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Consolidated peer review report (9 September 2022)

GENERAL ASSESSMENT

This interesting preprint by Suárez-Delgado et al. explores the mechanism by which activation of the Hv1 voltage-activated proton channel is dependent upon both the voltage and pH difference across the membrane. The authors are the first to incorporate the fluorescent unnatural amino acid, Anap, into the extracellular regions of the S4 helix of human Hv1 to monitor transitions of S4 upon changes in voltage or pH. The authors first checked that Anap is pH insensitive for practical use in Hv1, where changes in local pH are known to occur when the voltage sensor activates and the proton pore opens. Anap was incorporated at positions throughout the S3-S4 linker and the extracellular end of S4 (up to the 202nd residue) of hHv1 and some positions showed clear voltage-dependent changes in fluorescence intensity. The authors also obtained fluorescence spectra at different voltages and observed no spectral shifts, raising the possibility that voltage dependent changes in fluorescence intensity could primarily be due to fluorescence quenching. Upon mutation of F150, the Anap signal at the resting membrane voltage increased, suggesting dequenching upon removal of F150. The authors also discovered that the kinetics of Anap fluorescence upon membrane repolarization have two phases (rapid and slow) under certain pH conditions and that there is a pH- dependent negative shift of the conductance-voltage (G-V) relation compared with the fluorescence-voltage (F-V) relation in some mutants. The biphasic kinetics of the fluorescence decay upon repolarization were explained by modelling a slower transition of return from intermediate resting state to a resting state. The pH-dependent shift of the G-V relation from the F-V relation provides insight into mechanisms of ΔpH-dependent gating of Hv1, a longstanding enigma. Overall, the approaches are rigorous, the figures show important results, and this work paves the way for the use of Anap fluorescence to study Hv1 gating and modulation.

RECOMMENDATIONS

Revisions essential for endorsement:

1) In its current form, the narrative of the preprint has two threads. One on the mechanisms of Anap fluorescence changes (mainly quenching) and another on a previously unappreciated transition of the voltage sensor, as revealed by Anap. Our impression is that the preprint suffers somewhat from this split focus, which could be resolved by explaining why Anap was used to explore voltage sensor activation in Hv1 in the introduction. Perhaps the authors could also explain the advantage of smaller sized fluorophores compared to other maleimide-based fluorophores earlier in the introduction, or the utility of being able to insert Anap into transmembrane segments. The authors should more clearly point out how they exploited the advantages of Anap as a tool in this study. It would furthermore be helpful to discuss previous studies using nongenetic tools for VCF and spell out how they have delineated key aspects of Hv1, which would help to emphasize how several positions studied here (for example, 201 and 202) could not be labelled with cysteine-based fluorophores.

2) We think the authors should be cautious about understanding the physicochemical nature of Anap using prodan as a model. It would be helpful to discuss the possibility that undetected spectral shifts due to a nonquenching mechanism could be overlooked, even though major signal changes can be explained by fluorescence quenching in their data. Regarding the mechanisms of remaining voltage-dependent fluorescence changes of F150A-A197Anap, it would be helpful for the authors to suggest possible ideas about which residues might account for remaining signals.

The beautiful spectral data for Anap is impressive. However, the physicochemical basis of the fluorescence change of Anap cannot be understood by simple extension of findings for prodan, which shows structural similarity to Anap. Our understanding is that changes in Anap fluorescence can only reveal a change in the structural relationship between Anap and one of its neighbors because the physicochemical basis of Anap fluorescence is complicated. For example, fluorescence could also be affected by the electrostatic environment, stretch of peptide bond, etc. Previous studies, including those of TRP channels, showed that the kind of environmental changes that Anap faces in ion channels do not necessarily induce large spectral shifts, unlike in cell-free spectral analyses using distinct solvents. Further, only minor shifts in spectra occur upon local structural change, as seen in previous work including Xu et al. Nat. Commun. 2020 11:3790. Such minor shifts could be perhaps overlooked even when Anap is incorporated into S4 and exposed to environmental change. Therefore, it is not easy to decode the physicochemical basis of Anap fluorescence changes. F150A-A197Anap has increased fluorescence and no change in spectral pattern, leading the authors to conclude that F150 quenches Anap fluorescence of A197 position. However, a significant amount of fluorescence change still occurs upon changes in membrane potential after F150 is changed to alanine (Figure 4). It is very likely that quenching is not the only mechanism underlying the observed voltage induced change of Anap fluorescence of Hv1. The authors suggest that remaining voltage-dependent fluorescence change of F150A-A197Anap could be due to interaction with other aromatic residues, but this has not been tested.

3) The current version of the preprint is missing important control experiments, ideally performed using western blots to measure protein expression or, if that is not possible, proton current and fluorescence measurements, to demonstrate that protein expression or functional channels are not seen for all mutants in the absence of ANAP (but in the presence of the tRNA and Rs construct).  A similar control for imaging would be to use ANAP alone without encoding. 

4) Aromatics in the S4 segment were ruled out as potential quenchers on the assumption that they would move together with Anap during gating. It should be noted, however, that Hv1 is a dimer and therefore a fluorophore attached to S4 in one subunit could be quenched by S4 aromatics in the neighboring subunit if were close to the dimer interface. In Fujiwara et al. J. Gen. Physiol. 2014 143:377-386, for example, W207 does not appear very far from labeled positions in the adjacent S4. This possibility should be mentioned in the discussion.

5) It is not clear whether the Anap spectra purely represent Hv1 incorporated into the plasma membrane or perhaps include signals from the cytoplasm or channels in internal membranes (whether assembled or incompletely assembled). It would be helpful to provide a more complete presentation of the data obtained and to provide more information in the Methods Section. In the Methods section, it is stated “The spectra of both fluorophores (Anap and mCherry) were recorded by measuring line scans of the spectral image of the cell membrane, and the background fluorescence from a region of the image without cells was subtracted”. How are signals from cell membranes specified in this method being discriminated from those associated with the cytoplasm and intracellular membranes? If spectral data include signals from free Anap in the cytoplasm or Hv1 in intracellular membranes, spectral shifts upon membrane potential changes will be difficult to detect, even when Anap is incorporated into Hv1 and senses environmental change by voltage-induced conformational change. In Figure 3E, wavelength spectra were shown as standardized signals for different voltages. Amplitude change would be demonstrated (spectrum at different voltages without standardization would be shown). In Figure 4, spectra were compared between A197Anap and F150A-A197Anap, showing increases of fluorescence in F150A-A197Anap. Was this signal measured at resting membrane potential? How does the spectrum change when the membrane potential is changed?

Rationales for the confirmation of signals originating from the cell surface for Hv1 Anap might include the observations that: a) some mutants showed slightly different spectral patterns (in particular, Q191Anap showed a small hump at longer wavelengths, which is proposed to represent FRET between mCherry and Anap) and b) signal intensity was voltage dependent (if signals originate  from endomembranes, they should not be voltage dependent). Mentioning these two points earlier in the text might help to alleviate concerns about the location of the protein that contributes to the measured signals.

6) In Fig 5, the fluorescence kinetics do not really match the current activation kinetics for panels A, B, and C.  Is there an explanation for this mismatch?  It would be helpful to have the fitted data in the figure. A more thorough comparison of the kinetics of currents and fluorescence would be helpful throughout the study. 

7) Which construct of hHv1 was used to obtain the data in Figure 6? Unless we missed it, this information is not provided in the text or figure legend. Is it for L201Anap? This figure also shows an intriguing finding that the G-V relationship is negatively shifted from the F-V relationship at pHo7-pHi7 but not at pHo5.5-pHi5.5. A shifted G-V relation with the same ΔpH contrasts with what has been reported in other papers. However, the authors did not really discuss this surprising finding in the light of previous references. Could the shift of the G-V relation between two pH conditions with the same ΔpH be due to any position-specific effect of Anap? If Figure 6 represents L201Anap mutant, the presence of Anap at L201 probably makes such shift of G-V curve in Figure 6C? The authors should openly discuss this finding in relation to what has been reported in the literature.

8) The authors suggest that the small hump near 600 nm in Figure 1E represents FRET between Anap and mCherry. It is surprising that FRET can take place across the membrane. Can the authors point to another case of FRET taking place across a cell membrane? One possibility might be that misfolded proteins place mCherry and Anap close to each other. It is also curious that only A191Anap did not show such a FRET-like signal. Also, if there is FRET, why wouldn’t this also contribute to the voltage-dependent changes in fluorescence?

9) F150A-A197Anap shows a leftward shift of the F-V relation compared with the G-V relation only when ΔpH=1. Another unusual finding with F150A-A197Anap is the very small shift of the G-V relation between ΔpH=0 and ΔpH=1, when other reports in the literature suggest it should be 40 mV or more. Are these peculiar properties simply due to the absence of Phe at position 150, which might play a critical role in gating as one of the hydrophobic plugs of Hv1?  To address this possibility, it would be ideal to compare different ΔpH values with and without F150 when Anap is incorporated at a different position (such as L201Anap). Regardless, it would be helpful to discuss this point.

10) In Figure 1E, I202Anap exhibits a blue shift in its spectrum suggesting the environment of Anap on I202 is more hydrophobic than the other sites. We presume these spectra were obtained at a negative membrane voltage, but the text or legend should clearly state how these were obtained. The authors should also explain whether the whole cell or edge was imaged. If these are at negative membrane voltages, might the Anap spectrum shift to higher wavelengths (i.e. more hydrophilic) when the membrane is depolarized? Did the authors find any spectral shift for I202Anap when doing a similar test as depicted in Figure 3E?

11) In Figure 3E, spectra are shown as normalized signals for different voltages, but an amplitude change should also be demonstrated by providing raw spectra at different voltages.

12) In Figure 4, spectra are compared between A197Anap and F150A-A197Anap, showing increase of fluorescence in F150A-A197Anap. Were these obtained at a negative membrane voltage? How do these spectra change when membrane potential is changed?

Additional suggestions for the authors to consider:

1) The authors propose that Anap fluorescence tracks an S4 movement involved in the opening of the channel. They also argue that the existence of more than one open state could explain why the increase in florescence upon depolarization lags the proton current in most cases. While they convincingly show that Anap is not pH sensitive per se, when incorporated into the protein, the fluorescence efficiency of the fluorophore could still be affected by protonation of channel residues in the immediate environment when the channel opens, even after S4 has completed its movement. To address this alternative explanation, the authors could use Hv1 mutants with strongly reduced proton conductance. Channels bearing mutations corresponding to N214R or D112N were used successfully to isolate Hv1 gating currents from the much larger proton currents (De La Rosa & Ramsey, Biophys. J. 2018 114:2844-2854; Carmona et al. PNAS 2018 115:9240-9245; Carmona et al. PNAS 2021 118: e2025556118). Perhaps, they could be used with patch clamp fluorometry as well?

2) The data showing that Hv1-197Anap is quenched by Phe at position 150 are very nice. Yet, it would be useful to show that the quenching is specific to F150 using a negative control. F149, for instance, is just next to F150 but points in a different direction, so its mutation to alanine should not affect Hv1-197Anap fluorescence.

3) A major finding of this work is the identification of a slow kinetic component that is highly sensitive to ΔpH. Earlier studies found that the ability of Hv1 to sense ΔpH is altered by some channel modifications, e.g., in the loop between TMH2 and TMH3 (Cherny et al. J. Gen. Physiol. 2018 150:851-862). Did the authors check whether any of these modifications alter the transition responsible for the slow kinetic component? For instance, a suppression of the transition resulting from a H168X mutation would help tighten the link to ΔpH sensing.

4) We understand that it is difficult to tightly control intracellular and extracellular pH when Hv1 is heterologously expressed in mammalian cells. The G-V relation is not always reliable because accumulation of protons or depletion of protons upon Hv channel activity will alter gating, as the authors have previously published (De La Rosa et al., J. Gen. Physiol. 2016 147:127-136). Could the kinetic analysis of Anap fluorescence be affected by similar alterations to proton concentration in the vicinity of Hv1? It would be helpful for the authors to comment on this specifically.

5) Quenching of Anap by Phe could be verified in cell free conditions using a spectrophotometer with different concentrations of Phe, or citing the literature if it has already been reported.

6) The authors did not cite any example of Anap incorporation into S4 helices, but there are several recent papers where Anap was utilized to probe motion of S4 in other channels. Examples include Dai et al., Nat. Commun. 2021 12:2802 and Mizutani et al. PNAS 2022 119:e2200364119.

7) In the Anap-free negative control (with only A197TAG plasmid transfection), the mCherry signal seems positive (Supplementary Figure 1, left row, second from the top). Is this due to unexpected skipping of the TAG codon to make mCherry-containing partial polypeptides? It would seem like an explanation is needed.

8) The data of Figure 3E are shown as data with different membrane voltages. But there is no information about membrane voltage for Fig. 1E and Fig. 2A and Fig. 4B. Are these from unpatched cells? Please clarify.

9) G-V relations are shown for F150A-A197Anap, but current traces of F150A-A197Anap are missing.

10) On Page 11, Line 303 says “experimental F-V relationship is positively shifted by 10 mV with respect to the G-V curve”. But looking at the data Fig5D, the shift at ΔpH=2 seems the opposite. Perhaps “positively” should be “negatively” in this sentence?

REVIEWING TEAM

Reviewed by:

Yasushi Okamura, Professor, Osaka University, Japan: voltage-sensing proteins, electrophysiology and fluorescence spectroscopy

Francesco Tombola, Associate Professor, University of California, Irvine, USA: ion channel mechanisms, electrophysiology and fluorescence spectroscopy

Christopher A. Ahern, Professor, University of Iowa, USA: ion channel mechanisms, non-canonical amino acidic mutagenesis

Curated by:

Kenton J. Swartz, Senior Investigator, NINDS, NIH USA

(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)

Author Response

Reviewer #1 (Public Review):

High resolution mechanistic studies would be instrumental in driving the development of Cas7-11 based biotechnology applications. This work is unfortunately overshadowed by a recent Cell publication (PMID: 35643083) describing the same Cas7-11 RNA-protein complex. However, given the tremendous interest in these systems, it is my opinion that this independent study will still be well cited, if presented well. The authors obviously have been trying to establish a unique angle for their story, by probing deeper into the mechanism of crRNA processing and target RNA cleavage. The study is carried out rigorously. The current version of the manuscript appears to have been rushed out. It would benefit from clarification and text polishing.

We thank the reviewer for the positive and helpful comments that have made the manuscript more impactful.

To summarize the revisions, we have resolved the metal-dependence issue, updated the maps in both main and supplementary figures that support the model, re-organized the labels for clarity, and added the comparison between our and Kato et al.’ structures.

In addition, we describe a new result with an isolated C7L.1 fragment that retains the processing and crRNA binding activities.

Reviewer #2 (Public Review):

In this manuscript, Gowswami et al. solved a cryo-EM structure of Desulfonema ishimotonii Cas7-11 (DiCas7-11) bound to a guiding CRISPR RNA (crRNA) and target RNA. Cas7-11 is of interest due to its unusual architecture as a single polypeptide, in contrast to other type III CRISPR-Cas effectors that are composed of several different protein subunits. The authors have obtained a high-quality cryo-EM map at 2.82 angstrom resolution, allowing them to build a structural model for the protein, crRNA and target RNA. The authors used the structure to clearly identify a catalytic histidine residue in the Cas7-11 Cas7.1 domain that is important for crRNA processing activity. The authors also investigated the effects of metal ions and crRNAtarget base pairing on target RNA cleavage. Finally, the authors used their structure to guide engineering of a compact version of Cas7-11 in which an insertion domain that is disordered in the cryo-EM map was removed. This compact Cas7-11 appears to have comparable cleavage activity to the full-length protein.

The cryo-EM map presented in this manuscript is generally of high quality and the manuscript is very well illustrated. However, some of the map interpretation requires clarification (outlined below). This structure will be valuable as there is significant interest in DiCas7-11 for biotechnology. Indeed, the authors have begun to engineer the protein based on observations from the structure. Although characterization of this engineered Cas7-11 is limited in this study and similar engineering was also performed in a recently published paper (PMID 35643083), this proof-of-principle experiment demonstrates the importance of having such structural information.

The biochemistry experiments presented in the study identify an important residue for crRNA processing, and suggest that target RNA cleavage is not fully metal-ion dependent. Most of these conclusions are based on straightforward structure-function experiments. However, some results related to target RNA cleavage are difficult to interpret as presented. Overall, while the cryo-EM data presented in this work is of high quality, both the structural model and the biochemical results require further clarification as outlined below.

We thank the reviewer for the positive and helpful comments that have made the manuscript more impactful.

To summarize the revisions, we have resolved the metal-dependence issue, updated the maps in both main and supplementary figures that support the model, re-organized the labels for clarity, and added the comparison between our and Kato et al.’ structures.

In addition, we describe a new result with an isolated C7L.1 fragment that retains the processing and crRNA binding activities.

1. The DiCas7-11 structure bound to target RNA was also recently reported by Kato et al. (PMID 35643083). The authors have not cited this work or compared the two structures. While the structures are likely quite similar, it is notable that the structure reported in the current paper is for the wild-type protein and the sample was prepared under reactive conditions, resulting in a partially cleaved target. Kato et al. used a catalytically dead version of Cas7-11 in which the target RNA should remain fully intact. Are there differences in the Cas7-11 structure observed in the presence of a partially cleaved target RNA in comparison to the Kato et al. structure? Such a comparison is appropriate given the similarities between the two reports. A figure comparing the two structures could be included in the manuscript.

