to bind to A3Bctd and this interaction is additionally strengthened by L1 residues (Figures 2 & 4). Taken together with our prior cryo-EM data, these results point to a model in which EBV BORF2 and KSHV ORF61 bind to different surfaces of A3B (albeit near the active site and likely due to evolutionary “wobbling”). We therefore believe it to be unlikely that this mechanism is allosteric given our prior structural studies and the likely common evolutionary origin of this A3B antagonism mechanism.

3) Similar to point 1, the authors repeatedly discuss the "most critical determinant of EBV BORF2 binding" and other "most critical" interactions. This is not supported by the data and should be changed to something along the lines of 'the site of largest effect among the sites we analyzed'.

Response: We have endeavored to change this text as suggested except in cases referring to the interactions between EBV BORF2 and A3Bctd, since the results presented here together with our cryo-EM structure of the BORF2-A3Bctd complex (PMID 35476445) allow us to confidently say that L7 and L1 are the most critical determinants.

4) All microscopy figures need an A3 only panel (no RNR) to be able to judge relocalization.

Response: Changed as suggested.

5) The matrix of labels above each IP blot is excessive since each lane only has one component that differs from the other lanes. A single label for each lane would make the plot easier to discern. These figures would also benefit from clearer labels including which virus each blot panel corresponds to (these could be along the left side of each blot; currently, the RNR gene name is provided, but this is a bit hard to find within the figure). Figures 2-4 would benefit from a label above each panel A indicating "L1" "L3" "L7".

Response: Changed as suggested.

6) If the authors comment on pg9 ln 8 about intermediate relocalization effect, they should also mention 1C A3B L7G against BORF2

Response: Changed as suggested (page 8, lines 16-19).

7) Why is there no quantification of 6D relocalization? Could be supplemental if needed.

Response: We have performed quantification of the relocalization phenotypes in Figures 2, 3 & 4 in order to allow direct comparison between WT and chimeric A3 enzymes in the presence of the same viral RNR (EBV BORF2 or KSHV ORF61). On the other hand, the images in Figure 6D are representative of the A3B/A relocalization phenotypes elicited by a larger panel of different viral RNRs. These representative images should be interpreted together with the co-IP and ssDNA deaminase activity assay data in Figures 6C & 6E, respectively.

8) Pg 6 ln 13 and Pg 8 ln2-4, ICP6 doesn't coIP w A3B; this should be clarified.

Response: A similar concern was also raised by reviewer #2, and we agree that the ICP6 data present in the original version of this manuscript are not as easily interpretable compared to results with the RNRs from gamma-herpesviruses such as EBV and KSHV. For the sake of clarity and cohesion, we decided to remove all of the HSV-1 ICP6 data from the revised version of our manuscript and focus on the A3B interactions with gamma-herpesviruses.

9) Pg 8 ln21-23, the authors assume loss of function for A3G, but this swap could be functionally equivalent, but necessary for binding; it should be clarified that this is different than changing sequence and still binding.

Response: Rephrased (page 9, lines 13-16).

10) Pg 9, ln 11, what is an anchor region?

Response: We have removed the term “anchor region” and rephrased our text to more clearly describe the importance of L3 in KSHV ORF61 binding (page 10, lines 4-8).

11) Pg 9, ln 23, speculative - this might be explained by this 3 AA motif but it has not been tested.

Response: Changed wording (page 10, lines 17-20).

12) Pg 10 ln 8, this doesn't show that this region is dispensable for binding, only that there is equivalent contribution or lack of contribution of function by the A and B loops, again assuming that the G loop is LOF

Response: Removed the phrase “indicating that this region may be dispensable for the interaction” (page 11, 1-2).

13) Pg 10 ln 10 - "can be explained by presence of bulky tryp" - this should be reworded to 'could or is likely caused by'.

Response: Changed as suggested (page 11, lines 4-6).

14) Pg 11 ln 21, "can be explained by our cryo-EM" should be reworded to 'is supported by these contacts in cryo-EM'

Response: Changed as suggested (page 12, lines 15-18).

15) Pg 13 ln 10 (and other places) dissociation rates are only part of affinity, Ka is equally important (pg 18 ln 8 also)

Response: We agree and have revised our text account for this suggestion (page 13, lines 22-23; page 14, lines 1-2; page 22, lines 12-15).

