Author Response

Reviewer #2 (Public Review):

“To describe LLPS or to distinguish between polymer-polymer phase separation and LLPS, recent studies have used single particle tracking, a technique allowing to follow the dynamics of individual proteins in living cells (https://doi.org/10.7554/eLife.60577; https://doi.org/10.7554/eLife.69181; https://doi.org/10.7554/eLife.47098). The authors should mention that such an approach can be a good alternative to avoid the artefact of fixation. Using techniques such as single particle tracking or FCS, it is possible to estimate the effective diffusion coefficient of protein-living cells. When a liquid phase separation is formed, it is also possible to estimate the diffusion coefficient of the protein of interest (POI) inside versus outside of the LLPS.”

We thank the reviewer for their insight and fully agree that live-cell techniques like SPT and FCS are valuable for investigating LLPS while avoiding fixation artifacts. We have added discussion emphasizing this fact and incorporated the citations recommended by the reviewer in Paragraph 1 on Page 15: “Live imaging techniques that allow estimation of protein diffusion coefficients within specific cellular compartments, e.g., SPT (Hansen et al., 2018 and Heckert et al., 2022) and fluorescence correlation spectroscopy (Lanzanò et al., 2017), can be useful alternative approaches for diagnosing LLPS in vivo without the potential artifact of fixation, as diffusion dynamics are recently shown to be affected by LLPS (Heltberg et al., 2021; McSwiggen et al., 2019a; Miné-Hattab et al., 2021; Chong et al., 2022; and Ladouceur et al., 2020).”

“The authors say that less dynamic interactions are better captured by PFA fixation. In the simulation part, would it be possible to predict from the diffusion coefficients of the POI inside a condensate the effect of the PFA fixation? […] In the simulation part, they could try to incorporate the diffusion coefficient of the protein of interest and see if it is possible to predict the effect of fixation as a function of the diffusion coefficient.”

We thank the reviewer for pointing out the absence of this critical piece that connects our experimental observations to our kinetic model. Our model considers association/dissociation rates rather than diffusion coefficients to describe interaction dynamics, but the reviewers’ point is still very insightful and important. As described in Response 2, we compared two proteins: Halo-TAF15(IDR), which is poorly preserved by fixation, and TAF15(IDR)-Halo-FTH1, which is well preserved by fixation. We used SPT to measure the dissociation rates of Halo-TAF15(IDR) and TAF15(IDR)-Halo-FTH1 and showed that the dissociation rate of Halo-TAF15(IDR) from its puncta is much faster than that of TAF15(IDR)-Halo-FTH1, demonstrating more stable homotypic interactions of the latter than the former. The observation that TAF15(IDR)-Halo-FTH1 has less dynamic interactions and is better preserved by fixation compared to Halo-TAF15(IDR) agrees with our model’s prediction that less dynamic interactions are better captured by fixation. Please see Response 2 for more details. Our new data and discussion have been added to the revised manuscript in Paragraph 3 on Page 13 and in Figure 3B, Figure 3E, Figure 6, and Video 2.

“Finally, the authors propose that in the future, it will be important to design novel fixatives with significantly faster cross-linking rates than biomolecular interactions to eliminate fixation artifacts in the cell. It would be even more interesting if the authors could propose some ideas of potential novel fixatives. Did they test several concentrations of PFA, for example? Did they test different times of PFA incubation? Did they test cryofixation and do they know what would be their effect on LLPS? Do they have novel fixatives in mind? […] To strengthen the manuscript, the authors should try more protocols of fixation.”

We thank the reviewer for these good questions. As described in Response 1, we have done additional quantification of the change of LLPS appearance in cells upon treatment of 0% PFA (only PBS buffer), 1% PFA, 2% PFA, and 8% PFA as well as 4% PFA supplemented by 0.2% GA. We saw statistically significant changes in the LLPS-describing parameters upon all the PFA and PFA/GA treatments except the 0% PFA control. To examine how fixation artifacts depend on the time of PFA incubation, we acquired a time-lapse movie of a cell overexpressing EGFP-FUS(IDR) immediately after 4% PFA treatment and quantified the number of puncta over time (Video 1). We showed that fixation is complete (the number of puncta becomes constant) by roughly 100 seconds (Figure 1 – figure supplement 2). Our new data also justified our choice of a 10-minute PFA incubation time for analyzing fixation-induced change of LLPS appearance in the rest of the paper. Please see Response 1 for more details. Our new data and discussion have been added to the revised manuscript in Paragraph 3 on Page 3 and in Figure 1 - figure supplement 2 (time dependence of fixation artifacts), Figure 1 - figure supplement 3 (fixation artifact at various PFA concentrations), and Figure 1 - figure supplement 4 (fixation artifact upon treatment of 4% PFA supplemented with 0.2% GA).

We agree that testing more cell fixation protocols such as cryofixation on LLPS appearance would be interesting. However, given the complexity of novel fixation protocols like cryofixation and highly specialized equipment and reagents they require, testing widely how different fixation methods might change LLPS appearance would be a tremendous amount of work that is enough to fill a separate paper. These experiments would be much more appropriate for a separate study in the future.

Reviewer #3 (Public Review):

“Understanding whether/how fixation methods affect the detection of biomolecular condensates is of broad interest given the importance of LLPS in regulating different aspects of cell biology. However, in this manuscript, the authors use only paraformaldehyde as a fixation method and study only fluorescently-labelled IDR proteins. The work would benefit from a comparison between living cells and cells fixed with other fixation methods.”

We appreciate the reviewer for this suggestion and agree that more fixation protocols should be investigated. As described in Response 1 and Response 18, besides examining PFA fixation, we have quantified how fixation using 4% PFA supplemented by 0.2% GA changes LLPS appearance in cells. We saw statistically significant changes in all the LLPS-describing parameters upon PFA/GA treatments. Please see Response 1 and Response 18 for details. Our new data and discussion have been added to the revised manuscript in Paragraph 3 on Page 3 and in Figure 1 - figure supplement 4.

“In addition, it would be useful to test the impact of these fixation methods on the detection of endogenous proteins or IDR proteins without fluorescent tag.”

We appreciate the reviewer for this suggestion and have now investigated an endogenous IDR-containing protein in the revised manuscript. Specifically, we quantified the effect of 4% PFA fixation on endogenously expressed EWS::FLI1 in an Ewing sarcoma cell line A673, which is an oncogenic fusion transcription factor that causes Ewing sarcoma (Grünewald et al., 2018) and known to form local, high-concentration hubs at target genes associated with GGAA microsatellites (Chong et al., 2018). We previously Halo-tagged endogenous EWS::FLI1 in A673 cells using CRISPR/Cas9-mediated genome editing (Chong et al., 2018). Here, we quantified the effect of PFA fixation on endogenous EWS::FLI1 puncta in this knock-in cell line and found no significant difference in the distribution of EWS::FLI1 upon fixation. This result suggests that PFA fixation does not change the intracellular distribution of all proteins. Our new data and discussion have been added to the revised manuscript in Paragraph 1 on Page 8 and in Figure 3C.

Unfortunately, testing fixation artifacts of IDR-containing proteins without a fluorescent tag has been infeasible as we rely on fluorescence from a tag on the protein of interest to quantitatively compare LLPS appearance in live and fixed cells. Although we have considered using non-fluorescent methods, e.g., phase contrast microscopy, to visualize putative LLPS in cells, its lack of specificity in imaging proteins or cellular structures makes the type of quantification we do for fixation artifact characterization inaccessible.

Reviewer #3 (Public Review):

The authors compare the detection of biomolecular condensates in living cells overexpressing fluorescently tagged IDR proteins and upon fixation with paraformaldehyde (PFA). Given that they observe differences in the number and size of the condensates in the fixed versus living cells the authors conclude that the fixation method can introduce an artifact in the visualization of these condensates. Next, through kinetic modeling simulations, the authors propose a model in which the extent of the artifact introduced by PFA fixation correlates with the strength of the protein-protein interaction: artifacts are lower when the protein‐protein interactions are stable and less dynamic compared with the overall fixation rate. Based on their comparative analysis of PFA fixation and the kinetic modeling the authors strongly recommend caution in the interpretation of data obtained in PFA-fixed cells and suggest that parallel studies with living cells should be performed.