We have added a paragraph on page 12 that describe the differences in preparation of the two complexes and their structures. We observed minor differences in the overall protein structure (r.m.s.d. 0.918 Å for 8114 atoms) but did observe quite different interactions between the protein and the first 5’-tag nucleotide (U(-15) vs. G(-15)) due to the different constructs in pre-crRNA, which suggests an importance of U(-15) in forming the processing-competent active site. We added Figure 2-figure supplementary 3 that illustrates the similarities and the differences.

2.The cryo-EM density map is of high quality, but some of the structural model is not fully supported by the experimental data (e.g. protein loops from the alphafold model were not removed despite lack of cryo-EM density). Most importantly, there is little density for the target RNA beyond the site 1 cleavage site, suggesting that the RNA was cleaved and the product was released. However, this region of the RNA was included in the structural model. It is unclear what density this region of the target RNA model was based on. Further discussion of the interpretation of the partially cleaved target RNA is necessary. Were 3D classes observed in various states of RNA cleavage and with varied density for the product RNAs?

We should have made it clear in the Method that multiple maps were used in building the structure but only submitted the post-processed map to reviewers. When using the Relion 4.0’s local resolution estimation-generated map, we observed sufficient density for some of the regions the reviewer is referring to. For instance, the site 1 cleavage density does support the model for the two nucleotides beyond site 1 cleavage site (see the revised Figure 1 & Figure 1- figure supplement 3).

However, there are protein loops that remain lack of convincing density. These include 134141 and 1316-1329 that are now removed from the final coordinate.

The “partially cleaved target RNA” phrase is a result of weak density for nucleotides downstream of site 1 (+2 and +3) but clear density flanking site 2. This feature indicates that cleavage likely had taken place at site 1 but not site 2 in most of the particles went into the reconstruction. To further clarify this phrase, we added “The PFS region plus the first base paired nucleotide (+1*) are not observed.” on page 4 and better indicate which nucleotides are or are not built in our model in Figure 1.

1. The authors argue that site 1 cleavage of target RNA is independent of metal ions. This is a potentially interesting result, but it is difficult to determine whether it is supported by the evidence provided in the manuscript. The Methods section only describes a buffer containing 10 mM MgCl2, but does not describe conditions containing EDTA. How much EDTA was added and was MgCl2 omitted from these samples? In addition, it is unclear whether the site 1 product is visible in Figures 2d and 3d. To my eye, the products that are present in the EDTA conditions on these gels migrated slightly slower than the typical site 1 product. This may suggest an alternate cleavage site or chemistry (e.g. cyclic phosphate is maintained following cleavage). Further experimental details and potentially additional experiments are required to fully support the conclusion that site 1 cleavage may be metal independent.

As we pointed out in response to Reviewer 1’s #8 comment, this conclusion may have been a result of using an older batch of DiCas7-11 that contains degraded fragments.

As shown in the attached figure below, “batch Y” was an older prep from our in-house clone and “batch X” is a newer prep from the Addgene purchased clone (gel on right), and they consistently produce metal-independent (batch Y) or metal-dependent (batch X) cleavage (gel on left). It is possible that the degraded fragments in batch Y carry a metal-independent cleavage activity that is absent in the more pure batch X.

We further performed mass spectrometry analysis of two of the degraded fragments from batch Y (indicated by arrows below) and discovered that these are indeed part of DiCas7-11. We, however, cannot rationalize, without more experimental evidence, why these fragments might have generated metal-independent cleavage at site 1. Therefore, we simply updated all our cleavage results from the new and cleaner prep (batch X) (For instance, Figure 3c). As a result, all references to “metal-independence” were removed.

With regard to the nature of cleaved products, we found both sites could be inhibited by specific 2’-deoxy modifications, consistent with the previous observation that Type III systems generate a 2’, 3’-cyclic product in spite of the metal dependence (for instance, see Hale, C. R., Zhao, P., Olson, S., Duff, M. O., Graveley, B. R., Wells, L., ... & Terns, M. P. (2009). RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell, 139(5), 945-956.)

We added this rationale based on the new results and believe that these characterizations are now thorough and conclusive

1. The authors performed an experiment investigating the importance of crRNA-target base pairing on cleavage activity (Figure 3e). However, negative controls for the RNA targets in the absence of crRNA and Cas7-11 were not included in this experiment, making it impossible to determine which bands on the gel correspond to substrates and which correspond to products. This result is therefore not interpretable by the reader and does not support the conclusions drawn by the authors.

Our original gel image (below) does contain these controls but we did not include them for the figure due to space considerations (we should have included it as a supplementary figure). We have now completely updated Figure 3e with much better quality and controls. Both the older and the updated experiments show the same results.

Original gel for Figure 3e containing controls.

Typo in the tag here; "transculsion" should be "transclusion".

i think you forgot to use the header tag.

I think you have added extra link tag for css file. gone through all code and i felt it you have added extra tags .

This is interesting that you used a 'mark' tag, because I have not heard about it before.

would recomend dropping this end tag to the line below, just to make it easier to see the end tag as

is a block level element.

Chalk talk is in my plans this week, so I will give this strategy a try!

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

Learn more at Review Commons

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Reply to the reviewers

1. General Statements [optional]

In our work, we quantified the abundance and positions of major kinetochore proteins within the metaphase kinetochore in budding yeast using single-molecule localization microscopy. Based on these measures, we revised the current model of the kinetochore and provided a nanoscale view of the complex.

We now revised our manuscript according to reviewers’ points. We performed new analyses to quantify the measurement errors and to justify our data analysis workflows. We further exploited the correlation-based analysis and found a correlation between the spreads of kinetochore proteins perpendicular to the spindle axis and their positions along the axis. We also discussed the potential non-centromeric pools and revised our model of the kinetochore. Further information on our analyses was now provided to improve the clarity. Changes to the text were implemented to better reflect our data. Information from relevant works was incorporated to better connect this work to the field.

We thank the reviewers for their points, which help us show the rigorousness of our analyses, further demonstrate the potential of our work, and improve clarity.

2. Point-by-point description of the revisions

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

The authors have developed a rigorous methodology for using single-molecule imaging of exogenously labeled kinetochore proteins to count and estimate their copy numbers and the average distance from the kinetochore protein Spc105. Although the method is technically sound, its application to the kinetochore raises some crucial questions below. My biggest concern is the effect of non-centromeric pools of the centromeric proteins Cse4, Cep3, and Ctf19 on the estimated copy number per kinetochore. The authors should be able to address most, if not all, questions by presenting a more in-depth data analysis.

Major points

1. Accounting for tilt of the yeast spindle relative to the image plane: It is not clear to me how the authors ascertain whether the spindle being imaged is nearly parallel to the image plane. In the companion fission yeast study, spindle poles are used for this purpose, but this study seems to rely only on the labeled kinetochore proteins. The criteria used to select the in-plane spindles should be clearly defined.

We thank the reviewer for pointing this out. We selected the in-plane spindles based on their average PSF size, which informs the z positions of the center of the kinetochore cluster (for simplicity, now all ’half-spindle’ was changed to ‘kinetochore cluster’). To calibrate the z position of kinetochore clusters, we first measured the width of the kinetochore cluster by fitting a cylindrical distribution. Overall, the kinetochores are likely symmetrically distributed around the spindle axes. Therefore, the height and the width of a kinetochore cluster should be the same. We then calibrated the z positions of the PSF size based on fluorescent bead data. Next, we plugged in the cylindrical distribution to the calibration curve to correlate the mean PSF size and position of the kinetochore cluster. We only took the kinetochore clusters with a mean PSF size

1. The effects of PSF depth on counting kinetochore proteins: The authors use a well-characterized nuclear pore protein as the reference to estimate kinetochore protein counts per half-spindle. Although this method appears rigorous in principle, I am unsure about the effect of the spatial distribution of kinetochores on the accuracy of the estimated number. Nuclear pore proteins are all localized within an 100 nm away from the focal plane even when the spindle is perfectly parallel to the focal plane. A discussion of this possibility, its effect on the protein count/distance estimates, and any mitigating factors is essential to highlight the caveats associated with the conclusions.

Based on the cylindrical distribution (see please the reply to point 1) of kinetochore clusters and their positions in z, we calculated the upper and lower boundaries of the distribution of kinetochore proteins in z, given a specific mean PSF size cutoff of a kinetochore cluster. Regardless of how stringent the cutoff is (130 and 135 nm), we made sure the boundaries do not exceed the imaging depth defined by our choice of the PSF size filtering (

1. Presentation of the cross-correlation analysis: The authors use cross-correlation for an unbiased calculation of the axial separation between a protein of interest and Cse4, but I am curious about the structure of the underlying data, and the intensity image in Figure 1 is not easy to examine. It will be helpful to include more analysis of the underlying data for at least a subset of the proteins (e.g., proteins at short, intermediate, and long distances from Cse4) as supplementary data.

2. The authors should include X and Y projections of the cross-correlation function.

3. Do the widths of cross-correlation functions (i.e., their spread perpendicular to the spindle axis) match across all proteins and experiments? This should be an almost invariant characteristic of the measurements, assuming that proteins within each kinetochore tightly cluster around the 25 nm microtubule. This line of thinking makes the large width of the cross-correlation shown in Figure 1 somewhat surprising.

4. It will also be interesting to test if the correlation between the positions of Spc105 molecules, especially perpendicular to the spindle axis, is comparable to the known separations between adjacent microtubules in the yeast spindle (the authors could use Winey et al. 1995 for serial-section EM of yeast spindles for comparison).

The reviewer is interested in the spread, or the size of the distribution, of a protein in a kinetochore along and perpendicular to the spindle axis. This is an interesting idea and can be done practically. However, the information can be more easily obtained based on auto-correlation instead of cross-correlation, due to its better signal-to-noise ratio along the dimension perpendicular to the spindle axis. Cross-correlations in that dimension are convoluted with background localizations and different localization precisions of the two channels. These factors are hard to interpret and disentangled. In auto-correlations, although the background is still present, it can be modeled and then removed easily, as now mentioned on page 15 lines 500-516.

Accordingly, we performed auto-correlation analysis on all the proteins and compared them to simulations representing different sizes. We find that the size of the distribution correlates to the position of the protein along the spindle axis. The results are now included as the new Fig. S5 and discussed on page 6 lines 169-176.

The cross-correlation analysis was based on only the position of the maximum value, not the projections. To keep the figure concise, we decided not to include the projections. However, the auto-correlation analysis was indeed based on projections, which we now included in Fig. S5.

Regarding the correlation between the positions of Spc105 molecules, we believe the reviewer actually refers to the correlation between the positions of kinetochores. Auto-/cross-correlations contain the information of the cluster sizes, based on the first peak (as shown in Fig. S5), and the relative distance (if the pattern is periodic). Unfortunately, the positions of kinetochores perpendicular to the spindle axis are not periodically distributed. Therefore, we cannot comment on the separations between adjacent microtubules.

1. Cse4 count (4 per kinetochore) and the model presented: One of the surprising conclusions of the study is that there are two nucleosomes associated with each microtubule attachment, with Mif2/CENP-C potentially interacting with both nucleosomes. There are two critical issues that the authors must consider.

(1) Fluorescent protein chimeras of Cse4 and CBF3 and COMA complex members do not exclusively localize to kinetochores. Biochemical studies show that both Cse4 and CBF3 proteins interact with non-centromeric DNA, e.g., see work from the Biggins lab regarding Cse4 over-expression and also from the Henikoff group that used ChIP-seq. I can't think of a similar reference for the CBF3 complex, but the DNA-binding proteins are also likely to interact with other parts of the genome. The non-centromeric protein is visible as a significant background fluorescence in wide-field microscopy, e.g., see Cep3 localization here: https://images.yeastrc.org/imagerepo/viewExperiment.do?id=202308&experimentGroupOffset=3&experimentOffset=0&experimentGroupSize=3

Similar background fluorescence can be detected for Cse4 and Ctf19. This extra-centromeric localization of Cse4, Cep3, and Ctf19 makes it possible that the protein counts included by the authors are "contaminated" to some extent by the extra-centromeric protein. The authors should discuss this possibility and how it might affect their counts.

After consideration, we agree with the reviewer that, specifically, a fraction of counted Cse4 molecules should be considered non-centromeric. We agree that the previous data is certainly sufficient to conclude it. The reviewer made a similar suggestion about COMA and CBF3 subcomplexes. In recent years a substantial portion of inner kinetochore components has been reconstituted. In Harrison et al. 2019, the Ctf19 complex structure has been solved. Two copies of the complex were observed. Therefore, the non-centromeric pool of COMA is certainly possible and we now made the adjustments to the text (page 8, lines 219-225) and Fig. 4. Accordingly, we now also modified the abstract (page 1, lines 26-27) and restructured the sections (page 10) to accommodate the different possibility of Cse4 copy numbers. While, fluorescence imaging of CBF3 presents a signal throughout the nuclear region we observed only four copies of Cep3 (part of CBF3). A CBF3 structure also has been resolved by Yan et al. 2018, in which the complex was proposed to exist as a dimer. This translates into four copies of Cep3. Therefore, we find it more suitable to leave all observed Cep3 (CBF3) molecules within a kinetochore model.

(2) The model drawn in Figure 4 makes explicit assumptions about the positioning of the four Cse4 molecules (or two nucleosomes) in each kinetochore relative to the rest of the kinetochore components. Yet, the data shown do not justify this specific arrangement. Lawrimore et al. 2011 claim that the non-centromeric Cse4 nucleosomes must be randomly distributed in the pericentromeric chromatin to evade detection in biochemical tests. Therefore, the nearest-neighbor analysis suggested above will be valuable for gaining new insights into the relative positioning of the centromeric- and non-centromeric Cse4 nucleosomes. A similar analysis for Cep3 and Ctf19 will also be helpful. If stereotypical positioning of these molecules cannot be detected, then the model should be revised accordingly (alternative models that are also consistent with the data can be included).

The reviewer has pointed out that Lawrimore et al. 2011 proposed and justified the existence of a non-centromeric Cse4 pool. This arrangement, also potentially along other inner kinetochore components, makes sense and our data did not indicate it otherwise. Therefore, we now revised our model accordingly by applying changes in the main text on page 10 lines 302-305 __as well as in __Fig. 4.

(3) I suggest one experiment that can help the authors better understand protein organization in one kinetochore. Joglekar et al. 2006 used a dicentric chromosome to isolate single kinetochores on the spindle axis to test the assumption that each kinetochore consists of approximately the same number of molecules of kinetochore proteins. The strains are easy to construct (transform existing strains with a linearized plasmid). Single kinetochores can be seen with a low but reasonable frequency. I leave the decision to perform the experiment to the authors' discretion depending on whether the experiment will be worth the effort in strengthening or enhancing their conclusions.

We performed the suggested experiment using the strain published in Joglekar et al. 2006 (kindly provided by Prof. Kerry Bloom) with Cse4 additionally tagged with mMaple. However, we always observed several super-resolved Cse4 clusters (likely of several kinetochores) overlapping with Nuf2-GFP diffraction-limited signal, therefore unable to assign a single isolated kinetochore to the lagging centromere.

1. Information regarding the degree of correction applied to calculate protein count per half-spindle: It will be helpful to include data regarding the degree of correction applied to the expected and measured numbers of NPC protein as supplementary data so that the readers can see the magnitude of this correction relative to the measured counts.

We would like to clarify that we did not correct the data. Instead, we calibrate the copy number, given that the copy number of Nup188 per NPC is known. We assume the same ratio between localization and copy number applies to both Nup188 and the kinetochore proteins. We now include a new Table S4 listing calibration factors of all experiments shown in Fig. 3.

Minor points:

1. McIntosh et al. JCB 2013 used microtubule plus-ends in serial section electron micrographs of yeast spindles to align the centromeric region and found a disk-shaped structure that roughly corresponds to the size of a single nucleosome ~ 80 nm away from the tip of the microtubule and centered the microtubule axis. The authors should refer to this finding in their discussion of the model that they present with two nucleosomes. In my opinion, this is compelling evidence for a nucleosome-like structure serving as the kinetochore foundation.

We agree with this reviewer's comment. The study, among others, present compelling evidence for a point-centromere. We now included the finding in the discussion on page 10, lines 293-294.

1. As discussed by the authors, the number of Cse4 molecules per kinetochore has been the subject of some controversy. Biochemical data from the Biggins group and ChIPseq data from the Westermann group (Altunkaya et al. 2016 Current Biology) strongly suggest that Cse4 molecules can only be found centered on the centromeric sequence. The latter reference should be included in the discussion.

Thank you for pointing this out. Indeed, this is important. We have now added the relevant reference in the discussion on__ page 10 lines 291-292__.

1. Although microscopy-based methods have estimated anywhere from 1, 2, to 6 Cse4 molecules per kinetochore, these studies generally agree on the stoichiometry between Cse4 and the rest of the kinetochore proteins, e.g., Ndc80 complex proteins are ~ 4-fold more abundant that Cse4, etc. The present study seems to disagree with protein stoichiometry. The authors may find it worthwhile to note this feature of their data.