16) Pg 14, ln 15 should be reworded to 'relocalize HUMAN cellular A3s'

Response: Changed as suggested (page 15, line 11).

17) Pg 16 Ln 16, this should be reworded as the data can't say it is completely dispensable without deletion of the loop.

Response: Changed as suggested (page 21, lines 10-11).

18) New World monkeys have high activity of transposable elements of distinct types relative to catarrhines. It would be useful to mention that A3s restrict endogenous elements as well and how this might be a factor in the proposed evolutionary model.

Response: This is a very interesting point that we plan to discuss in a future review. In addition, many future experiments will be needed to test the potential relationship between the birth of A3B and its potential impact on different classes of endogenous transposable elements.

19) Are the New World monkey viruses pathogenic in their native hosts? Perhaps not based on previous literature (reviewed in PMID: 11313011). This should be included in the discussion as it could certainly effect the evolutionary model for the birth/retention of A3 antagonism in these viruses.

Response: While interesting, the observation that NWM herpesviruses do not cause disease in their native hosts is not unusual. In fact, most gamma-herpesviruses (including human viruses like EBV and KSHV) have limited pathogenic potential when infecting their natural hosts (Fleckenstein B, Ensser A. Gammaherpesviruses of new world primates. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. 2007). Additionally, although the mentioned study (PMID: 11313011) reports asymptomatic infection of squirrel monkeys with SaHV-2, pathogenic infection/ oncogenic transformation have been reported following natural infection of marmosets with CalHV-3 (PMID: 11158621).

20) In previous papers on this topic, the lab has tested the effect of mutations on viral titers. While this may be beyond the scope of this paper, this would certainly elevate the paper and should be more clearly discussed.

Response: As noted by the reviewer, we have previously demonstrated that A3B restricts EBV replication though a mutation-dependent mechanism and that this is counteracted by EBV BORF2 (PMID 30420783). While we completely agree that investigating the effect of A3B-catalyzed mutations on the titers of different gamma-herpesviruses would be interesting, this would be technically challenging as we are currently not equipped to work with KSHV or any non-human primate herpesvirus.

21) What is the degree of sequence similarity among these and other RNRs? Is there any sense of what region of RNR binds A3s from the CryoEM structures and differences within these regions that might explain the functional differences?

Response: We thank the reviewer for raising this important point. We have now included a new Figure 1 where we leverage the cryo-EM structure of the EBV BORF2-A3Bctd complex to make inferences about which regions of KSHV ORF61 may be involved in binding A3B/A. As described above, we also graft a short helical loop structure (HLS) from EBV BORF2 into the marmoset CalHV-3 RNR and showed that this small change enables the chimeric protein to bind to bind both human A3B and A3A (likely through L7), though not to the natural host marmoset A3A protein (Figure 7). Many additional interspecies chimeras could be constructed but we feel these are better suited for future studies (and specially to accompany future structural work in this area).

---

Reviewer #3 (Significance (Required)):

Significance

Previous work from the Harris lab showed that a subunit of the ribonucleotide reductase of some herpesviruses acts as an antagonist of several human APOBEC3s. Mechanistically, these viral protein block A3 inhibition by relocalizing nuclear A3s as well as inhibiting A3 deamination by binding and occluding the A3 active site. For Epstein-Barr virus, deletion of the antagonist (BORF2) results in a decrease in viral replication and accumulation of mutations likely introduced by host A3B that is no longer inhibited. However, deletion of the A3 antagonist from herpes simplex virus-1 (ICP6) had no effect on viral titers. Most recently, this group published a cryoEM structure of BORF2 in complex with the c-terminal half of A3B. This structure showed extensive contacts between BORF2 and two loops of A3B - L1 and L7.

The manuscript under review focuses on the previously suggested differences in the ability of different RNRs to bind A3A and A3B. This work provides an important contribution to this topic in defining specific regions of A3A and A3B and A3G that are necessary for viral RNRs to bind them. The variability in these interactions is surprising and likely testament to the impactful coevolution of herpesviruses and primate A3s. This manuscript will be of particular interest to virologists studying A3s or herpesviruses as well as evolutionary biologists interested in the rules of engagement between host restriction factors and viruses.

Response: We thank you again for these thoughtful comments and for appreciating the overall significance of our work.

---