Understanding whether/how fixation methods affect the detection of biomolecular condensates is of broad interest given the importance of LLPS in regulating different aspects of cell biology. However, in this manuscript, the authors use only paraformaldehyde as a fixation method and study only fluorescently-labelled IDR proteins. The work would benefit from a comparison between living cells and cells fixed with other fixation methods; in addition, it would be useful to test the impact of these fixation methods on the detection of endogenous proteins or IDR proteins without fluorescent tag.

Are the additional bands from the cleaved GFP tag? Again, a WB against HY5, SPA, and GFP may elucidate these bands.

Author Response

Reviewer #3 (Public Review):

The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.

Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:

(i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.

(ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.

(iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).

In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.

We thank the reviewer for the time dedicated to our manuscript. We are thankful for the critical and specific comments, which allowed us to further improve our manuscript. We agree that it would have been beneficial to have higher frame rates and there instead of two imaging channels. However, this would have further added technical complexity to an already complex experimental setup resolving fluorescent puncta with sizes below the resolution limit. And we are convinced that all our main conclusions are justified based on the imaging settings in the current data sets.

Reviewer #3 (Public Review):

The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.

Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:<br /> (i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.<br /> (ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.<br /> (iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).

In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.

unnecessary use of br tag (breaks)

i believe there should be more space between the heading tag and the content use after it. and the text size need to bigger for the content like month names, as i see in reference image.

its not look same as image reference its better if you use heading tag to make month name as heading.

Alternate thesis for why countries and cities vie to host money-losing events like the World Cup and the Olympics: grift.

With the necessary need for building infrastructure, there's easy and ample opportunity for cooking the books and pushing cash flow into the pockets of contractors and political figures as well as into the pockets of the governing bodies and their officials.

Cross reference FIFA bribery

Some of the money may go into the local economy and workers which is good, but who's really benefitting here? Where is the money going? Who is footing the loss? It can't all be written off to goodwill.

looks and nice and you have mentioned nicely

most of the time I use the "Div" tag instead of "article". I feel it gets easy to work with "Divs". "article " is not the issue still

You can use small tag to make this sentence smaller.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

You can use h3 tag instead of strong tag.

you can use the "main" tag here for the web content. It'll be good to have both header and footer too.

You can use this h1 tag in header section.

ArtículoHDBIOcolores

Zotero

Hello

Author Response

Reviewer #2 (Public Review):

Grasses develop morphologically unique stomata for efficient gas exchange. A key feature of stomata is the subsidiary cell (SC), which laterally flanks the guard cell (GC). Although it has been shown that the lateral SC contributes to rapid stomatal opening and closing, little is known about how the SC is generated from the subsidiary mother cell (SMC) and how the SMC acquires its intracellular polarity. The authors identified BdPOLAR as a polarity factor that forms a polarity domain in the SMC in a BdPAN1-dependent manner. They concluded that BdPAN1 and BdPOLAR exhibit mutually exclusive localization patterns within SMCs and that formative SC division requires both. Further mutant analysis showed that BdPAN1 and BdPOLAR act in SMC nuclear migration and the proper placement of the cortical division site marker BdTANGLED1, respectively. This study reveals a unique developmental process of grass stomata, where two opposing polarity factors form domains in the SMC and ensure asymmetric cell division and SC generation.

The findings of this study, if further validated, are novel and interesting. However, I feel that the data presented in the current manuscript do not fully support some crucial conclusions. The lack of dual-color images is the weakest point of this study. If it is technically impossible to add them, alternative analyses are needed to validate the main conclusions.

1) Is BdPOLAR-mVenus functional? Although the authors interpret that weak BdPOLAR-mVenus expression partially rescued the bdpolar mutant phenotype in Fig. S4D, the localization pattern visualized by BdPOLAR-mVenus may not be completely reliable with this partial rescue activity.

This is indeed a valid point. The partial complementation of weakly expressing translational reporters (Figure 3–figure supplement 1D) and the weak effect of BdPOLAR-mVenus overexpression lines (Figure 3–figure supplement 1J) at least suggest partial functionality which is strongly dependent on dosage. Yet the localization pattern and the temporal dynamics might indeed not fully reflect the spatiotemporal dynamics of the endogenous BdPOLAR. This criticism is, however, true for any transgenic reporter line–even when fully complementing–as the requirement for dosage, stability, and turnover likely varies strongly between different protein classes and functions.

Nonetheless, we have added a sentence on p. 7, which mentions this potential caveat.

2) Regardless of the functionality of the tagged protein, the authors need to provide more information on their localization. For example, is there a difference in polarity pattern depending on expression level? Does overexpressed BdPOLAR-mVenus invade the BdPAN1 zone? In such cases, might the loss of BdPOLAR polarity in the bdpan1 mutant be a side effect of overexpression, not PAN1 exclusion? Does BdPOLAR expression (no tag) show a dose-dependent effect, similar to the mVenus-tagged protein?

The difference in polarity patterns in bdpan1 mutants and wild-type does not depend on expression level. BdPOLAR-mVenus was crossed into bdpan1 and mutant and wild-type siblings in the F2 generation were analyzed. This means that the data presented in Fig. 3E and F show exactly the same transgene insertion line in wt and bdpan1 and were imaged with the same setting for comparability. Therefore, the difference in localization is not due to different expression levels but indeed reflects a PAN1-dependent effect.

To address if BdPOLAR without a tag is also sensitive to dosage, we have generated an untagged complementation line that includes the untagged, genomic locus of BdPOLAR including promoter (-3.1kb) and terminator (+1.1kb). Yet, even though this construct is much better at rescuing the mutant, we still see remaining defects in T0 lines (Figure 3–figure supplement 1K) suggesting that even without a tag we cannot fully recapitulate wild-type functionality. Yet, to actually measure protein levels of untagged BdPOLAR, we would need to raise an antibody against BdPOLAR, which we think is clearly out of the scope of this study.

3) A major conclusion of this study was that the polarity domains of BdPOLAR and BdPAN1 are mutually exclusive. However, not all the cells in the figures were consistent with this statement. For example, the BdPOLAR signals at the GMC/SMC interphase appear to match BdPAN1 localization (compare 0:03 s in Video 1 and 0:20 s in Video 2 [top cell]). The 3D rendered image in Fig. 2F shows that BdPOLAR is excluded near the GMC on the front side of the SMC, where BdPAN1 is not localized. Some cells did not exhibit polarization (Fig. 3A, bottom left; Fig. 3E, bottom left). The most convincing data are the dual-color images of these two proteins. Otherwise, a sophisticated image analysis is required to support this conclusion.

We agree that dual-color image analysis would have provided the most convincing data. As mentioned in our answers to the reviewing editor and reviewer 1, we have generated a dual marker line (BdPAN1p:BdPAN1-CFP; BdPOLARp:BdPOLAR-mCitrine), yet the BdPAN1-CFP signal (compared to mCitrine signal) was too weak to visualize the proximal BdPAN1 domain.

This issue was also raised by reviewer 1 and deemed an essential revision. To determine how BdPOLAR and BdPAN1 relate spatially to each other, we have added data in Figure 2E where we manually traced mature SMC outlines to determine BdPOLAR-mVenus and BdPAN1-mCitrine occupancy along the SMC’s circumference. This confirmed that the polarization is indeed opposite yet not perfectly reciprocal (see details above, Essential Revisions #1).

Finally, we realized that the 3D image renderings were more confusing than helpful and we removed them from the revised version.

4) Another central conclusion was that BdPOLAR was excluded at the future SC division site, marked with BdTANGLED1. However, these data are also not very convincing, as such specific exclusion cannot be seen in some figure panels (e.g., Fig. 3A, bottom left; Fig. 3E, all three cells on the left). If dual-color imaging is not feasible, a quantitative image analysis is needed to support this conclusion.

As for point 3, this was also criticized by reviewer 1 and deemed an essential revision by the reviewing editor.