We now discuss the stoichiometry difference between our results and others on page 11 lines 322-324.

1. Omission of the Dam1 complex from this study is disappointing to me personally, but I am sure that the authors have good reasons for this. They should briefly comment on the absence of the Dam1 complex in this study.

To provide information on the Dam1 complex, we imaged Ask1, a component of the complex. The measured positioning and copy number of the protein are now included in Fig. 2 and Fig. 3 respectively, and described and discussed in respective parts of the manuscript.

Reviewer #1 (Significance (Required)):

Cieslinski and colleagues present a single-molecule localization-based study to define the copy numbers and relative organization of kinetochore proteins in budding yeast. These numbers confirm and significantly refine prior measurements of the same aspects of the kinetochore. They also raise new questions and point to new research directions. The measurements also reveal a model of the protein organization of the budding yeast kinetochore in metaphase. For these reasons, the manuscript is of significant interest to the cell division field.

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

In this study, Cielinski and colleagues have applied single molecule localization microscopy to map the positions of proteins in the yeast kinetochore. This has not been reported previously and this study is both well-conducted and the data appear solid. They also use a modification of this technique to assess the stoichiometry of kinetochore proteins. The results that they obtain are broadly in line with several previous studies that use other methodology. There may be an improvement in accuracy using this new approach that has not been obtained previously and there are some important novel conclusions from this work. I would like the authors to address the following concerns prior to publication:

Major points

1. One interesting finding is that there is a discrepancy in the length of both the MIND and NDC80 complexes (from crystallographic data) with their relative positions. The authors suggest that the outer complexes could be twisted or rotated in respect of the spindle axis. It would be great if the authors could illustrate this in their model (or discuss it in the text), to demonstrate the required angle of twist/rotation of both complexes to account for the discrepancy. A twisted filament structure to the outer kinetochore does have some implications for its response to tension - a key determinant of kinetochore-microtubule attachment. It also may provide some flexibility to the structure under tension.

The discussion about this discrepancy has now been incorporated in the main text, page 9 lines 263-267. For clarity, we only partially reflect this in our schematic model (Fig. 4A; the MIND complex) but we already reflected this in the illustrative structural model in Fig. 4B.

1. For the experiment with cycloheximide, the authors state "Although we observed minor changes in copy numbers, the overall effect of CHX was small." For some proteins, Cse4i for example, there appears to be a significant decrease in intensity (30-40%) after cycloheximide treatment, see Figure S3. While the conclusion that tag maturation does not affect copy number measurements is sound, I suggest modifying this section to reflect the data.

We now modified the section accordingly by pointing out that Cse4i under CHX measurements led to reduction of the signal. The modification can be found on page 8 lines 207-211.

1. Page 5. The statement "These data agree reasonably well with previous diffraction-limited dual-color microscopy studies ..." provides readers with little ability to compare the data. I would like to see a supplementary figure comparing these new data with previous studies, especially those of Joglekar et al 2009, see Figure 3 in this paper.

We thank the reviewer for suggesting such a table. This will allow readers a direct comparison of the data between our study and Joglekar at al. 2009. The comparison can be found in new Table S1 __and __Fig. S4, which are now mentioned on page 5.

1. In terms of the distances quoted, are they in one dimension (as per Jogelkar et al 2009) or in three? The results section is entitled "...positions of kinetochore proteins along the metaphase spindle axis", which suggests a single dimension. Please make this very clear in the results section. In the discussion, is the statement "we mapped the relative positions of 15 kinetochore proteins along the kinetochore axis", which is not entirely clear. It seems from the methods that this is one dimension "...we determined the average distance between the two proteins along the spindle axis. “I suggest clarifying the results section briefly and clearly to indicate that this is a single dimension being measured and also using consistent wording of the axis measured throughout the text.

We agree the previous description may not be clear to the viewers. We now changed the text accordingly in the results section, page 5 lines 129-130.

Minor points:

Abstract: I would drop "all" from "For all major kinetochore proteins...", since full characterisation was performed on 14 proteins (9 in terms of copy number).

We now deleted “all” in the abstract as the reviewer suggested__.__

Page 2: "trough" to through.

Corrected.

Page 2 "S. cerevisiae" to italics

Corrected.

Methods p11. How do the MKY strains relate to common yeast genetic backgrounds? (e.g. are they S288C?).

MKY strains are derivative of S288C. The information was now updated in the Methods section and in Table S2.

Reviewer #2 (Significance (Required)):

This manuscript, together with an accompanying one from Virat et al., are nice complementary studies that provide the first single molecule localization studies of the yeast kinetochore. Although other labs have used super-resolution methods to study individual kinetochore proteins; both of these new studies map distances between many proteins at the kinetochore and thus are able to produce maps of the overall kinetochore structure. Like the previous study using standard resolution methods (Joglekar et al, 2009. Current Biology 19, 694-699); these studies will likely provide a benchmark for future studies on eukaryotic kinetochore architecture, including those in mammalian systems. Additionally, this work will appeal to super-resolution microscopists.

My expertise is as a yeast kinetochore cell biologist.

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 #2

Evidence, reproducibility and clarity

In this study, Cielinski and colleagues have applied single molecule localization microscopy to map the positions of proteins in the yeast kinetochore. This has not been reported previously and this study is both well-conducted and the data appear solid. They also use a modification of this technique to assess the stoichiometry of kinetochore proteins. The results that they obtain are broadly in line with several previous studies that use other methodology. There may be an improvement in accuracy using this new approach that has not been obtained previously and there are some important novel conclusions from this work. I would like the authors to address the following concerns prior to publication:

Major points

1. One interesting finding is that there is a discrepancy in the length of both the MIND and NDC80 complexes (from crystallographic data) with their relative positions. The authors suggest that the outer complexes could be twisted or rotated in respect of the spindle axis. It would be great if the authors could illustrate this in their model (or discuss it in the text), to demonstrate the required angle of twist/rotation of both complexes to account for the discrepancy. A twisted filament structure to the outer kinetochore does have some implications for its response to tension - a key determinant of kinetochore-microtubule attachment. It also may provide some flexibility to the structure under tension.
2. For the experiment with cycloheximide, the authors state "Although we observed minor changes in copy numbers, the overall effect of CHX was small." For some proteins, Cse4i for example, there appears to be a significant decrease in intensity (30-40%) after cycloheximide treatment, see Figure S3. While the conclusion that tag maturation does not affect copy number measurements is sound, I suggest modifying this section to reflect the data.
3. Page 5. The statement "These data agree reasonably well with previous diffraction-limited dual-color microscopy studies ..." provides readers with little ability to compare the data. I would like to see a supplementary figure comparing these new data with previous studies, especially those of Joglekar et al 2009, see Figure 3 in this paper.
4. In terms of the distances quoted, are they in one dimension (as per Jogelkar et al 2009) or in three? The results section is entitled "...positions of kinetochore proteins along the metaphase spindle axis", which suggests a single dimension. Please make this very clear in the results section. In the discussion, is the statement "we mapped the relative positions of 15 kinetochore proteins along the kinetochore axis", which is not entirely clear. It seems from the methods that this is one dimension "...we determined the average distance between the two proteins along the spindle axis."I suggest clarifying the results section briefly and clearly to indicate that this is a single dimension being measured and also using consistent wording of the axis measured throughout the text.

Minor points:

Abstract: I would drop "all" from "For all major kinetochore proteins...", since full characterisation was performed on 14 proteins (9 in terms of copy number).

Page 2: "trough" to through.

Page 2 "S. cerevisiae" to italics

Methods p11. How do the MKY strains relate to common yeast genetic backgrounds? (e.g. are they S288C?).

Significance

This manuscript, together with an accompanying one from Virat et al., are nice complementary studies that provide the first single molecule localization studies of the yeast kinetochore. Although other labs have used super-resolution methods to study individual kinetochore proteins; both of these new studies map distances between many proteins at the kinetochore and thus are able to produce maps of the overall kinetochore structure. Like the previous study using standard resolution methods (Joglekar et al, 2009. Current Biology 19, 694-699); these studies will likely provide a benchmark for future studies on eukaryotic kinetochore architecture, including those in mammalian systems. Additionally, this work will appeal to super-resolution microscopists.

My expertise is as a yeast kinetochore cell biologist.

(Could not highlight what attended to) Another good addition to making their web much more accesible to all the people that might go on to there website. And that is a descriptive tag for images on the website, for screen readers to be able to identify and describe the photo that is placed there. This allows people with visual impairments to identify everything that is going on, all with just listening to the Text To Speech.

This is optional we can add optional tag in bottom at all place

the formatting here makes it difficult to identify where the paragraph tag closes.

It would be better if you place the content next to the opening tag and the close it there, not in a new line.

p tag is not used. Few inline tags are used.

More minor nit-picking, the 'li' element is missing its closing tag

no errors

keep the tag along with the content it is related to

No error. good

This looks good! Only improvement could be to format the elements in such a away that would communicate the hierarchy. For example each 'li' element could be on its own line and further indented from the 'ul' tag.

again just needs opening and closing 'p' tags

I have tried to find mistakes but i am not able to do so. This is just example of perfect coding.

no errors

related, but not drawn from as I've been thinking about the continuum of taxonomies and subject headings for a while...

On the Spectrum of Topic Headings in note making

Any reasonable note one may take will likely have a hierarchical chain of tags/subject headings/keywords going from the broad to the very specific. One might start out with something broad like "humanities" (as opposed to science), and proceed into "history", "anthropology", "biological anthropology", "evolution", and even more specific. At the bottom of the chain is the specific atomic idea on the card itself. Each of the subject headings helps to situate the idea and provide the context in which it sits, but how useful within a note taking system is having one or more of these tags on it? What about overlaps with other broader subjects (one will note that "evolution" might also sit under "science" / "biology" as well), but that note may have a different tone and perspective than the prior one.

This becomes an interesting problem or issue as one explores ideas in a pre-designed note taking system. As a student just beginning to explore anthropology, one may tag hundreds of notes with anthropology to the point that the meaning of the tag is so diluted that a search of the index becomes useless as there's too much to sort through underneath it. But as one continues their studies in the topic further branches and sub headings will appear to better differentiate the ideas. This process will continue as the space further differentiates. Of course one may continue their research into areas that don't have a specific subject heading until they accumulate enough ideas within that space. (Take for example Daniel Kahneman and Amos Tversky's work which is now known under the heading of Behavioral Economics, a subject which broadly didn't exist before their work.) The note taker might also leverage this idea as they tag their own work as specifically as they might so as not to pollute their system as it grows without bound (or at least to the end of their lifetime).

The design of one's note taking system should take these eventualities into account and more easily allow the user to start out broad, but slowly hone in on direct specificity.

Some of this principle of atomicity of ideas and the growth from broad to specific can be seen in Luhmann's zettelkasten (especially ZK II) which starts out fairly broad and branches into the more specific. The index reflects this as well and each index heading ideally points to the most specific sub-card which begins the discussion of that particular topic.

Perhaps it was this narrowing of specificity which encouraged Luhmann to start ZKII after years of building ZKII which had a broader variety of topics?

Author Response

Reviewer #3 (Public Review):

1) Validation of reagents: The authors generated a pY1230 Afadin antibody claiming that (page 6) "this new antibody is specific to tyrosine phosphorylated Afadin, and that pY1230 is targeted for dephosphorylation by PTPRK, in a D2-domain dependent manner". The WB in Fig 1B shows a lot of background, two main bands are visible which both diminish in intensity in ICT WT pervanadate-treated MCF10A cell lysates. The claim that the developed peptide antibody is selective for pY1230 in Afadin would need to be substantiated, for instance by pull down studies analysed by pY-MS to substantiate a claim of antibody specificity for this site. However, for the current study it would be sufficient to demonstrate that pY1230 is indeed the dephosphorylated site. I suggest therefore including a site directed mutant (Y1230F) that would confirm dephosphorylation at this site and the ability of the antibody recognizing the phosphorylation state at this position.

We would like this antibody to be a useful and freely accessible tool in the field and have taken on board the request for additional validation. To this end we have significantly expanded Supplementary Figure 2 (now Figure 1 - figure supplement 2) and included a dedicated section of the results as follows: 1. We have now included information about all of the Afadin antibodies used in this study, since Afadin(BD) appears to be sensitive to phosphorylation (Figure 1 - figure supplement 2A). 2. We have demonstrated that the Afadin pY1230 antibody detects an upregulated band in PTPRK KO MCF10A cells, consistent with our previous tyrosine phosphoproteomics (Figure 1 - figure supplement 2B). This indicates that the antibody can be used to detect endogenous Afadin phosphorylation. 3. We have included two new knock down experiments demonstrating the recognition of Afadin by our antibody (Figure 1 - figure supplement 2C). There appear to be two Afadin isoforms recognised in HEK293T cells by both the BD and pY1230 antibody, consistent with previous reports (Umeda et al. MBoC, 2015). We have highlighted these in the figure. 4. We have performed mutagenesis to demonstrate the specificity of the antibody. We tagged Afadin with a fluorescent protein tag, reasoning that it would cause a shift in molecular weight that could be resolved by SDS PAGE, as is the case. We noted that the phosphopeptide used spans an additional tyrosine, Y1226, which has been detected as phosphorylated (although to a much lower extent than Y1230) on Phosphosite plus. The data clearly show that Afadin cannot be phosphorylated when Y1230 is mutated to a phenylalanine (compared to CIP control), indicating that this is the predominant site recognised by the antibody. In addition, the endogenous pervanadate-stimulated signal is completely abolished by CIP treatment (Figure 1 - figure supplement 2D). 5. We have included densitometric quantification of the dephosphorylation assay shown in Figure 1B, which was part of a time course and shows preferential dephosphorylation by the PTPRK ICD compared to the PTPRK D1. The signal stops declining with time, which could indicate antibody background, or an inaccessible pool of Afadin-pY1230 (Figure 1 - figure supplement 2E). 6. To further demonstrate that this site is modulated by PTPRK in post-confluent cells, we have used doxycycline (dox)-inducible cell lines generated in Fearnley et al, 2019. Upon treatment with 500 ng/ml Dox for 48 hours PTPRK is induced to lower levels than wildtype, however, normalized quantification of the Afadin pY1230 against the Afadin (CST) signal clearly indicates downregulation by PTPRK WT, but not the catalytically inactive mutant (Figure 1 - figure supplement 2F and 2G). Together these data strengthen our assertion that this antibody recognises endogenously phosphorylated Afadin at site Y1230, which is modulated in vitro and in cells by PTPRK phosphatase activity. For clarity, we have highlighted and annotated the relevant bands in figures. We have also included identifiers for each Afadin total antibody was used in particular experiments.

2) The authors claim that a short, 63-residue predicted coiled coil (CC) region, is both necessary and sufficient for binding to the PTPRK-ICD. The region is predicted to have alpha-helical structure and as a consequence, a helical structure has been used in the docking model. Considering that the authors recombinantly expressed this region in bacteria, it would be experimentally simple confirming the alpha-helical structure of the segment by CD or NMR spectroscopy.

To clarify, the helical structure in the docking model was independently predicted by several sequence and structural analysis programmes including AlphaFold2, RobettaFold, NetSurfP and as annotated in Uniprot (as a coiled coil). We did not stipulate prior to the AF2 prediction that it was helical. Isolated short peptides frequently adopt helical structure, therefore prediction of a helix within the context of the full Afadin sequence is, in our opinion, stronger evidence than CD of an isolated fragment.

3) Only two mutants have been introduced into PTPRK-ICD to map the Afadin interaction site. One of the mutations changes a possibly structurally important residues (glycine) into a histidine. Even though this residue is present in PTPRM, it does not exclude that the D2 domain no longer functionally folds. Also the second mutation represents a large change in chemical properties and the other 2 predicted residues have not been investigated.

The residues that were selected for mutation are all localised to the protein surface and therefore are unlikely to be involved in stable folding of PTPRK. In support of the correct folding of the mutated PTPRK, we include in Figure 1 below SEC elution traces for wild-type and mutant D2 showing that they elute as single symmetric peaks at the same elution volume as the WT protein. This is consistent with them having a similar shape and size, and not being aggregated or unfolded.

Figure 1. PTPRK-D2 wild-type and mutant preparative SEC elution profiles. A280nm has been normalised to help illustrate that the different proteins elute at the same volume. The main peak from these samples was used for binding assays in the main paper.

Furthermore, the yield for the double mutant was very high (4 mg of pure protein from a 2 L culture, see A280 value in graph below), whereas poorly folded proteins tend to have significantly reduced yields. This protein was also very stable over time whereas unfolded proteins tend to degrade during or following purification.

Figure 2. Analytical SEC elution profile for the PTPRK-D2 DM construct showing the very high yield consistent with a well-folded, stable protein.

Finally, we have carried out thermal melt curves of the WT and mutant PTPRK D2 domains showing that they all possess melting temperatures between 39.3°C and 41.7°C, supporting that they are all equivalently folded. We include these data as an additional Supplementary Figure (Figure 4 - figure supplement 3) in the paper.

4) The interface on the Afadin substrate has not been investigated apart from deleting the entire CC or a central charge cluster. Based on the docking model the authors must have identified key positions of this interaction that could be mutated to confirm the proposed interaction site.