To determine whether the absence of BdPOLAR signal and the presence of BdTAN1 signal colocalize, we again manually traced mature SMC outlines to determine BdPOLAR-mVenus and BdTAN1-mCitrine occupancy along the SMC’s circumference. We plotted the relative average fluorescence intensity in Figure 4G-I nicely showing that BdTAN1 indeed resides in the BdPOLAR gaps above and below the GMC (again, details above, Essential Revisions #2).

5) I could not find detailed imaging conditions and data processing methods. Are Figs. 2B and 2E max-projection or single-plane images? If they are single-plane images, which planes of the SMC are observed? In addition, how were Figs. 2C and 2F rendered? (e.g., number of images, distance intervals, processing procedures). This information is important for data interpretations.

We agree that we might not have provided sufficient imaging condition details and have added more details regarding image acquisition in the method part (p. 20). We always use a consistent depth and show the midplane of SMCs. As mentioned above, we removed Figs. 2C and 2F and the supplemental movies as these data did not seem to be helpful.

6) [Minor point] The authors should clearly describe where BdPAN1 is expressed and localized. Is it expressed in the GMC and localized at the GMC/SMC interface? Alternatively, is it expressed and localized in the SMC?

BdPAN1 is expressed throughout the epidermis but starts to strongly accumulate at the GMC/SMC interface. According to the literature (Cartwright et al 2009 with immunostainings against ZmPAN1 and Sutimantanapi et al. 2014 with PAN1 and PAN2 reporter) and our own observations (Fig. S3), this accumulation occurs in the SMC rather than in the GMC. In Fig. S3A, third panel, second GMC from the top, for example, one can see that the early PAN1 polarity domain expands beyond the GMC/SMC interface suggesting that it is indeed forming in SMCs rather than in GMCs. We have specified this in the text more clearly now (p. 5).

Reviewer #2 (Public Review):

Grasses develop morphologically unique stomata for efficient gas exchange. A key feature of stomata is the subsidiary cell (SC), which laterally flanks the guard cell (GC). Although it has been shown that the lateral SC contributes to rapid stomatal opening and closing, little is known about how the SC is generated from the subsidiary mother cell (SMC) and how the SMC acquires its intracellular polarity. The authors identified BdPOLAR as a polarity factor that forms a polarity domain in the SMC in a BdPAN1-dependent manner. They concluded that BdPAN1 and BdPOLAR exhibit mutually exclusive localization patterns within SMCs and that formative SC division requires both. Further mutant analysis showed that BdPAN1 and BdPOLAR act in SMC nuclear migration and the proper placement of the cortical division site marker BdTANGLED1, respectively. This study reveals a unique developmental process of grass stomata, where two opposing polarity factors form domains in the SMC and ensure asymmetric cell division and SC generation.

The findings of this study, if further validated, are novel and interesting. However, I feel that the data presented in the current manuscript do not fully support some crucial conclusions. The lack of dual-color images is the weakest point of this study. If it is technically impossible to add them, alternative analyses are needed to validate the main conclusions.

1. Is BdPOLAR-mVenus functional? Although the authors interpret that weak BdPOLAR-mVenus expression partially rescued the bdpolar mutant phenotype in Fig. S4D, the localization pattern visualized by BdPOLAR-mVenus may not be completely reliable with this partial rescue activity.<br /> 2. Regardless of the functionality of the tagged protein, the authors need to provide more information on their localization. For example, is there a difference in polarity pattern depending on expression level? Does overexpressed BdPOLAR-mVenus invade the BdPAN1 zone? In such cases, might the loss of BdPOLAR polarity in the bdpan1 mutant be a side effect of overexpression, not PAN1 exclusion? Does BdPOLAR expression (no tag) show a dose-dependent effect, similar to the mVenus-tagged protein?<br /> 3. A major conclusion of this study was that the polarity domains of BdPOLAR and BdPAN1 are mutually exclusive. However, not all the cells in the figures were consistent with this statement. For example, the BdPOLAR signals at the GMC/SMC interphase appear to match BdPAN1 localization (compare 0:03 s in Video 1 and 0:20 s in Video 2 [top cell]). The 3D rendered image in Fig. 2F shows that BdPOLAR is excluded near the GMC on the front side of the SMC, where BdPAN1 is not localized. Some cells did not exhibit polarization (Fig. 3A, bottom left; Fig. 3E, bottom left). The most convincing data are the dual-color images of these two proteins. Otherwise, a sophisticated image analysis is required to support this conclusion.<br /> 4. Another central conclusion was that BdPOLAR was excluded at the future SC division site, marked with BdTANGLED1. However, these data are also not very convincing, as such specific exclusion cannot be seen in some figure panels (e.g., Fig. 3A, bottom left; Fig. 3E, all three cells on the left). If dual-color imaging is not feasible, a quantitative image analysis is needed to support this conclusion.<br /> 5. I could not find detailed imaging conditions and data processing methods. Are Figs. 2B and 2E max-projection or single-plane images? If they are single-plane images, which planes of the SMC are observed? In addition, how were Figs. 2C and 2F rendered? (e.g., number of images, distance intervals, processing procedures). This information is important for data interpretations.<br /> 6. [Minor point] The authors should clearly describe where BdPAN1 is expressed and localized. Is it expressed in the GMC and localized at the GMC/SMC interface? Alternatively, is it expressed and localized in the SMC?

Reviewer #2 (Public Review):

The study aims to characterize the role of lncRNA H19 in senescence and proposes a mechanism involving CTCF and the activation of p53. The authors suggest that H19 loss induces let7b-mediated repression of EZH2, which is a critical component in the regulation of senescence-associated genes. Additionally, the authors state that H19 is required for inhibition of senescence by the mTOR inhibitor rapamycin.

The experiments appear to be performed to a high standard, and the individual observations, and conclusions about the importance of the individual players in senescence appear solid. For example, the authors convincingly show that H19 decreases in expression in aged cells/tissues and that its knockdown leads to entry into senescence. These results are consistent with recent studies in other systems (e.g., ref 38). Also, the knockdown of CTCF convincingly leads to senescence. However, these observations are largely not very surprising/novel. The premise of the manuscript is a connection between these components into a particular "axis" that regulates entry into senescence. This connection between the different regulators studied (H19, CTCF, EZH2, p53), and in particular, their specificity, which is key to the proposed "axis" remains insufficiently supported, and many of the results, unfortunately, appear to be over-interpreted.

Major comments

1. In Figure 1, the authors claim that H19 levels are reduced during aging in vitro and in vivo and that H19 levels are maintained by rapamycin treatment. To state the connection between H19 and rapamycin and its relation to aging, there is a need to show what happens in "young" cells treated with rapamycin.

Furthermore, the authors state that H19 "is essential for the inhibitory effect of rapamycin on cellular senescence". There doesn't appear to be sufficient evidence to support such a claim; additional data emphasizing the direct connection between H19 and rapamycin is needed - e.g., show that in H19-null cells rapamycin does not affect senescence.

2. CTCF is a general regulator involved in various cellular processes and supporting progression through the cell cycle; therefore, its perturbation can lead to global effects on cell health that are not necessarily related to H19. The data shown in figure 2 is insufficient to indicate a direct correlation between CTCF and H19. This will require showing that mutating specifically the CTCF binding sites near H19 affects senescence.

The same applies to the connection between H19 and let-7b shown in Figure 5. It is not very surprising that let-7b, a general antagonist of proliferation, positively regulates senescence. Here as well, the direct connection to H19 is weak. Can the authors rescue the cells that enter senescence following H19 depletion by H19 expression? If so - is this rescue capacity lost when let-7 sites are mutated? Is it possible to rescue by expressing an artificial let-7 sponge instead of H19? Otherwise, let-7b could very well be another factor related to senescence and/or regulated, but not the main mediator of the effects of H19, or part of an axis that includes H19, as proposed in the manuscript.

3. In figures 2d,3f,5i/j the authors present only representative tracks and regions from CUT&Tag-experiments, and its not clear to what extent these changes are significant when considering genome-wide data, replicates etc., and so these data are uninterpretable. This is important, as these panels are used as evidence for specific connections between members of the axis. The authors should provide a statistical test for all the regions in the genome, based on replicates, and show that these changes are significant to use these data to support their model. Otherwise, the specific connection between CTCF and H19 remains weak, and the specific change in p53 regulation of CTCF in the context of senescence is not convincing. In any case, the number of replicates and the QC of the data should be presented, and the data should be made available to the reviewers.