We have now made and tested several additional mutations within both the Afadin-CC and PTPRK-D2 domains to further validate the AF2 predicted model of the complex.

For Afadin-CC we introduced several single and double mutations along the helix including residues predicted to be in the interface and residues distal from the interface. These mutations and the pulldown with PTPRK are described in the text and are included as additional panels to a modified Figure 3. All mutations have the expected effect on the interaction based on the predicted complex structure. To help illustrate the positions of these mutations we have also included a figure of the interface with the residues highlighted.

For the PTPRK-D2 we have also introduced two new mutations, one buried in the interface (F1225A) and one on the edge of the interface encompassing a loop that is different in PTPRM (labelled the M-loop). GST-Afadin WT protein was bound to GSH beads and tested for their ability to pulldown WT and mutated PTPRK. These new mutations (illustrated in the new Figure 4 – figure supplement 2) further support the model prediction. F1225A almost completely abolishes binding as predicted, while the M-loop retains binding. These mutations and their effects are now described in the main text and the pull-down data, including controls and retesting of the original DM mutant, are included as panel H in a newly modified Figure 4 focussed solely on the PTPRK interface.

5) A minor point is that ITC experiments have not been run long enough to determine the baseline of interaction heats. In addition, as large and polar proteins were used in this experiment, a blank titration would be required to rule out that dilution heats effect the determined affinities.

All control experiments including buffer into buffer, Afadin into buffer and buffer into PTPRK were carried out at the same time as the main binding experiment and are shown below overlaid with the binding curve. These demonstrate the very small dilution heats consistent with excellent buffer matching of the samples.

We were able to obtain excellent fits to the titration curves by fitting 1:1 binding with a calculated linear baseline (see Figure 2B,D). Very similar results were obtained by fitting to the sum (‘composite’) of fitted linear baselines obtained for the three control experiments for each titration.

"The <meta> tag defines metadata about an HTML document. Metadata is data (information) about data. <meta> tags always go inside the <head> element, and are typically used to specify character set, page description, keywords, author of the document, and viewport settings."

Our justice system in a nutshell. The one who fits the tag of a media defined criminal instantly made to look guilty without validation or solid evidences.

.code code css code, kbd, pre, tt, .ace_editor.ace-github-light, .blame-sha, .blob-code-inner, .blob-num, .branch-name, .commit .sha, .commit .sha-block, .commit-desc pre, .commit-ref, .commit-tease-sha, .default-label .sha, .export-phrase pre, .file-editor-textarea, .file-info, .gollum-editor .expanded textarea, .gollum-editor .gollum-editor-body, .hook-delivery-guid, .hook-delivery-response-status, .input-monospace, .news .alert .branch-link, .oauth-app-info-container dl.keys dd, .quick-pull-choice .new-branch-name-input input, .tag-references > li.commit, .two-factor-secret, .upload-files .filename, .url-field, .wiki-wrapper .wiki-history .commit-meta code { font-family: 'Fira Code' !important; }

joined :)

Historically, the related topics section of each GoutPal page identified relationships using the WordPress Tag feature. However, after I invented WordPress Transmigration, I need a better approach.

So I must replace this with a navigation map. And create new workflows that enable me to update these maps efficiently.

#BuildInPublic

My latest research summarries about edamame and gout are available to GoutPal Links Subscribers. So far, my research notes cover topics for new edamame pages. Including reviews of studies about edamame and uric acid. Also, I'm researching general health benefits of edamame in my Food Research Channel. So I'll re-purpose some of that for gout sufferers where there is an impact on inflammation.

Such a good tool that I wish I knew about earlier.

this also seems unfortunately like not the right solution, though. should nice clothes always cost a lot of money? how would a utopian society deal with this sort of weighty clothes? are we all making clothes for each other? but then we lose the idea of fashion/designers/luxury - in some sense i wonder how capital and design are intertwined

Text-Based Adventure Game

Taken from Maciej Cegłowski https://idlewords.com/talks/website_obesity.htm.

I'm not sure if there is supposed to be a forward slash before the closing bracket in order to properly close the tag

I'm not sure if there is supposed to be a forward slash before the closing bracket in order to properly close the tag

annotation meta: may need new tag: fear would be used for ... valid uses for it beyond ...

annotation meta: may need new tag: you don't have a _; you have a _

annotation meta: may need new tag: applies to siblings only or applies to same level only

Make sure every element has one opening and closing tag. (with the exception of some elements don't require closing tags).

So in this case, the middle "p" tag isn't doing anything.

• It's good practice to put new elements on a new line
• You have two opening paragraph tags (

  ) before a closing tag (

  ), paragraph content should be between

annotation meta: may need new tag:

dynamic behavior vs. static analysis [not quite parallel]

or can we reuse something else like?: lexical semantics vs. run-time semantics

annotation meta: may need new tag: - don't want to change or remove existing feature [because...] - solving problem B will reduce misunderstandings around feature A

annotation meta: may need new tag:

distinction between nothing, false, and empty array

verbiage that allows for ...

Context: Lindsey Graham had just proposed a bill for a nationwide abortion ban after 15 weeks of pregnancy.

McConnell's position seems to be one that choice about abolition is an option, but one which is reserved for white men of power over others. This is painful because that choice is being left to people without any of the information and nuance about specific circumstances versus the pregnant women themselves potentially in consultation with their doctors who have broad specific training and experience in the topics and issues at hand. Why are these leaders attempting to make decisions based on possibilities rather than realities, particularly when they've not properly studied or are generally aware of any of the realities?

If this is McConnell's true position, then why not punt the decision and choices down to the people directly impacted? And isn't this a long running tenet of the Republican Party to allow greater individual freedoms? Isn't their broad philosophy: individual > state government > national government? (At least with respect to internal, domestic matters; in international matters the opposite relationships seem to dominate.)

tl;dr:<br /> Mitch McConnell believes in choice, just not in your choice.

Here's the actual audio from a similar NPR story:<br /> https://ondemand.npr.org/anon.npr-mp3/npr/me/2022/09/20220914_me_gop_sen_lindsey_graham_introduces_15-week_abortion_ban_in_the_senate.mp3#t=206

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McConnell is also practicing the Republican party game of "do as I say and not as I do" on Graham directly. He's practicing this sort of hypocrisy because as leadership, he's desperately worried that this move will decimate the Republican Party in the midterm elections.

There's also another reading of McConnell's statement. Viewed as a statement from leadership, there's a form of omerta or silent threat being communicated here to the general Republican Party membership: you better fall in line on the party line here because otherwise we run the risk of losing power. He's saying he's leaving it up to them individually, but in reality, as the owner of the purse strings, he's not.

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Thesis:<br /> The broadest distinction between American political parties right now seems to be that the Republican Party wants to practice fascistic forms of "power over" while the Democratic Party wants to practice more democratic forms of "power with".

Pillar of Oenomaus should be a tag added. Wish there was a better location description

Another possible tag? How many alters should we consider important to tag?

Could this be a potential tag on the map that we dont have?

I can see how this statement could relate to the previous reading (Ch. Introduction The Digital Humanities Moment) about there being too many people practicing the digital humanities. When hashtags get used and everyone starts contributing information, misinformation can spread just as easily as facts. Maybe this is an example of the kind of situation Gold implied when he said people can ask "disruptive questions"?

Nachdem Gott den Menschen am sechsten Tag schuf, sagte er dass es sehr gut war. Nachdem er [der Mensch] dann im Garten Eden das einzige Gebot brach, dass seinen geistlichen Tod herbeiführte und ihn von Gott trennte, wurde es nötig, dass Gott den Menschen von dieser Trennung erlöst. Die Erlösung kommt nur durch den Glauben an den Erlöser (Jesus Christus) selber, er befreit uns vom geistlichen Tod und hat unsere Schuld auf sich selbst genommen. Er hat uns Erlöst von unserer Schuld, sie kommt nur durch ihn.

• SEE

THIS WILL HELP WITH ORGANIZATION! USE IT (talking to myself here)

black box

opaque score

Introduction: The introduction to a scholarly article describes the topic or problem the authors researched. Sometimes it's labeled, sometimes it's not. The authors will present the thesis of their argument or the goal of their research. The introduction may also discuss the relevance or importance of the research question. An overview of related research and findings, called a literature review, may appear in the introduction, though the literature review may be in its own section.

The idea of social cost of carbon (SCC) is fascinating: seemingly it aims to make the social costs of climate crisis objective by giving them a price tag. But then it becomes clear that the price tag depends on political / value judgements concerning the future, on which the idea of "discounting" depends.

Always start component names with a capital letter.

Author Response

Reviewer #3: (Public Review):

In this ms Li et al. examine the molecular interaction of Rabphilin 3A with the SNARE complex protein SNAP25 and its potential impact in SNARE complex assembly and dense core vesicle fusion.

Overall the literature of rabphilin as a major rab3/27effector on synaptic function has been quite enigmatic. After its cloning and initial biochemical analysis, rather little new has been found about rabphilin, in particular since loss of function analysis has shown rather little synaptic phenotypes (Schluter 1999, Deak 2006), arguing against that rabphilin plays a crucial role in synaptic function.

While the interaction of rabphilin to SNAP25 via its bottom part of the C2 domain has been already described biochemically and structurally in the Deak et al. 2006, and others, the authors make significant efforts to further map the interactions between SNAP25 and rabphilin and indeed identified additional binding motifs in the first 10 amino acids of SNAP25 that appear critical for the rabphilin interaction.

Using KD-rescue experiments for SNAP25, in TIRF based imaging analysis of labeled dense core vesicles showed that the N-terminus of SN25 is absolutely essential for SV membrane proximity and release. Similar, somewhat weaker phenotypes were observed when binding deficient rabphilin mutants were overexpressed in PC12 cells coexpressing WT rabphilin. The loss of function phenotypes in the SN25 and rabphilin interaction mutants made the authors to claim that rabphilin-SN25 interactions are critical for docking and exocytosis. The role of these interaction sites were subsequently tested in SNARE assembly assays, which were largely supportive of rabphilin accelerating SNARE assembly in a SN25 -terminal dependent way.

Regarding the impact of this work, the transition of synaptic vesicles to form fusion competent trans-SNARE complex is very critical in our understanding of regulated vesicle exocytosis, and the authors put forward an attractive model forward in which rabphilin aids in catalyzing the SNARE complex assembly by controlling SNAP25 a-helicalicity of the SNARE motif. This would provide here a similar regulatory mechanism as put forward for the other two SNARE proteins via their interactions with Munc18 and intersection, respectively.

We thank the reviewer #3 for the summary of the paper and for the praise of our work. The point-to-point replies are as follow:

While discovery of the novel interaction site of rabphilin with the N-Terminus of SNAP25 is interesting, I have issues with the functional experiments. The key reliance of the paper is whether it provides convincing data on the functional role of the interactions, given the history of loss of function phenotypes for Rabphilin. First, the authors use PC12 cells and dense core vesicle docking and fusion assays. Primary neurons, where rabphilin function has been tested before, has unfortunately not been utilized, reducing the impact of docking and fusion phenotype.

We have discussed these questions as mentioned in our response to Essential Revisions 3 and added this corresponding passage to the Discussion section (pp.18-19, lines 407-427).

In particular the loss of function phenotype in figure 3 of the n-terminally deleted SNAP25 in docking and fusion is profound, and at a similar level than the complete loss of the SNARE protein itself. This is of concern as this is in stark contrast to the phenotype of rabphilin loss in mammalian neurons where the phenotype of SNAP25 loss is very severe while rabphilin loss has almost no effect on secretion. This would argue that the N-terminal of SNAPP25 has other critical functions besides interacting with rabphilin. In addition, it could argue that the n-Terminal SNAP25 deletion mutant may be made in the cell (as indicated from the western blot) but may not be properly trafficked to the site of release

To test whether the N-peptide deletion mutant of SN25 can properly target to the plasma membrane, we overexpressed the SN25 FL or SN25 (11–206) with C-terminal EGFP-tag in PC12 cells and monitored the localization of SN25 FL-EGFP and SN25 (11–206)-EGFP near the plasma membrane by TIRF microscopy. We observed that the average fluorescence intensity of SN25 (11–206)-EGFP showed no significant difference with SN25 FL-EGFP as below, suggesting that the N-peptide deletion mutant may not influence the trafficking of SN25 to plasma membrane.

(A) TIRF imaging assay to monitor the localization of SN25-EGFP near the plasma membrane. Overexpression of SN25 FL-EGFP (left) and SN25 (11–206)-EGFP (right) using pEGFP-N3 vector in PC12 cells. Scale bars, 10 μm. (B) Quantification of the average fluorescence intensity of SN25-EGFP near the plasma membrane in (A). Data are presented as mean ± SEM (n ≥ 10 cells in each). Statistical significance and P values were determined by Student’s t-test. ns, not significant.

真的！ 很多筆記是當下的靈感，預計未來在某些適當時機要用的，通常是備課要增加資訊豐富度時用。所以存了一堆。但隨著閱讀不斷的增加，靈感筆記也隨之暴增，即便有粗略以資料夾區分（我不習慣用tag)，但是，這麼大量的靈感筆記，已經造成我內心裡極大的壓力，甚至想到要再次整理與組織它時，資料龐大與凌亂到造成我心裡很大的壓力，甚至隱隱地開始討厭筆記起來，但是又不能不使用它，我實在對自己不牢靠的記憶感到抱歉！所以又繼續懷抱著既恨又無法割捨的心態下，繼續囤積筆記。別人的囤物症是衛生紙、鞋子等等，我囤物症是筆記（掩面）。

that would be if decision was "yes" or "no", I guess.

Does the game provider know who is playing?

annotation meta: may need new tag: codify a team's norms

Another approach, inspired by the <label> element, would be to encode these selectors as separate <link> elements in the head. You could write your links as normal, and then add these <link rel="selector" for="foo" href="XXX[...]X" /> to your document (where foo is the ID of the <a> element, and the href value is selector).

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

Learn more at Review Commons

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Reply to the reviewers

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

In this paper, Staneva et al describe a novel complex found at RNA PolII promoters that they term the SPARC. The manuscript focuses on defining the core components of the complex and the pivotal role of SET27 in defining its function, and role in PolII transcription. This manuscript is a logical follow on from an initial paper (Staneva et al, 2021) by the same authors where they systematically analyzed chromatin factors, and their role in both transcription start and termination. What is also very clear, is that this complex is one made of histone readers and writers which suggests its function is to change the chromatin structure around a PolII promoters. The authors show that this complex is necessary for the correct positioning of PolII and directionality of transcription.

This was a well-designed study and well written and clear manuscript that provides fascinating insight transcription control in bloodstream form parasites.

I have no major comments only a few minor ones.

1) Localisation of the different SPARC components appears to be either nuclear or nuclear and cytoplasmic. - Both SET27 and CRD1 show a nuclear and cytoplasmic localisation in the bloodstream form IFA (Supplementary Fig 1B), but only a nuclear localisation procyclic form.

Did the authors attempt C terminally tagging SET27, CRD1 to see if this resulted in a change in the pattern?

We have not tagged either protein at the C terminus, however SET27 (Tb927.9.13470) has been tagged both N- and C-terminally in procyclic form (PF) cells as part of the TrypTag project (http://tryptag.org). In both cases, SET27 localized to the nucleus, suggesting that the differences in localization we observe for SET27 depend on the life cycle stage, and not on the position of the tag. One caveat is that in the TrypTag project proteins are tagged with mNeonGreen whereas in our study proteins were tagged with YFP. Based on our images, CRD1 appears to be predominantly nuclear in both bloodstream form (BF) and PF parasites. CRD1 (Tb927.7.4540) has been tagged only N-terminally in PF cells as part of the TrypTag project where it has also been classified as mostly nuclear with only 10% of cells showing cytoplasmic localization for CRD1.

We are well aware that tags can alter the behaviour of a protein. Absolute confirmation of location will require the generation of antibodies that detect untagged proteins. However, this is a longer-term undertaking. We have added the following statement to the Results section to address the point raised:

“We tagged the proteins on their N termini to preserve 3′ UTR sequences involved in regulating mRNA stability (Clayton, 2019). We note, however, that the presence of the YFP tag and/or its position (N- or C-terminal) might affect protein expression and localization patterns”.

• The point is made that JBP2 shows a 'distinct cytoplasmic localisation' in PF cells. by this logic, the SET27 localisation in BF is also distinctly cytoplasmic and a nuclear enrichment is not clear.

Indeed the reviewer is correct - we have inadvertently over accentuated the significance of this difference in the text. We had emphasized the predominantly cytoplasmic localization of JBP2 in PF trypanosomes as potentially related to its weaker association with other (predominantly nuclear) SPARC components in the mass spectrometry experiments. The presence of SET27 in the nuclei of both BF and PF cells is confirmed by a positive ChIP signal. We have revised the manuscript text by changing “distinct cytoplasmic” to “predominantly cytoplasmic” to describe JBP2 localization in PF cells. We hope that this resolves the issue.

• Why would the localisation pattern change between life cycle stages? Surely PolII transcription should remain the same?

Although our analysis suggests that there may be some shift in SET27 and JBP2 localization between BF and PF stages, sufficient amounts of these proteins may be present in the nucleus for proper SPARC assembly and RNAPII transcription regulation in both life cycle forms. The proportion of SET27 and JBP2 proteins that localizes to the cytoplasm may have functions unrelated to transcription.

2) Several of the images in Supplementary Fig 1B seem to show foci in the nucleus (CSD1, PWWP1, CRD1). Do you see foci throughout the cell cycle or just in G1/S phase cells as shown here?

We have not systematically investigated protein localization at different cell cycle stages, so we do not have microscopy images for all proteins at all stages of the cell cycle. However, the images we did collect suggest the punctate pattern is preserved for CRD1 in the G2 phase in both BF and PF cells (see below) as we showed in Supplemental Figure S1B for cells with 1 kinetoplast and 1 nucleus (G1/S phase cells). The significance of these puncta remains to be determined.

3) In Figure 6, what does 'TE' stand for?

TE denotes transposable elements. We have added this to the figure legend.

4) The authors show this interesting link between SPARC complex and subtelomeric VSG gene silencing. -In the CRD1 ChIP or RBP1 ChIP, are there any other peaks in telomere adjacent regions in the WT cells similar to that seen on chromosome 9A? And does the sequence at this point resemble a PolII promoter?

Apart from peaks located on Chromosome 9_3A, there are other CRD1 and RPB1 ChIP peaks in chromosomal regions adjacent to telomeres in WT cells. We observed broadening of RPB1 distribution in these regions upon SET27 deletion, similar to what we show for Chromosome 9_3A. In particular, wider RPB1 distribution on Chromosome 8_5A coincides with upregulation of 10 VSG transcripts. These two loci explain most of the differentially expessed genes (DEGs) detected, but other subtelomeric regions show a similar pattern. We have added the following statement to the Results section to highlight that the phenotype shown for Chromosome 9_3A is not unique:

“We also observed a similar phenotype at other subtelomeric regions, such as Chromosome 8_5A where 10 VSGs and a gene encoding a hypothetical protein were upregulated upon SET27 deletion (Supplemental Table S3)”.

Cordon-Obras et al. (2022) have recently defined key sequence elements present at one RNAPII promoter. We searched for similar sequence motifs but failed to identify them as underlying CRD1 and RPB1 ChIP peaks, highlighting the likely sequence heterogeneity amongst trypanosome RNAPII promoters. To address this point, we have added the following sentence to the Discussion:

“Sequence-specific elements have recently been found to drive RNAPII transcription from a T. brucei promoter (Cordon-Obras et al., 2022), however, we were unable to identify similar motifs underlying CRD1 or RPB1 ChIP-seq peaks, suggesting that T. brucei promoters are perhaps heterogeneous in composition”.

-In the FLAG-CRD1 IP (Figure 3B), the VSG's seen here are not represented (as far as I can tell) in Figure 6B and C. If my reading is correct could, is this a difference in the FC cut off for what is significant in these experiments?

The VSGs detected in the FLAG-CRD1 IP from set27D/D cells are indeed different from the ones shown in Figure 6 (even after setting the same fold change cutoffs). We have highlighted this by adding the following statement to the Results section: “Gene ontology analysis of the upregulated mRNA set revealed strong enrichment for normally silent VSG genes (Figure 6B-D) which were distinct from the VSG proteins detected in the FLAG-CRD1 immunoprecipitations from set27D/D cells (Figure 3B)”.

The VSGs in the mass spectrometry experiments likely represent unspecific interactors of FLAG-CRD1. To clarify this, we have added the following statement to the Results section: ”Instead, several VSG proteins were detected as being associated with FLAG-CRD1 in set27D/D cells, though it is likely that these represent unspecific interactions”.

Reviewer #1 (Significance (Required)):

Trypanosomes are unusual in the way that they transcribe protein coding genes. Recent advances have defined the chromatin composition at the TSS and TTS, and the recent publication of a PolII promoter sequence(s) further adds to our understanding of how transcription here is regulated. Defining the SPARC complex now add to this understanding and highlights the role of potential histone readers and writers. I think that this will be of interest to the kinetoplastid community especially those working on control of gene expression.

Our lab studies gene expression and antigenic variation in T. brucei.

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

In this manuscript, the authors identify a six-membered chromatin-associated protein complex termed SPARC that localizes to Transcription Start Regions (TSRs) and co-localizes with and (directly or indirectly) interacts with RNA polymerase II subunits. Careful deletion studies of one of its components, SET27, convincingly show the functional importance of this complex for the genomic localization, accuracy, and directionality of transcription initiation. Overall, the experiments are well and logically designed and executed, the results are well presented, and the manuscript is easy to read.

There are a few minor points that would benefit from clarification and/or from a more detailed discussion:

1) The concomitant expression of many VSGs (37) in a SET27 deletion strain is remarkable and has important implications for their normally monoallelic expression. It is well established that VSG expression in wild-type T. brucei can only occur from one of ~15 subtelomeric bloodstream expression sites, which include the ESAGs. This result implies that VSG genes are also transcribed from "archival VSG sites" in the genome, not only from expression sites. Are there VSGs from the silent BESs among the upregulated VSGs? Is there precedence in the literature for the expression of VSGs from chromosomal regions besides the subtelomeric expression sites?

Our analysis of differentially expressed genes (DEGs) revealed that 43 VSG genes (37 of which are subtelomeric) and 2 ESAG genes are upregulated in the absence of SET27. Both ESAGs but none of the upregulated VSGs in set27D/D cells are annotated as located in BES regions. While it is possible that recombination events have resulted in gene rearrangements between the reference strain and our laboratory’s strain, at least some of the upregulated VSGs are likely to be transcribed from non-BES archival sites. VSG transcript upregulation from non-BES regions was also recently described by López-Escobar et al (2022).

We note that the upregulated mRNAs in set27D/D are still relatively lowly expressed (Figure 6C). This is presumably insufficient to coat the surface of T. brucei, and expression from BES sites instead may be required to achieve this. We have revised the manuscript Discussion section to make these points more clear:

“Bloodstream form trypanosomes normally express only a single VSG gene from 1 of ~15 telomere-adjacent bloodstream expression sites (BESs). In contrast, in set27D/D cells we detected upregulation of 43 VSG transcripts, none of which were annotated as located in BES regions. Recently, López-Escobar et al (2022) have also observed VSG mRNA upregulation from non-BES locations, suggesting that VSGs might sometimes be transcribed from other regions of the genome. However, the VSG transcripts we detect as upregulated in set27D/D were relatively lowly expressed (Figure 6C) and may not be translated to protein or be translated at low levels compared to a VSG transcribed from a BES site”.

2) The role of SPARC in defining transcription initiation is compelling. It's less clear to the reviewer if the observed transcriptional silencing within subtelomeric regions can also ascribed to SPARC. Have the authors considered the possibility that some components of the SPARC may be shared by other chromatin complexes, which could be responsible for the transcriptional activation of silent genes in SET27 deletion mutants?

We cannot rule out indirect effects through the participation of some SPARC components in other complexes operating independently of SPARC. Indeed, the transcriptional defect within the main body of chromosomes appears to be somewhat different from that observed at subtelomeric regions, particularly with respect to distance from SPARC. We have added a statement in the Discussion section to highlight the possibility raised by the reviewer:

“However, an alternative possibility is that transcriptional repression in subtelomeric regions is mediated by different protein complexes which share some of their subunits with SPARC, or whose activity is influenced by it”.

3) The authors mention that the observed interaction of FLAG-CRD1 with VSGs in the immunoprecipitations (Fig. 3B) is evidence for the actual expression of normally silent VSGs on the protein level. This is true, but it should be spelled out that this interaction is nevertheless likely an artifact, at least the physiological relevance of these interactions is questionable.

We agree that these are likely background associations and have added the following statement to the Results section to clarify this point:

“Instead, several VSG proteins were detected as associated with FLAG-CRD1 in set27D/D cells, though it is likely that these represent unspecific interactions”.

To avoid unnecessary confusion we have also removed the following sentence from the revised Discussion:

“The interactions of FLAG-CRD1 with VSGs in the affinity selections from set27Δ/Δ cells indicate that some of the normally silent VSG genes are also translated into proteins in the absence of SET27”.

4) "ophistokont" is misspelled in the introduction

Thanks for noticing. We have corrected it to “Opisthokonta”.

Reviewer #2 (Significance (Required)):

The manuscript by Staneva et al. addresses the fundamental regulatory mechanism of gene transcription in the protozoan parasite Trypanosoma brucei, a highly divergent eukaryotic organism that is renowned for unusual features and mechanisms in gene regulation, metabolism, and other cellular processes. While post-transcriptional regulation is prevalent and relatively well established in T. brucei, much less is known about the mechanism of transcription initiation and transcriptional control, in part due to the general paucity of well-defined conventional promoter regions in this organism (only very few have been identified thus far). In this context, the work by Staneva et al. is highly significant and represents an important contribution to the field of gene regulation and chromatin biology in T. brucei and other related kinetoplastid parasites.

My GoutPal Research notes are similar to GoutPal Triage. But they're a response to my own interests and concerns. As such, I usually assign them lower priority than my notes on specific issues.

In fact, this note is an example. Because I'm researching how to best implement my new GoutPal Links services. Then I'm adding notes like this to describe my progress towards a complete set of documented resources.

As I move forward, I will publish extensive notes for subscribers and for members. That will give readers choices about extra information beyond these public gout research notes.

Please note that those public gout notes are from every Hypothes.is user who writes about gout. Currently, that's only me. But I hope other gout sufferers will join me. At which point, you can find my gout notes with my user tag.