4. The authors state in the Discussion that the mechanism that lead to decreased H19 expression as part of the senescence program consists of two phases: an acute response driven by p53 activation and a prolonged response dictated by the loss of CTCF. There doesn't appear to be enough evidence to support this claim, as the individual experiments don't measure any such bi-phasic phenomena.

please update the sample code. I have fixed it and am sending on slack

good call using the

This closing tag is unnecessary you can remove it.

missing head and closing head before the body tag

https://hypothes.is/search?q=tag%3A%27etc556+etcnau%27

Randomly ran across a great tag full of education resources...

Seems to be related to this class:<br /> ETC 556 - Contexts And Methods Of Technology In Adult Education

Description: This course is designed for adult educators in the various contexts, including: higher education, military, non-profit, health and business settings. Through research, readings and collaborative activities, students will gain an understanding of various adult learning methods that include, but are not limited to, training, professional development, performance improvement, online and mobile learning. Letter grade only.

https://catalog.nau.edu/Courses/course?courseId=011553&catalogYear=2223

comment tag again could be used to differentiate which closing div relates to which div class

Without using the <br> tag you can achive the new line by adjusting the width of the block.

It's better to have some space between each tag in order to understand the code easy(i.e., visibility must be good).

You may want to add small tag to this sentence.

take off the padding on the li and give it to the a tag so you can hover less over the word to select the link, makes it better for accessibility.

Reviewer #3 (Public Review):

This manuscript will be of interest primarily to researchers in the field of NADPH oxidases (NOXs) but also to those interested in the wider ferric reductase superfamily, also comprising members of the six-transmembrane epithelial antigen of the prostate enzymes (STEAPs). More limited interest may be expressed by investigators of ferredoxin - NADP reductases, resembling the dehydrogenase region (DH) of NOXs, expressing lesser "visibility" in the structure described in the paper. Considering the fact that NOXs are essentially electron transport machines from NADPH to dioxygen, along a multi-step redox cascade, those interested in hydride and electron transfer, at a more conceptual level, might also want to have a look at the paper. Elucidating structures of NOXs are still rare achievements, with only four published papers, so far (one coming from the group of the present main author) and, thus, any new publication profits from the aura of novelty.

Introduction<br /> This manuscript offers a detailed and in depth description of the structure of the catalytic core of the human phagocyte NADPH oxidase, NOX2, in heterodimeric association with the protein p22phox. The phagocyte NADPH oxidase is responsible for the production of reactive oxygen species (ROS), the primary molecule of which is the superoxide radical (O2.-), derived by the one-electron reduction of molecular oxygen by NADPH. NOX2 belongs to the NOX family, consisting of 7 members (NOX 1-5, and DUOX1 and DUOX2), sharing common structural characteristics but expressing a wide variety of functions. The principal but not the only function of NOX2 is as a source of ROS for the killing of pathogenic microorganisms (bacteria, fungi, protozoa) engulfed by phagocytes in the course of innate and acquired immunity.

The structures of C. stagnale NOX5, and that of murine and human DUOX1 were determined by X-ray crystallography (NOX5) and cryo-EM (DUOX1). As sources of potentially dangerous auto-toxic ROS, NOXs are subject to strict functional regulation. Whereas Nox5 and the DUOXs are regulated by Ca2+, NOXs 1, 2, and 3 are regulated by several cytosolic proteins, that associate with the Nox2-p22phox dimer forming the active O2.-generating complex. The paramount model of cytosolic regulation is Nox2 and the "dream" of structure investigators is to elucidate the structure of NOX2 in both resting and activated states.

Achievements<br /> Note: When this paper was received for review, this reviewer was not aware of any publication dealing with the structure of human Nox2. However, on October 14, 2022 a paper was published on line, dealing with the structure of Nox2 (S. Noreng et al., Structure of the core human NADPH oxidase Nox2, Nature Communications (2022)13:6079). This review will not discuss the present manuscript in relation to the paper by S. Noreng et al.

This manuscript is successful in describing the structure of the NOX2-p22phox heterodimer using cryo-EM methodology. In order to compensate for the small size of the complex, use was made of the Fab of a monoclonal anti-Nox2 antibody binding an anti-light chain tagged nanobody. In order to mimic as much as possible the milieu of NOX2-p22phox in the phagocyte membrane bilayer, the authors reconstitute the quaternary complex in a nanodisc, using soybean phosphatidylcholine (PC) and a membrane scaffold protein (MSP). To the best of my knowledge, this is the first report of studying a NOX in a nanodisc, for both function and structure. Peptidiscs were used in determining the structure of human DUOX1 by a group led by the main author of this paper, but nanodiscs offer the advantage of adding a phospholipid chosen by the investigator. The purified nanodiscs incorporating the quaternary complex led to successful structure determination of the transmembrane domain (TMD), extracellular and intracellular loops, inner and outer hemes, distances between hemes and FAD to inner heme, and a hydrophilic tunnel connecting the exterior of the cell to the oxygen-reducing center of NOX2. The structure of the dehydrogenase region (DH) was less well defined; the FAD-binding domain (FBD) was more visible than the NADPH-binding domain (NBD). The structure of p22phox and the interface between Nox2 and p22phox are well described.

The mutations in NOX2 and p22phox causative of the deficient bactericidal function in Chronic Granulomatous Disease are related in detail to the location and role of the mutated residues as revealed by the solved structure.<br /> The authors make it clear that the structure, as presented, is in the resting state. The distances between hemes are suitable for electron transfer but the distance between FAD, in the FBD, and the inner heme is too large for transfer. The poor quality of the obtained structure of the DH (especially, the NBD), even after local refinement focusing, suggests its flexibility (mobility?) relative to the TMD and that, in NOX2, the DH is "displaced" relative to the TMD, when compared to the situation in the activated (by Ca2+) DUOX1. The mobility of NBD in NOX2 also results in weak interaction with FBD, making hydride transfer from NADPH to FAD inefficient

A major achievement of the work described in this manuscript is what I believe to be the first description of the activation of recombinant NOX2-p22phox in a nanodisc, to generate O2.-, when activated by a trimeric fusion protein (trimera), consisting of the functionally important parts of the three cytosolic components, p47phox, p67phox, and Rac (see Y. Berdichevsky et al., J. Biol. Chem. 282, 22122-22139, 2007). This proves that the resting state structure of NOX2-p22phox has all that is needed to be converted to the activated state. The fact that the nature of the phospholipid in the nanodisc can be varied and that this is known to have a major effect on the affinity of the trimera for NOX2-p22phox, offers additional advantages.

Weaknesses<br /> A weakness of this, otherwise impressive work, is the difficulty for readers who are not sufficiently "structure educated" to fully understand the "displacement" of the DH of NOX2, shown in the NOX2/DUOX1 overlay (Figure 5). The meaning of "centers of mass" of FBD and FAD, in Figures 5C and 5D, respectively, is not properly explained.

Yet another weakness is the much too vague wording of the change in NOX2 conformation from the resting to the activated state by cytosolic factors as "the cytosolic factors might likely stabilize the DH of NOX2 in the "docked" conformation which is similar to that observed in the activated DUOX1 in the high-calcium state". First, the evidence from biochemical studies of NOX2 activation indicates clearly distinct targets of individual cytosolic components and not a "block" action. There is also support for the conformational change being the result of the action of a single cytosolic component (p67phox), with the other cytosolic components acting as carriers or activators of one cytosolic component by another, such as Rac-GTP acting as a carrier and inducer of a conformational change in p67phox (see J. El-Benna and P.M-C. Dang, J. Leukoc. Biol. 110, 213-215, 2021, and E. Bechor et al., J. Leukoc. Biol. 110, 219-237, 2021). Also, the concept of "docking of the DH to the TMD" seems like an oversimplification of the many locations and partners of such "docking" and ignores the possible multiple consequence of such docking. Even before the appearance of structural studies of NOXs, revealing precise distances between redox stations (NADPH-FAD; FAD-inner heme; inner heme - outer heme), as first reported for C. stagnale Nox5, by F. Magnani et al., Proc. Natl. Acad. Sci. U.S.A. 114, 6764-6769, 2017, a shortening of the distance between an electron donor and acceptor at specific locations in the redox cascade was proposed. The most popular was the NADPH - FAD hydride transfer, based on structural work by P.A. Karplus on Ferredoxin - NADP reductases, the accepted model for the DH of NOXs.