To get more gout research notes, join the waitlist for gout subscribers.

```html

<div class="select-container" data-content=""> <select class="select" id="words"> <option value="lorem ipsum dolor sit amet">Lorem ipsum dolor sit amet</option> <option value="lorem">Lorem</option> <option value="ipsum">Ipsum</option> <option value="dolor">Dolor</option> <option value="sit">Sit</option> <option value="amet">Amet</option> </select> </div>

css .select { color: transparent; appearance: none; padding: 5px; background: transparent url("https://cdn4.iconfinder.com/data/icons/ionicons/512/icon-arrow-down-b-128.png") no-repeat calc(~"100% - 5px") 7px; background-size: 10px 10px; }

.select-container { position: relative; display: inline-block; }

.select-container::before { content: attr(data-content); pointer-events: none; position: absolute; top: 0; right: 10px; bottom: 0; left: 0; padding: 7px; font: 11px Arial, sans-serif; white-space: nowrap; text-overflow: ellipsis; overflow: hidden; text-transform: capitalize; } js const selectContainer = document.querySelector(".select-container"); const select = selectContainer.querySelector(".select");

select.value = "lorem ipsum dolor sit amet"; selectContainer.dataset.content = select.value;

function handleChange(e) { selectContainer.dataset.content = e.currentTarget.value; }

select.addEventListener("change", handleChange); ```

1. 在阅读的时候记笔记：这个可以是在书本上进行高亮或者使用在书上写笔记。
2. 在读完书后，重新阅读你的笔记，并修改笔记：这一步是将书上的笔记转移到卡片上。
3. 对笔记进行分类和归档：可以给笔记卡片进行分类，或者打上 tag。

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

Learn more at Review Commons

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Referee #1

Evidence, reproducibility and clarity

This study shows that the ORF1 protein of the LINE-1 retroelement forms puncta in vivo that they define as cytoplasmic biomolecular condensates based on the characterization of the biophysical properties of ORF1p condensates in vitro.

Defective retrotransposition of some ORF1p mutants correlates with defects in puncta formation in vivo and alteration of biophysical properties of in vitro condensates leading the authors to conclude that condensation of ORF1p is required for retrotransposition.

The study combines biochemical reconstitution, biophysic analysis and live-cell imaging. In particular, the authors take advantage of a new powerful tool they have developed based on the tagging of ORF1 within a functional L1 reporter element. The fluorescent tag allows following the dynamics of ORF1p by live-cell imaging.

The key conclusion is that ORF1p condensation is important for L1 retrotransposition. The correlation is clearly shown but raises several questions: Is the defect in ORF1p condensation the only explanation for the retrotransposition defects of the ORF1p mutants analyzed here? Can we exclude that the mutations in ORF1p affect other functions of the protein such as its binding to RNA (as in the case of the R261 mutant) and cis-preference, or its binding to other factors involved in L1 replication? Could the loss of these functions affect L1 retrotransposition independently of ORF1p condensation?

Major comments:

On several occasions, the authors propose that ORF1p-HALO dynamics in vivo is linked to its co-translational association with L1 RNA. However, they never show the presence of L1 RNAs in ORF1p-HALO puncta in vivo. To strengthen the conclusion that the puncta observed in vivo are L1 RNPs, the authors should add experiments showing the presence of L1 RNA in the cytoplasmic puncta (by RNA FISH) or that the puncta are dependent on the presence of L1 RNA (expressing ORF1p-HALO alone should not be sufficient for puncta formation). These experiments seem to be realistic in few weeks with the tools already available in the laboratory.

Apart from this comment, the authors are cautious in their conclusions. It is clear, as they indicate in the Discussion, that showing that ORF1p condensation is also required for the mobility of other retroelements will strengthen the implication of ORF1p condensation in L1 replication.

The data are well presented and the methods described in detail so that others should be able to use them. The experiments seem to be adequately replicated and the statistical analysis adequate.

Minor comments:

Figure 1F: Having the pictures of cell nuclei (like in Figure 1D) would be nice to know how many cells we are looking at in this panel.

Figure 2E: it is surprising that there is no correlation between the ORF1p:RNA ratio and the number of individual fusion events (i.e. curves of ORF1p+RNA 10000:1 and 1000:1 overlap while 3000:1 is different). Could the authors discuss this point?

Previous studies are appropriately referenced. Text and figures are clear and precise.

Referees cross-commenting

The main critical points shared by all reviewers are: 1) the need to show the presence of LINE1 RNAs in ORF1p condensates in vivo and 2) the lack of evidence for causality between ORF1 condensate formation and L1 transposition efficiency (At this stage, the authors should moderate their conclusions, especially in the abstract). Regarding the other reviews, we noticed the need to cite additional relevant studies in the field (reviewer #2) and the interesting points raised by reviewer #3 to investigate the formation of ORF1 condensates in an endogenous situation, and whether other RNAs do affect ORF1p condensates.

Significance

The study is technically interesting in that it describes a new system for tracking ORF1p puncta formation in vivo. The findings are not unexpected because it comes after the publication of Newton et al. in 2021 (PMID : 33798566), describing that ORF1p does phase separation in vitro. Furthermore, the fact that RNPs form "membrane-less" structures is already established in other situations as the authors point out. Compared to Newton et al., condensates are better-defined biochemically, especially for RNA association features and in vivo dynamics.

The ORF1 protein is widely studied for its role in L1 retrotransposition. The protein forms a homotrimer in vitro, binds to L1 mRNA in a cis-preferential manner, and is required for retrotransposition. On the other hand, RNA-binding proteins are often involved in the formation of membrane-less organelles (stress granules, RNA processing bodies...). These observations suggest that ORF1p may also form RNP condensates required for L1 retrotransposition. A study published in Biophysical Journal in 2021 (Newton et al. PMID: 33798566) has already reported the phase separation of the LINE-1 ORF1p that is mediated by the N-terminus and coiled-coil domain. This former study was based on in vitro microscopy and NMR approaches and is cited in the submitted manuscript. The study submitted to Rev commons goes further by analyzing the biochemical properties of ORF1p condensates in the presence of L1 RNA and by following in vivo condensates of ORF1p (WT or mutants) expressed from a functional L1 reporter element by live-cell imaging. The findings will interest a wide audience investigating the biology of retroelements and more particularly scientists who study the L1 retrotransposon.

I am an expert in retrotransposon biology but I do not work on L1. I am not expert enough to assess the quality and relevance of the biophysical experiments in the paper. In particular, panels 2D, 3B and 3D were difficult to analyze.

• OK

Review coordinated via ASAPbio’s crowd preprint review

This review reflects comments and contributions by Ruchika Bajaj and Gary McDowell. Review synthesized by Bianca Melo Trovò.

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This study demonstrates the utility of an L-Methionine analog - ProSeMet - to tag and enrich proteins which have residues that are methylated in vivo, ex vivo and in vitro. Furthermore, the study demonstrates that this can be used in combination with mass spectrometry to identify these sites. Overall this is a useful, well-verified and well-described approach that will be helpful for future identification and investigation of methylation sites.

Major comments

It would be helpful if the manuscript could additionally discuss the reversibility of methylation generally, and the reversibility of the modification of protein residues by the alkyne group specifically, in the discussion, and whether that has any implications for their results. It may be that the dynamics of methylation and demethylation vary between the two; or it may be that they are the same - either way, that may affect how they suggest others use this method and interpret its results.

Perhaps related to the question of reversibility, it would be helpful if the manuscript would comment on whether these are “true” methylation sites or not; i.e. whether they consider all these methylation sites to be functional. Trying to determine this would be an interesting direction for future work, but for this study a reflection on whether these novel functional methylation sites are simply capable of being methylated, or are likely to be methylation sites that are meaningful biologically, would be helpful.

Results, ProSeMet competes with L-Met to pseudo methylate protein in the cytoplasm and nucleus: the manuscript claims that ProSeMet is not incorporated into newly synthesized proteins but rather converted to ProSeAM and used by native methyltransferases. There does appear to be some reduction in the labeling with ProSeMet on cycloheximide treatment in Figure 2D - could this suggest that it is incorporated into newly synthesized proteins as well as being converted to ProSeAM? If not, could the manuscript explain why not? This experiment clearly shows that in contrast to AHA labeling, there is still use of ProSeMet as a substrate when translation is inhibited; however, it is not clear how this demonstrates that it is not incorporated at all into newly synthesized proteins. If methyl has been incorporated in previously present proteins, perhaps this can be clarified in the text.

Results, ProSeMet competes with L-Met to pseudomethylate protein in the cytoplasm and nucleus: the conclusion that “Cell fractionation of the cytosolic and nuclear compartments followed by SDS-PAGE fluorescent analysis revealed no fluorescent labeling of the L-Met control” is correct but may be overstated as there appears to be some background in the cytosolic fraction.

Minor comments

• Introduction: Recommend including a mention to ProSeMet's permeability.
• Introduction, Figure 1: the last step with CuAAC and N3 labeling in the description of the Chemoenzymatic approach for metabolic MTase labeling is not clear. Please, add the description in the legend.
• Results, Figure 2D: the image suggests an overloaded gel, consider using an alternative gel image.
• Supplementary Material, Fig. S1: the data with L-met is only shown with T47D stacks.
• Supplementary Material, Fig. S3: please add the control for the no treatment condition.
• Results, Fig. 2A ‘ incubating for 30 m in L-Met free media’: Please confirm that the length of incubation was 30 minutes.
• Results, Enrichment of pseudo methylated proteins used to determine breadth of methyl proteome: Please provide some description for the SMARB1-deficient G401 cell line. Why smarb1 deficient?
• Results, Figure 3: Please define BP, MF, HP, NES, and label the x and y axes in panel D.
• Results, ProSeMet-directed pseudo methylation is detectable in vivo: Please, clarify if the administration was oral.

Comments on reporting

• Results, ProSeMet competes with L-Met to pseudo methylate protein in the cytoplasm and nucleus: Please verify the quantity reported: 5µg on SDS-PAGE gel seems low.
• Results, ProSeMet-directed pseudo methylation is detectable in vivo: the manuscript reports that “mice starved prior to ProSeMet injection had increased ProSeMet labeling in the heart, whereas mice fed prior to ProSeMet administration had increased labeling in the brain and lungs”. The error bars are large, it would be helpful to show the individual real data points for the graphs in Figure 4.
• Results, Figure 4C: please report the mathematical expression used to calculate the relative fluorescence.
• Supplementary Material, Fig. S7: please provide more details on the antibody employed.

Suggestions for future studies

Future studies could investigate the biological functionality of the novel methylation sites - but this is a great proof of principle.

ReconfigBehSci. (2021, November 26). RT @mvankerkhove: Following the meeting of the @WHO TAG-VE today, WHO classifies B.1.1.529 as a variant of concern named Omicron. We call… [Tweet]. @SciBeh. https://twitter.com/SciBeh/status/1464302372100976650

ReconfigBehSci [@SciBeh]. (2021, November 26). RT @mvankerkhove: I thank researchers from 🇿🇦 and 🇧🇼 for sharing information with @WHO & the world about B.1.1.529 variant that has been re… [Tweet]. Twitter. https://twitter.com/SciBeh/status/1464196599773642779

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Referee #1

Evidence, reproducibility and clarity

This study characterises a Plasmodium class I Histone deacetylase (PfHDAC1). The manuscript reports a wide range of experiments - some of them complex and involved, but not all of these experiments appear to be well controlled, and some are insufficiently described to know if they have been appropriately designed and interpreted. A link to HDAC1 regulation and artemisinin resistance is advanced, but the evidence here is very indirect and inconclusive.

The study shows that HDAC1 interacts with PfCKII- a homologue of the mammalian casein kinase known to interact with mammalian HDAC1. They also demonstrate that, at least in vitro, HDAC1 can serve as a substrate for phosphorylation by PfCKII, and that this phosphorylation impacts HDAC1's deacetylation of histones. Such assays where a kinase is provided with a single, abundant substrate in vitro, are not always rigourous tests for kinase specificity, but do in this case at least indicate that HDAC1 associated with its activity.

Major issues:

1. The authors conduct CHiP seq experiments on a GFP tagged HDAC. It is unclear from the methods and results section what control is used in these experiments. The ENCODE consortium has established minimum standards (Landt et al 2012) for conducting and reporting CHiP seq experiments, and states that the "recommended control for epitope-tagged measurements is an immunoprecipitation using the same antibody against the epitope tag in otherwise identical cells that do not express the tagged factor.". These experiments appear to lack that control and the enrichments reported should be approached with caution in the absence of such a control.
2. The genes with apparently altered ChiP seq were subjected to gene ontology enrichment analysis, and the authors report potential enrichments - which appear to impact a range of unconnected biological pathways throughout the parasite and throughout the lifecycle, despite the CHIP seq being conducted only at a single time stage. No mention is made of correction for multiple hypothesis testing, known to present a considerable problem for such analyses, and no correction is described for background GO distributions in the P. falciparum genome, so again it's unknown if or how that was performed. The reported enriched categories must be also treated with considerable caution given the absence of description of these crucial steps. The authors report from this section that HDAC1 is associated with stress responses, but really, by their criteria, HDAC1 is associated with 1/3 of the whole genome, so it's a bit selective to regard it as a stress regulator
3. The authors preform a well-designed series of transfection experiments with modulation of HDAC1 to show that an overexpression of HDAC1 leads to increased growth rate, and that this increase reduces when the overexpression of HDAC1 is inducibly repressed. However, I found the presentation of results from these experiments difficult to understand and there is considerable transformation of the data prior to plotting - they would be easier to understand if no background subtraction to normalise for GFP were conducted, and if all strains were plotted on the same axes. A potential confounding factor in this experiment is that many lines overexpressing GFP grow more slowly, and that this growth defect can be localisation dependent, so that over-expression of GFP alone may cause a different growth penalty than GFP on a nuclear protein. I am uncertain that the conclusion of 50% faster growth is a safe one based on these graphs - at some time intervals the over-expressor appears to grow just as slow or even slower (as a percentage of the previous timepoint) than the control, and these appear to have been based on technical replicates of a single biological experiment. The authors contend that the growth rate is due to changed expression of invasion genes (among many other substrate gene categories) giving rise to enhanced invasion - such a phenomenon is readily testable, and the authors should dissect this if they wish to substantiate the frankly surprising claim that overexpression of HDAC leads to increased growth rate.
4. The authors also report an apparent down regulation of HDAC abundance in artemisinin resistant parasites. This conflicts with previous global proteomic analyses of artemisinin resistant parasites which found no such change in HDAC1 regulation or abundance (eg Siddiqui et al 2017, Yang et al 2019). Stage matching is a particular challenge in such experiments given the differences in cycle progression between ARTR and ARTS parasites, and it isn't clear that this has been adequately controlled for to have confidence in these results, particularly given their contradiction of previous analyses. The abundance of PfHDAC1 changes considerably throughout the asexual intraerythrocytic cycle, (out of synch with the control used here actin), so potential stage-mismatch might contribute to apparent differences here. Again, explicit mention of replicates is lacking. The authors also mention genes regulated by HDAC1 as including genes related to processes related to artemisin resistance, but this is hard to sustain - indeed with so many genes apparently substrates of HDAC1 it would be highly surprising if there were no overlap with some genes in pathways related to artemisin resistance. An accompanying experiment demonstrating an increase in survival (of both ART resistant and ART sensitive lines) in an artemisinin ring stage survival assay is intriguing, after using a possible inhibitor of HDAC but these results are hard to reconcile with a dynamic transcriptional response. (Why was this done with an uncharacterised inhibitor, rather than the more specific HDAC1 overexpressor/knockdown system? An accompanying RNAseq analysis is described, but the analysis is piecemeal and selective, with the authors pointing out candidate genes representing categories plausibly linked to artemisinin resistance. I found this section unconvincing and indirect - lots of genes are changed in these experiments, and so they inevitably include some that are feasibly linked to artemisinin resistance, but the one gene convincingly known to modulate resistance, K13, isn't mentioned, and presumably wasn't specifically changed in this analysis.
5. A previous study by the laboratory of Christian Doerig (Eukaryot Cell. 2010 Jun; 9(6): 952-959.) reported that HDAC1 activity (unclear which of the HDACs) is associated with Pfcrk-3). This activity may not correspond to the HDAC1 characterised here, but deserves some discussion.
6. The Western blots are letterboxed and in some cases appear to crop out bands on the limit of the image (eg Fig 5, 6). Please provide fuller pictures of the blots and indicate the relevant bands if there are several background bands.

Minor issues

The text uses breaking spaces for the gap between genus abbreviation and species throughout. Replace with non-breaking spaces. Abstract: "is correlated with parasitemia progression" - Unclear meaning. Reword. Introduction "closes in on 400,000 deaths annually" Unclear meaning/vernacular usage. Reword. Very long paragraph on pages 3-4. Reorder logical flow and break into smaller paragraphs to make this more easily read. "Given the evidence of the role of HDAC inhibition in the emergence of chemotherapeutic resistance in mammalian system" - needs a reference - no mention of this phenomenon up until this point of the manuscript

Referees cross-commenting

I agree with the other reviewers comments. Although the manuscript contains a very large number of complex experiments, necessary controls, sufficient replicates, and appropriate analysis are missing from many of the experiments.

I appreciate that the experiments referred to would require a very substantial time and resource commitment to complete, but in their current form, many of these experiments are not safely interpretable.

Significance

This manuscript makes major claims for HDAC1, in particular for its role in artemisinin resistance. Such a link would be significant, but I regard few of these claims as having been robustly substantiated in this manuscript. The CHIP-seq evidence is of interest as a useful dataset, particularly if accompanied by relevant controls

annotation meta: may need new tag: the need to “find buy-in” for every little thing.

tag a specific moment in past commits

If you've not come across the examples, one of the precursors of the slip box tradition was the widespread use of florilegia from the 8th through the 13th centuries and beyond, and they were primarily used for religious study, preaching, and sermon writing.

• https://en.wikipedia.org/wiki/Florilegium
• https://en.wikipedia.org/wiki/Commonplace_book
• https://hypothes.is/users/chrisaldrich?q=tag%3Aflorilegium

A major example of early use was by Philip Melanchthon, who wrote a very popular handbook on how to keep a commonplace. He's one of the reasons why many Lutheran books are called or have Commonplace in the title.

A fantastic example is that of American preacher Jonathan Edwards which he called by an alternate name of Miscellanies which is now digitized and online, much the way Luhmann's is: http://edwards.yale.edu/research/misc-index Apparently he used to pin slips with notes on his coat jacket!

If I recall, u/TomKluender may have some practical experience in the overlap of theology and zettelkasten.

(Moved this comment to https://www.reddit.com/r/antinet/comments/wth5t8/bible_study_and_zettelkasten/ as a better location for the conversation)

I often rush into assessing new applications. Because I learn quicker by applying compared to reading. But one downside is that I frequently miss key features.

That's only a major drawback if I abandon the application where I can't see how it fits my project. And today I avoided that with serendipity. Because… 1. My application was a Chrome Extension 1. I wanted to test to see if was active and change webpage content accordingly 1. I found I could run the app without an extension - as fully described in the documentation that I skipped reading!

All of which is an idea for my next blog post. But the real point is I have established a process for starting ad-hoc Shrewd Learning projects "in the wild". Because normally, I start making notes somewhere. Then forgetting where I put them.

I think we all do that when we spot something interesting that might warrant future research. Now for my established subject areas, I always start annotating new topics within that subject area. So, I can prioritize it in my usual processes.

Today, I've extended this by tagging public notes with Shrewd Learning. So when I look at the Shrewd Learning Tag, I see all notes that present potential new learning topics. Which opens a great way to collaborate loosely with other people if I can establish some traction with Shrewd Learning.

For now, this is my reminder to do a personal blog entry based on this. More importantly, I should update this blog entry to reflect recent advances in Shrewd Learning and my other 2 online learning projects.

Author Response

Reviewer #1 (Public Review):