An unfair request for an unachieved task<br /> Of course, the dream of those hoping for a structure-based response to solving the molecular mechanism of NOX activation is to see the structure of the activated NOX2 in complex with three cytosolic components. The compelling finding in the present manuscript that a nanodisc-embedded recombinant NOX2-p22phox can be activated to ROS production by the use of a [p47phox-p67phox-Rac] trimera (replacing three cytosolic components) will provoke in all the readers the wish to see the structure of such a complex. The size of the trimera with a GFP tag (108 kDa) might make the use of the anti-Nox2 Fab and anti-light chain nanobody, unnecessary. Prenylation of the trimera at the Rac moiety is bound to markedly enhance its affinity for the phospholipids in the nanodisc and is likely to generate a more stable complex, most suitable for cryo-EM (see A. Mizrahi et al., J. Biol. Chem. 285, 25485-25499, 2010).

Is there a way to search for your replies to someone's public annotations?

Currently, they don't show up when I search my user name and the tag I used in the reply. Is there an elegant way to search for these annotations and my reply to them?

I agree with Sahil. I think creating a link to the homepage by placing the img inside an anchor tag would be ideal .

This probably could have been an h2 tag, there will be alot of contrast between Assignment E and your name with it being a paragraph.

you don't actually need the div class= "image" also I think it's missing a closing tag.

Not totally sure why you have an open and closed paragraph tag.

You can use h tag instead of p.

You can change this to h2 tag.

You may want to change this content to article and add h2 tag, so user can understand what the content describes.

comments for tag: undefined vs. null: Technically this is undefined (unset, !diff) vs. true (diff), but it's similar enough that don't need a separate tag just for that.

annotation meta: may need new tag: undefined/unset vs. null/set

MiguelAngelLópezRodríguez

FernandoMoctezumaSoto

you can use list tag instead of using too more br tag

This tag is used very often.

nav tag is good for list instead of p tag

A small tag would give more context to the function of the element

Both Jonathan and Mina keep their journals in shorthand, and yet what we read here is in complete sentences. And Dr. Seward's Diary is spoken into a phonograph. We are experiencing this text very differently from how it is created. We combined with the several posts that contain correspondence that was never opened by or delivered to the intended experience (see Unopened or Undelivered tag), there is much of this story that we experience differently from the characters within it.

This comment is a good idea. Just looks like a slight typo in the closing tag.

might wrap this in a header tag to indicate its your title section

missing its closing tag

I would pay attention to the semantic coding of this section. I'm not sure if a single image would make sense as an article, especially since article typically requires a heading. I would personally just tag it as a figure (that way you can also attach a caption in case it doesn't load) and if you're worried about styling apply a div.

Reviewer #2 (Public Review):

The goal of this study was to understand the molecular mechanism of how transcription factor DUX4, which has a role in cancer, inhibits the induction of genes stimulated by interferon-gamma. The authors achieved this goal, and their results mostly support their conclusions. They found that DUX4, in their experimental model, interacts with STAT1, thereby decreasing STAT1 and Pol-II recruitment to sites of gene transcription.

The present study has many strengths: The topic is of broad interest, the findings are novel and intriguing, the experiments are well-designed and controlled, the data, with one exception, is carefully interpreted, and the manuscript is very well-written.

Two major weaknesses were identified. One is that all experiments, except Figure 6, rely on one experimental setup, which is a human skeletal muscle cell line with an integrated doxycycline-inducible transgene. The concern is that both the treatment of cells with the drug doxycycline and the fact that signaling pathways could be disrupted in this (immortalized?) cell line could lead to artifacts that skew results. Indeed, results in Figure 4C indicate that total STAT1 is completely localized in the nucleus even prior to interferon stimulation when it should be in the cytoplasm. The other weakness is the use of the DUX4-C-terminal-domain (DUX4-CTD) mutant for the majority of the mechanistic experiments. The concern here is that although the phenotype of ISG repression is observed in this truncated mutant, important regulatory domains could be missing that modulate the interaction with STAT1 or other proteins. Is the NLS added after the flag tag identical to the endogenous NLS? Related, I disagree with the interpretation of Figure 4C that "this interaction happens within the nuclei of DUX4-CTC expressing cells". The interaction could happen prior to STAT1 shuttling to the nucleus.

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Referee #1

Evidence, reproducibility and clarity

This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.

Major points are the following:

1. Quantitation and presentation of the data throughout the manuscript should be improved.

The criteria used for identifying fluorescent spots as single motors are not given. This is typically based on photobleaching experiments and fluorescence intensity measurements - the authors should show these data to validate that the motility reported is due to single motors.

A table should be included that shows the single molecule motility parameters that were analyzed and compared for the three motors, rather than just the dwell times for the assays shown in Fig. 1. Other motility characteristics should include run lengths, binding rates, detachment rates, and velocity. The percentage of time that the single motors move directionally, diffuse, or remain stationary should also be given.<br /> The authors refer to imaging rates (1 frame/50ms, p. 5), but do not state the total time of the assays, making the statements uninterpretable, as it is not clear what would be expected without knowledge of the total assay time. The authors also state that a slower imaging rate (1 frame/2 sec) was used to detect slow processive motility, but the logic underlying this statement is not clear, as a longer assay time should reveal the slow processive movement irrespective of the imaging rate. These statements should be clarified.<br /> The authors give the data for the dwell times in single motor assays and velocities in multiple motor assays as the mean + SEM, but the SD rather than SEM should be reported for these assays, given that the data are for individual single motors or individual gliding microtubules. The authors state the number of replicate experiments for the assays, but they should also state the number of data points that were obtained for each replicate. Further, they should evaluate the significance of differences in their data by giving P values obtained using appropriate statistical tests and indicate whether the differences among the motors are significant.<br /> The percentages of processive events (p. 5) are most likely dependent on the amount of inactive or denatured protein in a given preparation, rather than a motility property of the motor protein - this could be determined by analysis of whether the percentages differ from preparation to preparation of each motor and whether the mean+SD of the preparations of a given motor differs from the other motors. The statements by the authors on p. 8 that "the majority of proteins do not undergo unidirectional processive motility as single molecules but rather diffuse along the surface of the microtubule for several seconds" and "It is presently unclear why only a subset of kinesin-6 molecules are capable of directional motility (Figure 1 ..." are not meaningful, as they do not take into account the percentages of the kinesin-6 proteins that are inactivated or denatured during protein preparation.<br /> Again, given that inactive motors are produced during preparation of the proteins, it is not clear what the frequency of processive motility events means. If the authors think that the frequency of processive motility events is informative and a characteristic of each motor, they should present controls showing frequencies of processive motility events for specific well characterized motors. For example, does a control of kinesin-1 show 100% or only 95% processive motility events?<br /> For the multiple motor gliding assays, velocities are shown in Fig. 2 without controls demonstrating the dependence of the velocities on motor concentration in the assays - the gliding assays require dilution experiments to show that the velocities are within the linear range of motor concentration and do not fall within the range of higher concentrations in which motor gliding velocity is inhibited or lower motor concentrations in which the density of motors on the surface is too low to support processive movement. These control experiments of motor concentration vs velocity for the gliding assays should be shown for each of the three motors that was assayed. The authors should state whether the gliding velocities that were determined correspond to the Vmax for each of the motors that was assayed.

Again, the velocities given on p. 6 should include the SD and evaluation of the significance of the differences among the motors by obtaining P values.

Proteins for motility assays: Western blots of the purified proteins should be shown as a supplemental figure.