Rasicci et al. have developed a FRET biosensor that is designed to light up when cardiac myosin folds. This structure is extremely important to understand, and its link to the super-relaxed (SRX) state has not been fully shown. Their study provides a comprehensive review of the literature and provides compelling data that the 15 heptad+leucine zipper+GFP construct does function well and that the DCM mutant E525K has a similar IVM velocity despite a reduced ATPase compared with HMM. They rely on the ionic strength-dependent changes in the rate of MantATP release to argue that the E525K mutation stabilizes the 'interacting heads motif' (IHM) state, which makes logical sense.

Strengths:

Well written and comprehensive.

Utilizes the appropriate fluorescence-based sensor for measuring the folding of the myosin structure. Provides a detailed range of techniques to support the premise of the study

Weaknesses:

Over-interpretation of the outcomes from this study means that the IHM and SRX are the same. Similar studies, e.g. Anderson 2018 and Chu 2021 support the opposite view that IHM and SRX are not necessarily the same, Anderson (and Rohde 2018) point out that S1 has some element of a reduced ATPase, this clearly cannot be due to folding of the molecule. Also, mavacamten was used in these studies to show that even S1 is inhibited suggesting that SRX and IHM are not connected. This is not to say that with enough supporting evidence that these observations cannot be over-ridden, it is just not clear that there is enough in this study to support this conclusion.

We have revised our discussion to emphasize that our results support a model in which the SRX state is enhanced by formation of the IHM, but given the S1 and 2HP data the IHM may not be required for populating the SRX biochemical state (see page 8).

I felt that the authors passed over the recent Chu 2021 paper too quickly, the Thomas group used a FRET sensor as well and provides a direct comparison as a technique, but with opposite conclusions. They also have supporting data in Rohde 2018 that their constructs were less ionic strength sensitive. It would be useful to understand what the authors think about this.

We have discussed the Rohde and Chu papers in more detail in the discussion (see page 8). In the Rhode paper they used proteolytically prepared HMM and S1. Rohde found 20% SRX at all KCl concentrations in S1, while HMM shifted from 50% to 20% SRX in low and high salt conditions, respectively. Our results are different in terms of the absolute fraction of the SRX state but the trend is similar in terms of S1 being salt-insensitive and HMM being salt-sensitive. The difference could be proteolytic HMM, which is a longer construct, and proteolytic S1, which is prone to internal cleavage that can impact ATPase activity. Another difference could be the mixed isoform of mantATP used in previous studies and the single isoform of mantATP used on our study (see page 5)

Reviewer #2 (Public Review):

The paper by Rasicci et al. examines the impact of the DCM mutation E525K in beta-cardiac myosin on its function and regulation by autoinhibition. The role of the auto-inhibited state of beta-cardiac myosin in fine-tuning cardiac contractility is an active and exciting area of current research related to muscle biology and cardiomyopathies. Several studies in the past have linked the destabilization of the autoinhibited, super-relaxed (SRX) state of myosin to the pathogenesis of hypertrophic cardiomyopathy. This timely study provides one of the first few examples where the hypocontractile phenotype of a DCM mutation has been linked to the stabilization of the SRX state.

One of the strengths here is the utilization of a wide variety of both pre-existing and novel biochemical and biophysical assays for the study. The authors have characterized a new two-headed long-tailed myosin construct containing 15-heptad repeats of the proximal S2 (15HPZ), which they show allows myosin to form the SRX state in vitro using single ATP turnover assays. The authors go on to compare the E525K and WT proteins using the 15HPZ myosin construct in terms of their steady-state actin-activated ATPase activity, in-vitro actin-sliding velocity and single ATP turnover measurements. These assays reveal that the predominant effect of this mutation is the stabilization of the SRX state which is maintained even at 150 mM salt concentration where the WT SRX is largely disrupted. This is an important observation because DCM mutations so far have been believed to only affect the force-generating capacity of myosin.

One of the biggest strengths of this study is the attempt to develop a FRET-based approach to directly ask if the biochemical SRX state here correlates well with the structural IHM state, which is an important unresolved question in the field. The authors have designed a FRET pair (C-terminal GFP and Cy3ATP bound to the active site) that is sensitive to the relative position of the heads and the tail, allowing them to distinguish between the low-FRET closed IHM conformation and the no-FRET open conformation. Remarkably, the authors show that the salt dependence of the FRET efficiency values closely follows their results from the salt dependence of the percent SRX for both WT and E525K proteins. The authors then attempt to substantiate their FRET results by a direct visual analysis of the conformational states populated by both WT and E525K proteins at low salt using negative staining EM analysis. The authors have optimized conditions to allow the deposition of the IHM state on grids without adding the small molecule mavacamten, which was found to be necessary in an earlier study to visualize the closed state using EM. The authors conclude that the SRX state correlates well with the IHM state and that the E525K mutation indeed stabilizes the folded-back conformation of myosin.

This study significantly strengthens the previously illustrated correlation between the SRX and IHM states and provides methodological advances (especially visualization of the IHM state by negative EM in the absence of cross-linking agents) that will be very useful to the field going forward. The observation that a DCM mutation can lead to stabilization of the folded back state is a novel insight that should spark interest in the field to test how broadly this applies to other DCM mutations. The conclusions of the paper are mostly supported by the data; however, some clarifications and qualifications are needed.

Weaknesses:

The extremely low enzymatic activity of the M2β 15HPZ myosins as compared to the WT S1 control (which is a historical control not assayed in parallel with the 15HPZ proteins), is concerning for the low protein quality of the 15HPZ myosins. The authors attribute the low kcat to the high proportion of SRX population in their ensembles. However, the DRX rates reported for the WT and E525K 15HPZ proteins in the single ATP turnover assay are ~3-4 fold lower than those of their S1 counterparts. These rates reflect basal turnover of ATP in the open state and thus should not be affected by the presence of the S2 tail, which leads to concerns about the 15HPZ protein activity. In addition, the very high percentage of stuck filaments in the in vitro motility assay for the 15HPZ constructs (despite the use of dark actin) is also concerning for significant amounts of enzymatically inactive protein.

We thank the reviewer for pointing out the differences in the S1 and HMM DRX rates. We performed additional single turnover measurements with S1, adding two sets of measurements from one additional preparation (N=3), and we demonstrate that there is a significant increase in the DRX rates of WT S1 compared to WT HMM (see pages 4-5, Table 3, Figure 3- figure supplement 3). A faster rate in S1 was also reported in Rohde et al. 2018. Indeed, the DRX rates of E525K S1 are significantly higher than WT in S1, which we also now report in the results (see page 5, Figure 3 – figure supplement 3). We addressed the concerns about 15HPZ activity by performing NH4+ ATPase assays to demonstrate that the number of active heads was similar in S1 and 15HPZ HMM (see page 4). It is possible that the higher percentage of stuck filaments in the HMM motility is due to myosin heads in the IHM state on the motility surface, which generate a drag force by non-specifically interacting with actin, but further study is necessary to examine this question.

The authors assert that the E525K mutation represents a new mechanism by which DCM-causing mutations lead to decreased contractility - by stabilizing the sequestered state rather than affecting motor function. However, there is no evaluation of the motor function (actin-activated ATPase activity or in vitro motility) of the E525K S1, which would reveal the effects of the mutation without confounding effects due to the sequestering of heads. Interestingly, in the single ATP turnover assay, the DRX rate of the E525K S1 is >2-fold higher than the WT control, suggesting that the mutation may have effects beyond stabilization of the SRX state. The conclusion that the E525K mutation's effect on myosin function is mediated via stabilization of the SRX state would be strengthened if the effects of the mutation on the motor domain alone were also known.

We thank the reviewer for this suggestion. We performed actin-activated ATPase assays with WT and E525K S1 and found that E525K increases kcat and lowers KATPase, demonstrating enhanced intrinsic motor activity in the mutant S1 construct (see page 4, Figure 2B). This adds an interesting dimension to the manuscript because we report a mutant that enhances the intrinsic motor activity but stabilizes the SRX/IHM (see Discussion page 10). We did not perform in vitro motility, because this assay depends on the surface attachment strategy, and we would like to compare all constructs with the same attachment strategy using a C-terminal GFP tag (mutant and WT S1 and 15HPZ HMM). Therefore, we are making the S1 construct with a C-terminal GFP tag for this purpose, to be examined in a future study.

While the authors show strong qualitative correlations between the SRX and IHM states using single ATP turnover, FRET, and EM experiments, attempts to quantitatively compare the fraction of heads in the IHM state using the various experimental approaches is problematic. For example, the R0 value of the FRET pair used here doesn't allow precise measurement of the distances being probed here to be made, but the distances are reported and compared to predicted distances. The authors report that the R0 for their FRET pair is 63 Å. Surprisingly the authors go on to use the steady-state FRET efficiency values to determine the average D-A distance (Fig 5B) which is 100 Å when all heads are in the IHM configuration and becomes larger than that when heads open. R0 of 63 Å allows a precise distance measurement to be made in the 31.5-94.5 Å range which corresponds to 0.5-1.5 R0. It is therefore technically incorrect to use the steady-state FRET efficiency values to determine the D-A distance here. Besides, there are several unknown factors here like orientation factor (κ2) which further complicate these calculations. Similarly, the quantification of IHM state molecules from the negative stain EM experiments is significantly hampered by the disruptive effect of the grid surface on the structure of the IHM state. The authors find that limiting the contact time with the grid to ~ 5s is necessary to preserve the IHM state.

Despite that, only ~15% WT molecules were seen in the IHM state at low salt (Fig. 6B). In contrast, ~56% E525K molecules were in the IHM state. Both these proteins have similar SRX proportions (Fig. 3C) and similar FRET efficiency values (Fig. 5A) at this salt concentration. This mismatch highlights the problem arising due to not having a measure of the populations from the FRET data. It is not clear if the hugely different proportions of the IHM state in EM experiments are indicative of the relative stability of this state in the two proteins or a random difference in the electrostatic interactions of WT vs mutant with the grid. These experiments do not provide a correct idea of the %IHM in the two proteins. In the absence of any IHM population measurement, it is important to proceed with caution when quantitatively correlating the SRX and IHM.

We thank the reviewer for pointing out that measuring precise distances by FRET can be difficult. We agree that the low FRET efficiency makes precise distance determination even more challenging. However, FRET is quite good at measuring a change in distance given a specific donor-acceptor pair. We feel our FRET biosensor clearly demonstrates FRET efficiencies that are salt-insensitive in E525K but a clear decrease in FRET at higher salt concentrations in WT. In order to compare the trend in the predicted FRET, based on the single turnover measurements, and the actual FRET we thought it was important to plot the two together on the same graph. We understand that this could have been misleading that we were reporting actual distances. We have now plotted the FRET efficiency instead of distance as a function of KCl concentration (Figure 5B), to prevent any confusion with reporting distances. In addition, we have emphasized that the data are plotted to allow for a comparison of the trend in the single turnover and FRET data (see page 6, 10, Figure 5B).

We agree that it is important to proceed with caution when comparing the EM to the FRET and single turnover data. The EM does not give a quantitative estimate of the fraction of IHM molecules, due to the disruptive effect of the grid surface on protein conformation. However, it does provide direct (though qualitative) evidence that the conformation underlying SRX and enhanced FRET is the IHM, and it is consistent with our interpretation that the E525K mutation enhances FRET and SRX by stabilizing the IHM. To consolidate this result, we have performed EM experiments now with a total of 3 preparations of WT and mutant (see page 6-7 and Figure 6D). We find that while there is variability from experiment to experiment, likely because the grid surface is slightly different each time the experiment is performed, in all cases there was a ~4-fold higher fraction of folded molecules in the mutant. Since each WT/mutant experimental pair was studied in parallel, using identically prepared grids, the results provide further evidence that the mutant stabilizes the IHM. However, we agree that a quantitative, direct visual correlation of the SRX and IHM is not possible based on the current EM data.

Finally, the utility of the methods described in the paper to the field would be greatly enhanced if they were described in more detail. As currently written, it would be difficult for others to replicate these experiments.

Thank you for the comment. We have made significant changes in the methods to clarify the details of the experiments (see pages 11-14). In addition, we have added details to the results and figure legends.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The authors used CRISPR engineering to drop a V5 or GFP tag into teh PGRP-LE locus (protein fusions) to monitor the behavior of this intracellular peptidoglycan sensing receptor in the drosophila midgut. They show that upon immune stimulation with Ecc that PGRP-LE forms some sort of aggregate or punctae that is dynamic during the 24 hour of infection monitored. A similar response is not seen with live E. coli but a week and smaller response is observed with heat killed E. coli, for unclear reasons. These punctae appear to form independent of the classic IMD signaling components, suggesting it is upstream event in the pathway which is consistent with early studies showing the PGRP-LE multimerizes (infinitely) upon binding PGN and also that it forms amyloid fibrils doing signaling. The Ecc punctae tightly colocalize with Rab5 but not Rab7 or other early endosome markers, but in the absence of Rab5 the PGRP-LE punctae are greatly enlarged. Rab5 was found to critical for induction of PGRP-SC1 but not the classic IMD pathway AMP, Attain.

While the conclusions of the report are intriguing and the development of these tools is very exciting, the conclusions are not fully convincing. To start, the author wish to conclude that PGRP-LE localization is altered with Ecc infection but they have not excluded that the expression of the protein is sharply upregulated. I.e. in the uninfected animals there is not really any PGRP-LE observed (1D). The try to tackle this by looking at mRNA expression, but this data lacks the unaffected control. [In fact, the uninfected control is missing on most of the gene expression data, which is a troubling omission and makes it hard to really understand what the data shows.]. Moreover, the mRNA levels do not necessarily corresponding to the protein levels, i.e. there could be post translation control. So, overall, the authors need to provide more compelling evidence that PGRP-LE is relocalized upon Ecc challenge rather than upregulated.

Moreover, the paper contains some seeming contradictory findings that the authors make little effort explain. For example, they conclude "These results suggest that although smaller PGRP-LE aggregates can form normally in the absence of Rab5, the latter is required for proper bigger E.cc mediated PGRP-LE aggregates" because E. coli induced PGRP-LE clusters don't colocalize with Rab5, yet in the absence of Rab5, the Ecc cluster are super-enlarged (4F). This makes no sense with the conclusions.

Finally, the interaction and function of the Rab5 interaction is underdeveloped and lacks insight. For example, why is Rab5 required for the induction of one target gene but not another? And, why not characterize this more completely? Why is there not Rab5 vesicle with E. coli feeding or even uninfected? The cell biology requires more in-depth consideration. From 4E, the authors wish to conclude that the Rab5 vesicle are induced by Ecc (even in the absence of PGRP-LE) yet the uninfected control is not shown. IN a simple world, would not one would expect Rab5 endosomes in all cells, at least to some level?

And, focusing on the big picture, the authors claim that it is "not easily testible" if the PGRP-LE aggregates are amyloidal, as suggested by earlier publications. This could actually be tested by staining with amyloid specific dies and/or suitable mutants engineered int he RHIM domain. This would be very informative if the authors could extend this work to examine this question.

Minor comments:

All the colocalization data should be quantified as in 4B. It is not true that DAP = Gram negative. Gram-positive bacilli also have DAP PGN. The wording in the Introduction should be adjusted. The text needs a careful proofreading.

Referees cross-commenting

I think the comments from #2 and myself are aligned. Working is interesting, tools are especially exciting, but the studies are descriptive and under-developed. I will further add, I found the absence of uninfected controls for many assays a major problem.

Significance

The significance of this work lies in the development of powerful tools to track an intracellular innate immune receptor in an intact animals. The connection to Rab5 is curious and likely an important advance in our understanding of the cell biology of this pathway, but is under-developed. The significance is this difficult to know for certain. The Drosophila immunity field, and the insect immunity field more broadly, will be keenly interested in this study. The wider NF-κB/innate immune field will also be interested in these findings, given teh similarity between this pathway and NOD1/NOD2 immune sensing in mammals.

My area of expertise is the Drosophila immune response and this manuscript is very much in my wheelhouse.

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Reply to the reviewers

August 17, 2022

RE: Review Commons Refereed Preprint #RC-2022-01442

Dear Editor of the EMBO Journal,

Please find our updated manuscript and response to the reviewers’ comments. We appreciate the effort that the reviewers have put into the evaluation of our manuscript.

We are happy with the potential importance the reviewers realise in the study:

Reviewer 1: The finding that ubiquitination occurs inside mitochondria would be an important conceptual advance, which would open new perspectives both for ubiquitination and mitochondrial biology

Reviewer 2: This work would represent a significant/exceptional discovery if supported by compelling data.

Reviewer 3: the results are interesting and very important, as mentioned in the major comments section…

With regard to the major comments raised by the reviewers, you will find below our specific response point by point with explanations and suggested novel experiments (highlighted in yellow). In summary we suggest the following actions to fully support our model:

• We will perform a-complementation with ubiquitin (lacking the GG motif) fused at its C-terminus to the short fragment of b-galactosidase (a). Blue colonies with ωm will indicate import.
• As shown in Figure S2, now added to the manuscript, we show detection of ubiquitinated proteins and mono ubiquitin in extracts of mitochondria pre-treated with trypsin.
• A bio-archives address of our other manuscript will be provided.
• The use of a-complementation for protein localization was developed by us 15 years ago and since then has been used by us and other groups verifying its use as a screening tool. One point is clear, ωm or ωc do not leak into other subcellular compartments. Nevertheless, in the research of specific genes validation is important. Yes!!! ωm and ωc are exclusively located in mitochondria or the cytosol respectively.
• We will highly purify mitochondria on gradients and treat them with protease.
• We cannot be sure that we will be able to detect a protein with ubiquitin modifying activity which functions solely on certain proteins in mitochondria, so publication cannot rely on this.
• Repeat mass spectrometry with careful editing will be undertaken as suggested by the reviewer.
• We will attempt to perform protease protection assays in the presence of specific detergents.

Before tackling the very tough revision, we would like to know if EMBO Journal would positively consider acceptance of our manuscript based on the review and planned revision.

Prof. Ophry Pines Microbiology & Molecular Genetics Hebrew University of Jerusalem Jerusalem 91220 Israel

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

Summary:

In this manuscript, Zhang et al. investigate whether ubiquitination occurs inside mitochondria of the budding yeast S. cerevisiae. They first observe thanks to a sensitive complementation assay that several components of the yeast ubiquitination (and deubiquitination) machinery can localize inside mitochondria. To be able to specifically probe ubiquitin conjugates assembled inside mitochondria they fused HA-tagged ubiquitin to a mitochondrial targeting sequence. Using this construct, they demonstrate that ubiquitin conjugates can be assembled in mitochondria. A series of elegant experiments demonstrates that the pattern of ubiquitin conjugates depends on the mitochondrial localization and the activity of the ubiquitin conjugating enzyme Rad6. Altogether, these results convincingly demonstrate that ubiquitination can occur inside yeast mitochondria when ubiquitin is intentionally targeted inside this organelle. It however remains unclear whether mitochondrial ubiquitination occurs in endogenous conditions (without targeting ubiquitin into this compartment) and whether it affects mitochondrial functions.

Response: Regarding the question whether mitochondrial ubiquitination occurs in endogenous conditions, we feel that this is obvious based on our results. We detect numerous ubiquitination related enzymes (E1, E2, E3, DUB) eclipsed in mitochondria but none of the proteasome subunits. As pointed out by the reviewer “these results convincingly demonstrate that ubiquitination can occur inside yeast mitochondria”. With that said, additional data will be incorporated into the manuscript as suggested by the reviewer and can be seen below.