How are the motility characteristics of the three motors related to their spindle functions? This is the central point of the manuscript but is not clearly stated.

1. Functional assays should be relevant to motor function.

Given that the kinesin-6 motors under study are mitotic spindle motors that do not normally transport vesicles, it is not clear why the authors chose to show load dependence using peroxisome and Golgi dispersion assays, rather than assays of spindle function. The authors interpret peroxisomes and Golgi to differ in dispersion load, but this appears to be based on interpretations from assays of highly processive motors, kinesin-1 and myosin V, that function in vesicle trafficking, rather than quantitative data from appropriate controls showing that peroxisomes and Golgi can be dispersed by spindle motors that bear different loads. The problems inherent in the use of these assays for spindle motors are evidenced by the authors' observations on p. 6 that MKLP1- mNG-FRB and KIF20-mNG-FRB in midbodies could not be localized to peroxisomes by rapamycin. There are no data presented showing the dependence of dispersion on protein expression/presence in the cytoplasm, making the dispersion assays difficult to interpret.

The kinesin-6 motor functional tests would be more relevant if they involved mitotic spindle assays, rather than peroxisome or Golgi dispersion assays. It is not clear how the loads involved in peroxisome or Golgi dispersion are related to kinesin motor function in the spindle. What are the implications of low- vs high-load motors in the spindle? How do the authors envision that motor loads in spindles relate to loads borne by vesicle transport motors?

Minor points needed for clarity and reproducibility of the data:

Methods

Plasmids<br /> "MKLP1(1-711) lacks the insert present in KIF23 isoform 1" - the insert present in KIF23 isoform 1 but missing in MKLP1 (1-711) should be depicted/pointed out in Fig. S1 and information provided as to its predicted or actual structure.

"KIF20B contained the protein sequence conflict E713K and natural variations N716I and H749L "- the sites of these changes should be indicated in Fig. S1 and information provided as to their effects on predicted or actual structure.<br /> Protein purification: "MKLP1(1-711)-3xFLAG-Avi was cloned by stitching four oligonucleotide primer sequences together into a digested MKLP1(1-711)-Avitag plasmid" - please explain what this means: what do the four oligonucleotide primer sequences correspond to? if they are the 3xFLAG-Avi tags, why were four sequences stitched together instead of three?<br /> The figures showing the kymographs should include labeled X and Y axes, rather than scale bars.

The significance of the statement that "All motors displayed similar behaviors when tagged with Halo and Flag tags" is not clear, as the Halo and Flag tags were also C-terminal tags, like the 3xmCit tag.

The figures (Fig. 3-5) that contain grey-scale cell depictions would be more readily interpretable by others if they were labeled with the authors' classification of the dispersion phenotype.

Significance

This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.

My expertise: motors, motor function in division, motility assays, microtubules

Because there is no maximum width specified, paragraph elements that are very long like this one show up as very long on the user's browser. This is not very readable.

When you use <span> tags here as an example, they do not render on the user's browser. Character entities must be used if you want to display reserved characters like < and >. See this for more information.

When you talk about how they use the align attribute wrong, they can't see the code you're talking about.

probably keeps the tag together.

annotation meta: may need new tag: no exact translation in other language

annotation meta: may need new tag: how could they know / how would one find out?

the css part is done very great and they have right space in between the codes , like we can easily see the each tag and what is used in it. i also saw new thing like we can use h1,h2 tags combine and edit it together

This has to be a close tag.

i loved it how you write the code its very clean and easy to read and every tag is used appropriate.

You might want to consider consistency in formatting. In the first one the closing tag of list item is placed on a new line and in the next one it is placed right after the content.

Given your talents, if you've not explored some of the experimental fiction side of things (like Mark Bernstein's hypertext fiction http://www.eastgate.com/catalog/Fiction.html, Robin Sloan's fish http://www.robinsloan.com/fish/ or Writing with the Machine https://www.robinsloan.com/notes/writing-with-the-machine/, or a variety of others https://hypothes.is/users/chrisaldrich?q=tag%3A%22experimental+fiction%22), perhaps it may be fun and allow you to use some of your technology based-background at the same time?

Another example of marketing serving badly for the concepts being easily studied and used. Positioning and differentiation backfires here. Lack of sources linking is a huge issue in a popular non-fiction.

Unlike HTML XML is more useful and flexible when adapting to other applications while HTML is restricted to only one set of tags

An understandable concern/desire: compatibility

Added new tag for this: allowing full syntax to be used, not just subset

annotation meta: may need new tag: "definable __"?

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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

-- the Impact Tag Library is where we define the list of tags for our materials.

i.e.: Plastic, Glass, Concrete...etc

Tags

DESPUES DE LA GUERRA

DURANTE LA GUERRA

Added 3 different google fonts in the head section by the tag LINK

i think you have to put this in h1 tag and make it more bold

Reviewer #1 (Public Review):

This paper has many strengths that support its conclusions. Specifically, the use of natively expressed Piezo1 engineered to carry the HA tag allowed the authors to explore the distribution of the protein from primary cells isolated from a mouse at native expression levels. Thus, over-expression effects could be avoided. The super-resolution imaging is nicely controlled and convicting in its analysis of the distribution of the channel in 3D. The supporting EM data also supports the findings from fluorescence. Likewise, the theory is convincing in proving a mechanistic reason why the channel distributes into this region of the cell. While the data are quite nice and well analyzed, the paper is lacking in an exploration of what function this distribution of the channel would provide to the cell. Likewise, if this distribution was disturbed, would the red blood cell's behavior change? For example, would calcium signals in response to an osmotic challenge or squeezing change if the channel was not concentrated in the dimple? As it stands now, the paper presents a structural view of the distribution of piezo1 in a primary cell plasma membrane but lacks direct experimental evidence for the mechanism of this concentration or mechanistic insight into the effects of this spatial distribution on red blood cell physiology.

The individual element tags can be formatted as

opening tag

                   content

closing tag

for better readability.

This closing tag can be together for better readability.

Closing tag not required. Everything else seems perfect.

closing tag not required for img.

Had no idea there was an address tag. Good use of proper semantic labelling.

Alt tag should contain information describing image

Having all of these as H4 will not allow individual styling. Try enclosing each in a span or button tag then you can add background colour to each.

make sure to add alt tag

The <br /> tag showed warning for me when I tried to validate my code. So I used <br>.. Everything else looks great.

From my understanding, EAD is used only for archival files, and uses the components mentioned before.

Tags must be correctly spelled and in the right position for them to work. A combination of tags cannot be used. This means that beginning tags cannot be used with empty tags or closing tags.

no closing tag

new tag: pressure to understate the real cost/estimate

in reply to los2pollos<br /> https://www.reddit.com/r/antinet/comments/y5un81/comment/it4jy3c/?utm_source=reddit&utm_medium=web2x&context=3

You're not the only one to think of a card index as diary. Roland Barthes practiced this as well. His biographer Tiphaine Samoyault came to call it his fichierjournal.

nice work

The reliance upon tag-like keywords in physical note-taking of this type seems to be a limitation compared to digital systems that allow full text search. That said, the benefits of full text search might be somewhat overblown, as found search terms say nothing of the context and would need either tags or a quick read of the text to provide that context.

I don't see any issues. Great job, Ravi.

Looks great!

This could also be a way in which artists could strategize the use of specific words in songs to attract a larger audience. They could look at the similarities between top hits and find certain words that were used in all of them and then include it when advertising the music. For example, they could use it as a tag when posting on instagram or twitter and it may attract more attention.

Use padding and margin for space instead of br tag.

You can use div tag as well

the opening p tag needs to be brought down to where it begins

What paragraph is this closing tag for?

u/LBHO https://www.reddit.com/r/antinet/comments/y77414/worried_about_paper_cards_being_lost_or_destroyed/

As a firm believer in the programming principle of DRY (Don't Repeat Yourself), I can appreciate the desire not to do the work twice.

Note card loss and destruction is definitely a thing folks have worried about. The easiest thing may be to spend a minute or two every day and make quick photo back ups of your cards as you make them. Then if things are lost, you'll have a back up from which you can likely find OCR (optical character recognition) software to pull your notes from to recreate them if necessary. I've outlined some details I've used in the past. Incidentally, opening a photo in Google Docs will automatically do a pretty reasonable OCR on it.

I know some have written about bringing old notes into their (new) zettelkasten practice, and the general advice here has been to only pull in new things as needed or as heavily interested to ease the cognitive load of thinking you need to do everything at once. If you did lose everything and had to restore from back up, I suspect this would probably be the best advice for proceeding as well.

Historically many have worried about loss, but the only actual example of loss I've run across is that of Hans Blumenberg whose zettelkasten from the early 1940s was lost during the war, but he continued apace in another dating from 1947 accumulating over 30,000 cards at the rate of about 1.5 per day over 50 some odd years.

Reccomend to change h3 tag

Reccomend to change h3 tag.

just use a header tag, dont nest a header tag inside a paragraph tag.

don't make each list item a paragraph, add a list tag

no closing tag

<ul> is a parent tag of <li> you need to add a <ul> Additionally, in this situation I would recomment not using a list, instead just use the header and paragraph tags.

You must be more specific while selecting tag because it affects all images in the page.

used capital letter in closing tag

I believe you put the h1 inside the main tag

everything seems well structured. But the image tag could be given the value in both html and css.

you can keep this as a paragraph tag, but it may be better to turn it into a <span>

Perhaps small tag could have been used for a smaller amount of text placed on a separate line instead of an entire paragraph for the sake of readability.

Review coordinated via ASAPbio’s crowd preprint review

This review reflects comments and contributions by Ruchika Bajaj, Sree Rama Chaitanya Sridhara and Sara El Zahed. Review synthesized by Ruchika Bajaj.

This study has developed a novel one-step methodology for the incorporation of membrane proteins from cells to lipid Salipro nanoparticles for structure-function studies using surface plasmon resonance (SPR) and single-particle cryoelectron microscopy (cryo-EM), which is a profound technology in the field of membrane protein structural biology. We raise some points that may strengthen the manuscript below:

• Main section, 4th paragraph “resuspended in digitoxin-containing buffer”- Does the sentence mean that membrane proteins were solubilized by detergent before reconstitution into salipro particles? Are salipro and digitoxin added at the same step? If this is the case, it is unclear how one can distinguish between the step wise solubilization and reconstitution or direct reconstitution into salipro particles. Further discussion on the mechanism of reconstitution would be helpful. In the same paragraph, the fragment “to increase membrane fluidity and render lipids” raises the question of whether the concentration of digitonin was optimized to balance the increase in membrane fluidity but not rendering the solubilization of membrane proteins.
• Main section, 4th paragraph, “the formation of saponin-containing mPANX1-GFP particles was assessed by analytical size exclusion chromatography using fluorescence detector” - It is assumed that fluorescence is detected from GFP. As the construct expressed is PANX1-GFP, GFP fluorescence signal will be received from reconstituted as well as not reconstituted PANX1. Is saponin specific signal being used as a signal for measuring the reconstitution of PANX1-GFP? In the same paragraph, “PreScission protease for on-column cleavage” is mentioned. Is GFP still intact in the expressed PANX-1 or is it cleaved? A diagram of these procedures showing the various steps will be helpful for readers.
• Main section, 4th paragraph “SDS-PAGE revealed the formation of pure and homogeneous Salipro-mPANX1 nanoparticles”- However, extra bands are present above the major band in Figure 1E, can some comment be provided on this point. Possible explanations for the additional bands could be post translational modifications or degradation of mPANX1.
• Methodology section, “membrane protein reconstitution screening using fluorescence-detection size exclusion chromatography (FSEC)” - The amount of salipro is given in ug. A comment on the ratio of protein to salipro particles would be important to decide the concentration of salipro with respect to the mass of the cell pellet.
• Figure 1G: The molecular weight of Salipro-mPANX1 particles is mentioned to be approximately 466kD. mPANX1 weighs about 48kD and heptamer will be 336kDa. A discussion on comparison of experimental and actual molecular weight would be interesting.
• hPANX1 was expressed in sf9 insect cells. A description regarding trials of expression of this construct in expi293 cells would be informative.
• Supplemental Figure 1B: The gel is overloaded and shows multiple bands for hPANX1, recommend selecting an alternative image for hPANX.
• Paragraph 6A phrase, “challenged with bezoylbenzoyl-ATP(bzATP), spironolactone and cabenoxolone” - Please explain the meaning of ‘challenged’ here.
• Supplementary Figure 2: Paragraph 6 mentions “binding constant could not be determined”. Please provide an explanation for this. Is it about the saturation phase not being approachable because of the feasibility of the binding experiment at higher concentration of cabenoxolone?
• The last summary sentence in Paragraph 6 is not clear, recommend rephrasing it.
• Figure 2A shows that Salipro particles have His tag. This suggests that an additional step of affinity purification with His tag could have been used to distinguish or separate reconstituted and un-reconstituted PANX1.
• Supplementary figure 4: Please explain whether the datasets for samples in the presence and absence of fluorinated lipids were combined together.
• Paragraph 8, “intracellular helices were not well resolved” - Please comment on a possible explanation. Does the Salipro scaffold contribute to the resolution? Please mention any future possibilities regarding improving the resolution by modifying the salipro scaffold or alternative scaffold. In the same paragraph, rmsd is mentioned at promoter level, please comment on how this value changes at heptamer level and why is it important to report the rmdd value to appreciate the direct reconstitution methodology.
• Last paragraph 10, “future membrane protein research” - Please comment on the utility of this methodology on prokaryotic membrane proteins, bacterial outer or inner membrane proteins or eukaryotic membrane proteins. Some more examples of reconstitution with the same method will support the applicability of this methodology on diverse kinds of membrane proteins. A discussion section comparing this methodology to other methods would also be useful for readers.

needs a tag

needs to have a tag. <a href="tel:+2503387467">to add a telephone, <a href="mailto:salon.hairpins@gmail.com"> to add email

needs to have a tag

needs to have a tag

needs to have a tag.

I would recommend using a p or heading tag (maybe even article?) here, I don't think div is the best option for this

might be because you've closed your h2 before your div. remember opens and closes should mirror eachother. ex) h2, div, content, /div, /h2

not sure you need the span tag here, I think the h3 would have been enough to separate it from the rest.

make sure to close your p tag before your div or you will have issues.

This closing tag can be at the end of line 34 (?)

Oh no! I thought the flag meant post & accidentally reported your thoughtful feedback to moderators instead of replying. Hopefully they figure it out (no obvious way to contact them or "unflag"

Comment Figure 2C → please include indication of statistical significance Figure 3C → please include indication **of statistical significance Figure 6A → please include indication of statistical significance Figure 8B → please include indication of statistical significance Figure S1B → please include indication of statistical significance Figure S3B → please include indication of statistical significance

Response Easy to add

Comment For your overexpression experiments, do the overexpressed proteins have a tag? It would be helpful to have Western blot data showing that the particular proteins are actually being overexpressed. I think the phenotypes that you observe are very compelling so I don’t doubt the conclusions. Western blot data would just provide some additional confirmation that you are actually achieving overexpression of UppS, MraY, and BcrC.

Response The proteins are untagged. For the UppS and BcrC the cell shortening occurs with addition of inducer, , so strong indication expression is occurring. A western would provide information about degree of overexpression, but we don’t think is necessary to support conclusion drawn. Do you think there is an alternative possibility that needs to be excluded? We note that in another preprint (https://www.biorxiv.org/content/10.1101/2022.02.03.479048v1) the authors delete the native uppS in their inducible Phy-uppS strain (Fig S4) and at 100 uM IPTG (10X less than what we used in experiment) the cells have wt growth on LB plates, so we at least know the Phy-uppS is functional and made (or they would die!). We are introducing the uppS deletion into our strain to see if we can identify a concentration of IPTG that doesn’t affect cell growth but still induces shortening.

For MraY, the result is negative, so you are spot on – it is impossible to tell if due to lack of overexpression from data shown. We only know the strain is correctly made from sequencing. We will investigate if there is an antibody or functional fusion available. The reason we were not sure was worth doing is because the MraY reaction is reversible (15131133). This means that without a phenotype, there is no simple way to know the reaction can even be pushed forward even if the overexpression is confirmed (more negative data). We actually overexpressed some other proteins that act downstream (MraY, MurJ, AmJ) and they were also negative for shortening. Probably we should remove the negative data or reword to make the caveats of the negative result clear.

Question Based on your data, there are definitely differences in gene expression when you compare cells grown in media with and without magnesium. Because the majority in cell length increase occurs in such a short time though (the first 10min), I was wondering if you think that some or most of it is not due to gene expression?

Response The shortening is even faster than 10 min (not only statistically significant, but also obvious qualitatively if we mount immediately after adding Mg2+ ). We did not include the first timepoint because original purpose was to check everything was ready with microscope – did not expect shortening so fast! We can definitely add that data in. When we saw, we tried to capture the transition on pads, but going from culture to pad seems to stress the cells too much in the small window where the cool stuff happens. Since growth rate doesn’t appear to be a big factor in those initial divisions, we might be able to grow at lower temp and shift to pads for adjustment period before adding Mg2+. Did not play with it much due to lack of resources atm, but a flowcell setup would probably be best. In short, we think rapid divisions right after transition do not require transcription or translation. It really “smells” more like a biophysical thing.

Question Do you have any hypotheses what is most likely to be affected by magnesium? Do you think if the membrane may be affected?

Response We have a lot of hypotheses, but all speculative. There could be an extracytoplasmic enzyme involved in envelope synthesis is sensitive to Mg2+ availability, and that at lower concentrations, its activity is affected. There is some old literature with membrane preps that suggests PG synthesis requires higher Mg2+ than teichoic acid synthesis. If Und-P is limiting, higher Mg2+ may shift make the pool more available to make the septum. Tingfeng initially hypothesized there might be a receptor/signal mechanism but has not been able to identify one. Und-P seems to be important, but “availability” is not just pool, but how fast (and where!) the flipping across the membrane occurs. If Und-PP needs to be dephosphorylated to Und-P before being flipped back to cytoplasmic side, anything that effects the PP/Pi equilibrium would be predicted to affect the reaction rate, with lower Pi (in periplasm or pseudoperiplasm in case of G+) favoring the dephosphorylation. Cell wall associated Mg2+ could shift equilibrium to be more favorable for a Und-PP phosphatase more closely associated with the divisome. I could go all day… In short, we don’t know enough!

Question Why do you think less magnesium activates this program of less division and more elongation? Additionally why is abundant magnesium activating a program of increased cell division and less elongation? Do you think there is some evolutionary advantage, especially considering how important magnesium is for ATP production?

Response In the window we looked at, the elongation rate is constant (not less or more) and only the division frequency changes. Some bacteria (like Caulobacter and to lesser extent E. coli) clearly elongate and divide simultaneously, so model there is some competition for substrate (like Lipid II) makes sense. Septators like Bacillus seem to delineate the two processes more, though we have found conditions where even Bacillus invaginates during division, so it’s not absolute. Like eukaryotic cells, bacterial undoubtedly have mechanisms not only commit to a round of DNA replication when there is some signal that resources are sufficient. Clearly with some bacteria, this is not the case with cell division. The alternative would be that every cell cycle there is an opportunity to divide if some threshold of something(s) is reached. There is a hypothesis from Mtb literature that it may be GTP, but it’s not at all clear that is sufficient. In yeast, size at cell division is affected by perturbing 1-C pool.

Question Related to this previous question, I also wonder if this magnesium-dependent phenotype would extend to other unicellular organisms, may be protists or algae? That would be a really exciting direction to explore!

Response It’s a great question – lots to do! We didn’t even look at another Gram-positive, but we plan to. It’s trickier to limit Mg2+ in Gram-negatives (see 27471053 – we tried Bsub homolog for those wondering – it’s not responsible for phenotype we see).

Question Regarding the zinc and manganese experiments, why do you think they lead to additional phenotypes compared to magnesium? Do you have any hypotheses?

Response We have hypotheses, but if my (Jen’s @rosh_ba) twitter engagement is any indication, way too speculative for public consumption at present. Need acquire preliminary data/write grant.

Question Regarding your results that Lipid I availability may be a major a problem for the cell division in the absence of magnesium, do you think that is due to effects magnesium has on the enzymes directly, or do you think magnesium affects the substrate availability/conformation by coordinating the phosphate groups? Or something else, may be membrane conformation?

Response Several proteins involved in envelope synthesis (like UppS) are Mg2+ dependent enzymes. But at least for intracellular players, levels of Mg2+ should be more than high enough to support enzyme activity (0.8 – 3.0 mM is Bsub range I recall off top of head). Could have impact extracytoplasmically by lowering pool sponged into the cell wall, but intuition (for what that is worth) is that it is not the coordination of an enzyme with a metal that is impacted rather the equilibrium with other ions like Pi and H+ and their impact on net ATP synthesis. Lots to think about and do, and no simple answers. When Tingfeng started project idea was to find mechanism – didn’t realize we were asking “How does the cell work?” Turned out to be a bit much for a dissertation project :)

General comments:

This study carefully delineates the role of magnesium in cell division versus cell elongation. The results are really important specifically for rod-shaped bacteria and also an important contribution to the broader field of understanding cell shape. Specifically, I love that they are distinguishing between labile and non-labile intracellular magnesium pools, as well as extracellular magnesium! These three pools are really challenging to separate but I commend them on engaging with this topic and using it to provide alternative explanations for their observations!

A major contribution to prior findings on the effects of magnesium is the author’s ability to visualize the number of septa in the elongating cells in the absence of magnesium. This is novel information and I think the field will benefit from the microscopy data shown here.

I completely agree with the authors that we need to be more careful when using rich media such as LB. It is particularly sad that we may be missing really interesting biology because of that! It’s worth moving away from such media or at least being more careful about batch to batch variability. Batch to batch variability is not as well appreciated in microbiology as it is for growing other cell types (for example, mammalian cells and insect cells).

For me, the most exciting finding was that a large part of the cell length changes within the first 10min after adding magnesium. The authors do speculate in the discussion that this is likely happening because of biophysical or enzymatic effects, and I hope they explore this further in the future!

I love how the paper reads like a novel! Congratulations on a very well-written paper!

Kudos to the authors for providing many alternative explanations for their results. It demonstrates critical thinking and an open-mind to finding the truth.

Specific comments:

Figure 2C → please include indication of statistical significance

Figure 3C → please include indication of statistical significance

Figure 6A → please include indication of statistical significance

Figure 8B → please include indication of statistical significance

Figure S1B → please include indication of statistical significance

Figure S3B → please include indication of statistical significance

For your overexpression experiments, do the overexpressed proteins have a tag? It would be helpful to have Western blot data showing that the particular proteins are actually being overexpressed. I think the phenotypes that you observe are very compelling so I don’t doubt the conclusions. Western blot data would just provide some additional confirmation that you are actually achieving overexpression of UppS, MraY, and BcrC.

Questions:

Based on your data, there are definitely differences in gene expression when you compare cells grown in media with and without magnesium. Because the majority in cell length increase occurs in such a short time though (the first 10min), I was wondering if you think that some or most of it is not due to gene expression? Do you have any hypotheses what is most likely to be affected by magnesium? Do you think if the membrane may be affected?

Why do you think less magnesium activates this program of less division and more elongation? Additionally why is abundant magnesium activating a program of increased cell division and less elongation? Do you think there is some evolutionary advantage, especially considering how important magnesium is for ATP production?

Related to this previous question, I also wonder if this magnesium-dependent phenotype would extend to other unicellular organisms, may be protists or algae? That would be a really exciting direction to explore!

Regarding the zinc and manganese experiments, why do you think they lead to additional phenotypes compared to magnesium? Do you have any hypotheses?

Regarding your results that Lipid I availability may be a major a problem for the cell division in the absence of magnesium, do you think that is due to effects magnesium has on the enzymes directly, or do you think magnesium affects the substrate availability/conformation by coordinating the phosphate groups? Or something else, may be membrane conformation?

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

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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.

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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.