Major comments:

1) The materials and methods section is lacking important information (western blot protocol, details of antibodies, strains, plasmids...). It is thus difficult to evaluate how several experiments were performed and how their design (e.g. the promoters chosen to express tagged proteins) could impact the interpretation of the results. This is a major issue that needs to be corrected. The main text should also explicitly indicate whether tagged proteins used in the alpha-complementation assay are overexpressed or not.

Response: The materials and methods section will be updated accordingly.

2) Despite the previous comment, the data presented in the manuscript convincingly demonstrate that multiple components of the ubiquitination machinery can localize within mitochondria and that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is modified to be intentionally targeted into this compartment. However, little data is shown to support the hypothesis that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is not fused to a mitochondrial targeting sequence. Thus, in my opinion, the evidences presented in the current manuscript are not sufficient to conclude that ubiquitin conjugates are assembled in mitochondria in endogenous conditions (as this is done implicitly). Additional evidences are needed to draw this conclusion (see some experimental suggestions hereafter). Without further evidences, the speculative aspects of the claim that "ubiquitination occurs in the mitochondrial matrix" should be discussed explicitly.

Response: See the discussion above why we are confident that ubiquitination occurs in mitochondria. Our major problem with ubiquitin and the ubiquitination enzymes is that they are eclipsed in mitochondria. We propose as suggested by the reviewer (item 4 of his review) to perform a-complementation with ubiquitin fused at its C-terminus to the short fragment of b-galactosidase (a). Blue colonies with ωm will indicate import.

3) The authors used a mass spectrometry approach to identify mitochondrial ubiquitination substrates. However, they have not yet succeeded in identifying a substrate whose modification is specifically regulated by a given component of the mitochondrial ubiquitination machinery. They have also not identified a phenotype or process impacted by mitochondrial ubiquitination. Thus, at this stage, the biological consequences of mitochondrial ubiquitination remain elusive.

__Response: __We have not identified a substrate whose modification is dependent on a given component of the mitochondrial ubiquitination machinery, even though we have tried. Again, the problem is low levels of these proteins eclipsed in mitochondria. Even when we do find a protein that is ubiquitinated (e.g. Aco1) its ubiquitination is not exclusively dependent on Rad6. Thus, different ubiquitin enzymes may have the same substrates.

4) The authors have not directly investigated whether ubiquitin itself (without a mitochondrial targeting sequence) localizes in mitochondria. I encourage them to address this question since it would provide an important piece of evidence suggesting that mitochondrial ubiquitination can occur in endogenous conditions. This could be done using the alpha-complementation assay and the results could be presented within Figure 1. Ideally this experiment should be performed without overexpressing ubiquitin. Note that if the authors decide to use a C-terminally tagged form of ubiquitin for this experiment, the GG motif of ubiquitin should be mutated to avoid cleavage of the alpha tag by cellular DUBs. This form of ubiquitin will not be conjugatable, but this is not an issue for this experiment since its aim is to determine whether ubiquitin can be targeted to mitochondria, not to probe conjugates.

Response: We will perform experiments as suggested by the reviewer including ubiquitin fused at its C-terminus to the short fragment of b-galactosidase (a), see item 2. We have previously made a PreSu9-Ubi lacking a GG motif but now will look at a different combination of this and other constructs.

5) In the top panels of Figure 2 and S1, free ubiquitin is well detectable in the total and cytosolic fractions. It is however not clear to me whether it is also detectable in the concentrated mitochondrial fraction. If yes and if it would be resistant to trypsin digestion, it would provide additional evidence that endogenous ubiquitin can be targeted to the mitochondrial matrix (see previous comment).

Response: See Item 6.

6) The data shown in the top panel of Figure 2 and S1 also suggest that free ubiquitin is less concentrated in mitochondria than in the cytosol (since it is more difficult to detect in the concentrated mitochondrial fraction than in the cytosolic fraction, see previous comment). It is thus possible that the use of preSu9-HA-Ubi (or preFum1-HA-Ubi) lead to an artificially high intra-mitochondrial concentration of free ubiquitin. As the concentration of free ubiquitin is known to impact ubiquitination processes, I encourage the authors to compare the relative levels of free ubiquitin present in the mitochondrial fraction prepared from WT and preSu9-HA-Ubi (or preFum1-HA-Ubi) expressing cells. If free ubiquitin is detectable in mitochondrial fractions and resistant to trypsin (see previous comment), this could be done by repeating the experiment shown in Figure 3B and probing the blot with an antibody that recognizes free ubiquitin.

Response to 5 and 6: Detection of ubiquitin in mitochondria is extremely difficult even when mitochondria are 15-fold concentrated versus the cytosol and when HA-Ubi is overexpressed. Thus, ubiquitin is eclipsed in mitochondria. Nevertheless, as shown in the Figure below which was not part of the submitted manuscript yet was performed in parallel to experiments done early on, shows detection of very weak bands of free ubiquitin in extracts of mitochondria pre-treated with trypsin.

Endogenous ubiquitination pattern in mitochondria of _Δrad6 _cells is restored to normal by Rad6-α. __WT or Δrad6 cells containing a Rad6-α construct or an empty plasmid were subjected to subcellular fractionation. Mitochondrial fractions with or without trypsin treatment, were probed for ubiquitin by WB. Aco1 is a matrix mitochondrial protein, and Tom70 is a mitochondrial outer membrane protein (MOM) facing the cytosol.

7) I strongly encourage the authors to provide more data indicating that "ubiquitination occurs in mitochondria" by performing experiments that do not rely on the use of the preSu9-HA-Ubi or other forms of ubiquitin that are intentionally targeted to mitochondria. For instance, they could analyse the pattern of HA-Ubi conjugates of trypsin digested mitochondrial fractions prepared from wt, rad6-delta, and rad6-delta complemented with preSu9-Rad6-alpha-SL17. Note that if trypsin digested mitochondrial fractions are too contaminated by ubiquitinated proteins present outside mitochondria to perform this experiment, the authors may use the unspecific DUB Usp2 as an alternative protease to strip ubiquitinated proteins from the mitochondria periphery.

Response: Concentrated mitochondrial extracts from WT and Δrad6 cells untreated or treated with trypsin were probed with anti-ubiquitin antibodies (Figure above). A very weak band corresponding to free ubiquitin can be detected in extracts of mitochondria treated with trypsin but these are very weak and are on the limit of detection.

Minor comments:

1) Overall, the manuscript is well organized and easy to follow. The text is clearly written; the figures are well annotated.

2) The authors should provide full images of all the blots with anti-ubiquitin and anti-HA antibodies so that one can see the bands corresponding to free ubiquitin (or free HA-Ubi). For instance, in Figure 3B, it is not possible to see the presence (or absence) of the band corresponding to free HA-Ubi because the very bottom of the image is cut.

3) The authors should indicate whether the MTS of Su9 (and Fum1) are expected to be cleaved after import of preSu9-HA-Ubi (and preFum1-HA-Ubi) in mitochondria. They should also label on the corresponding immunoblots the presence (or absence) of the band corresponding to the free preSu9-HA-Ubi (and preFum1-HA-Ubi) (or HA-Ubi if the MTS is expected to be cleaved from these constructs).

4) In Figure 3B, the ubiquitin conjugates produced with preSu9-HA-Ubi and preFum1-HA-Ubi have different migration patterns. I think this should be explicitly mentioned and discussed. Could it be due to the presence of lysine residues in the Su9 or Fum1 MTS that could lead to the assembly of artificial ubiquitin chains?

5) The authors indicate that "endogenous Rad6 [...] is expressed at very low levels and can hardly be detected in the mitochondrial fraction by WB (Figure S5)". I did not manage to observe the band corresponding to endogenous Rad6 in the mitochondrial fraction in the pdf. The authors should provide a more contrasted or better quality image.

CROSS-CONSULTATION COMMENTS I agree with reviewer 2 that proper validation of the complementation assay is crucial for this manuscript. I was myself wondering whether it uses endogenously tagged proteins or whether it is based on an overexpression system. I imagine this information will be detailed in the manuscript in preparation mentioned by the authors. I am therefore wondering whether it would be possible to ask the authors to provide the draft of this manuscript (or at least the validation part).

Response: A bio-archives address of our other manuscript will be provided upon resubmission. See other issues referred to the response Reviewer 2.

I agree with most comments of reviewer 3. Regarding the hypothesis that preSu9-HA-Ubi could form aggregates on the cytosolic surface of the mitochondria, I think that the results presented on Figure 7B rather argue against it (since they indicate that Rad6 localized inside mitochondria can restore the pattern of ubiquitin conjugates). That's why (in my opinion) the major question the author now need to adress is whether intra-mitochondrial ubiquitination occurs in endogenous conditions (ie without forcing ubiquitin into this compartment and without E2 or E3 overexpression).

Response: See response to the other reviewers

Reviewer #1 (Significance (Required)):

The finding that ubiquitination occurs inside mitochondria would be an important conceptual advance, which would open new perspectives both for ubiquitination and mitochondrial biology research. However, the significance of the current manuscript is limited because the presented evidences heavily rely on the use of artificial conditions (ubiquitin tagged with a mitochondrial-targeting sequence) that may trigger irrelevant ubiquitination events. The significance would be much higher if the authors would provide further evidences indicating that intra-mitochondrial ubiquitination occurs in endogenous conditions and/or if they had identified a mitochondrial process specifically impacted by mitochondrial ubiquitination.

Expertise of the reviewer: Ubiquitination, Yeast biology, protein-protein interactions. No specific expertise in mitochondrial biology

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

In the manuscript by Yu et al., the authors test the concept that certain proteins are unevenly distributed within distinct cell compartments. Due to this localization discrepancy, protein detection in some subcellular compartments can be "eclipsed" by a predominant subset of specific protein localizing in another cell compartment their actual distribution. Therefore, tiny amounts of physiologically relevant proteins could be biologically relevant. Still, their function in some locations can be overlooked (or eclipsed) because of the high expression level of the same protein in another subcellular compartment(s). Although, this concept is not particularly novel. For example, it is already known that many different proteins can localize to distinct cellular locations (e.g., permanent mitochondrial and peroxisomal localization of many proteins or transient localization of particular proteins to separate cell compartments). The authors apply a yeast system and an α-complementation assay to test further the role of such eclipsed proteins in mitochondrial biology. Specifically, they focus on the ubiquitin (Ub, or as abbreviated incorrectly in this manuscript; Ubi) conjugation pathway, components of which have never been convincingly shown to localize inside the mitochondria. This work proposes that certain ubiquitination events can occur inside yeast mitochondria. This work would represent a significant/exceptional discovery if supported by compelling data. However, the major problem with this work is that the conclusions are based on the ectopic expression of distinct proteins. This approach is not failproof in precise protein expression/delivery to the specific subcellular locations and is likely to result in a non-specific localization. Thus, the problem of eclipsed proteins is addressed by the methodology that may lead to the artificial generation of eclipsed overexpressed proteins. A more effective approach would be if the authors found a way to study this issue with endogenous proteins. The need for overexpression of mitochondria-targeted ubiquitin makes it challenging to reconcile the physiological role of these fundings. In addition, some critical technical issues and omissions further reduce the potential impact of this work (see Specific comments below). For example, strong evidence of mitochondria fraction purity and additional evidence that all the essential constructs used in this work are not misdirected to a different compartment are needed.

Response: “Although, this concept is not particularly novel” is a very disappointing remark by the reviewer!! While dual targeting of proteins has been known for many, many years, how widespread the phenomenon was unknown and thought to be negligible. We are leaders for the last 30 years in the field of dual targeting and distribution and in particular distribution of single translation products. We coined the terms “echoforms” and “eclipsed distribution” and developed methods to detect and screen for dual targeting. The concept of eclipsed distribution and in particular eclipsed targeting to mitochondria is very new, and is leading to a novel perception of the mitochondrial proteome (see MS submission). While the reviewer appears to be an expert on ubiquitination, we are experts on dual targeting.

• Ub was abbreviated incorrectly in this manuscript, Ubi. __Response: __This will be corrected.

Other comments will be referred to in the response to Specific comments.

Specific comments 1. The authors should demonstrate beyond doubt that the ω components of their assay (ω-C, which supposedly stays in the cytosol-ONLY and the ω-M component, which seemingly remains in the mitochondria-ONLY) are in the compartment that the authors claim. These two proteins are transfected into yeast cells and overexpressed. Therefore it is possible that they leak to other, not intended, subcellular compartments. The authors assume that ω-M and ω-C are exclusively located either in the mitochondria or the cytosol. However, this should be shown as validation of the assay. The indicated reference from 2005 (Ref.13) and others are irrelevant since assays have variations and are often researcher/lab dependent. This validation is very important since a misallocation of the overexpressed ω-M or ω-C, leaking into other subcellular compartments, may cause misdetection of the α-constructs.

Response: The use of a-complementation for protein localization was developed by us 15 years ago and since then has been used by us and other groups verifying its use as a screening tool. One point is clear, ωm or ωc do not leak into other subcellular compartments. Nevertheless, in the research of specific genes validation is important. Yes!!! ωm and ωc are exclusively located in mitochondria or the cytosol respectively.

It is not surprising that Ub conjugates are detected in mitochondrial fractions. It could be due to ubiquitination of the OMM (coming from the cytosol) or perhaps since the subcellular fractions were not pure mitochondria free from contamination (the likely culprit could be the ER). The mitochondrial fractions in this work were obtained by 10,000 g separation between cytosolic and mitochondrial crude fractions. Indeed, these 10,000 g crude fractions are highly impure with membranes from other compartments (i.e., microsomes, lysosomes, and so on). Therefore, more sophisticated purification methods should be used. In addition, the authors should also test these fractions for non-mitochondrial proteins from other membrane organelles.

Response: We agree with the reviewer and therefore will take the following approaches:

1. i) We will treat isolated mitochondria with protease in order to remove adhering proteins and digest OMM proteins…… see attached figure.
2. ii) We will highly purify mitochondria on gradients and this will be straight forward since we are now employing such methods in other projects in the lab. iii) Matrix protein enrichment (by mass spec) is associated with IP for preSu9-HA-Ub conjugates which is three-fold higher than for HA-Ub. In any case the fact that we identify conjugates of proteins not known to be mitochondrial, strongly supports our thesis.

Figure 2. Coomassie blue staining does not show any signal in the "M" fraction. It can be interpreted that the authors do not get any mitochondria there, and therefore the lack of Ub signal is due to the absence of the protein in the samples. Using the same amount of protein from each fraction would probably reduce the necessity of 15x enrichment.

Response: The Coomassie blue staining does show a signal in the "M" fraction which is weak yet when a 15x enrichment is run, the protein level by Coomassie blue staining is similar to the cytosolic fraction.

Figure 3. It is puzzling why the HA-UBQ presence is so strong in the crude mitochondrial fraction, but the preSu9-HA-Ub signal (mito-matrix) is comparatively weak. These data suggest that the crude mito-fraction could be highly contaminated with OTHER membranes. On the other hand, the preSu9-HA-UBQ signal is no more than 1-5% of the total mitochondrial signal. The high enrichment of the HA-Ubi in both cytosols and the mitochondria could indicate the OMM ubiquitination or (again) contamination by other compartments. The constructs with MTS are detected in the mitochondria. However, the localization of tagged MTS-Ubi in a non-targeted compartment (e.g., cytosol) should be excluded by additional exposure times. Because the manuscript talks about eclipsed proteins, this is important.

Response: The HA-Ub is strong in the mitochondrial fraction, in the absence of trypsin, but is very weak in the presence of the protease indicating that most of the ubiquitinated proteins are externally attached to mitochondria. In contrast, PreSu9-HA-Ub is imported into the mitochondrial matrix and is protected from trypsin. This manuscript refers to “eclipsed in mitochondria” (not the cytosol) and this is true for ubiquitination enzymes as well as for ubiquitin.

Figure 3C-E. These data indeed suggest that the Ub-conjugates could be formed inside the mitochondria. However, the above-discussed possibility that other than mitochondria compartments co-sediment in the 10,000g fractions makes the data interpretation highly challenging.

__Response: __We will highly purify mitochondria on gradients and this will be straight forward since we are now employing such methods in other projects in the lab.

Figure 4. Unsurprisingly, mitochondrial targeting of Ub leads to detecting some co-immunoprecipitating mitochondrial proteins. However, these data do not support the notion that Ub conjugation machinery acts inside the mitochondria and that the target proteins are indeed conjugated with Ub (the interaction with Ub is not equal to being conjugated). At the minimum, the authors should provide a validation that some of the detected mitochondrial matrix proteins are indeed ubiquitinated. To this end, purified mitochondria could be used for the candidate protein IP under denaturing conditions and then blotted for the candidate protein and Ub.

__Response: __As shown in Table S2 and figure S7, forms of Ilv5, a mitochondrial protein, are ubiquitinated in WT and Drad6 cells. These modified forms of Ilv5 can be eluted from mitochondrial extracts of WT and Drad6 cells. However, the ubiquitination of ilv5 is not dependent or effected by the Drad6 mutation. We cannot be sure that we will be able to detect a protein with ubiquitin modifying activity which functions solely on certain proteins in mitochondria.

Figure 5. The knock-out of the E2 Rad6 causes a change in the mitochondria ubiquitination pattern. This is an interesting observation, but again it does not prove that the change in the mitochondrial ubiquitination is due to the activity of Rad6 inside of the mitochondria, as opposed to ubiquitination of the OMM proteins or contaminating fractions. One also wonders why overexpression of mitochondria-targeted Ub would be necessary to detect the ubiquitination if this process was physiologically relevant, especially given that detecting endogenous Ub is not challenging. Furthermore, the apparent increase in ubiquitination in E2 mutant cells (Fig. 5) should also be addressed in more detail. Finally, data from one WB is shown, and quantification of several independent experiments should also be provided.

__Response: __We show in the MS that RAD6 is exclusively targeted to mitochondria (Su9MTS) while unimported molecules are degraded (SL17; degron). This hybrid Rad6 can restore the WT ubiquitin pattern, while a rad6 active site mutant cannot.

Figure 6. Can the authors provide Western blot data showing the expression of Rad6? Furthermore, quantifying these rescue experiments is necessary to make this conclusion more solid.

Response: Even though we did not succeed in making good Rad6 antisera, we can clearly detect Rad6-a fusion proteins (Figure 7B).

Figure 7. The authors found that preSu9-Rad6-α have problems being imported into the mitochondria matrix; therefore, they rebuild it as a preSu9-Rad6-α-SL17 protein. SL17 is a degron that targets the cytosolic protein (not imported into the mitochondria) to the proteasome and degraded (Figs. 7A-B-C). These issues could be a red flag for the rest of the manuscript, suggesting that other constructs (that were not critically evaluated for their localization in this work) could leak to different cellular compartments.

Response: The wording used by the reviewer is particularly disturbing since current understanding in cell biology of eukaryotic cells does not accept “leaking” of proteins to different cellular compartments. One wouldn’t want DNAses, RNAses, Proteases etc leaking from one compartment to another. The localization of proteins to different cellular compartments involves very precise signals on the proteins, and specific cellular components, such as translocases, are required to target proteins to their exact destination. This is true for Rad6; it contains an MTS like sequence which when removed blocks import of the protein into mitochondria. Rad6 according to our analysis is an eclipsed dual targeted protein, so it no surprise that it is in two compartments and the trick with the SL17 degron solves the problem.

The manuscript needs to be carefully edited, some references are in the not correct format, and there are issues with figure labels.

Response: Careful editing will be undertaken as suggested by the reviewer.

CROSS-CONSULTATION COMMENTS I agree with a great summary by reviewer 1. This discovery should be validated by top-quality data.

Reviewer #2 (Significance (Required)):

In the manuscript by Yu et al., the authors test the concept that certain proteins are unevenly distributed within distinct cell compartments. Due to this localization discrepancy, protein detection in some subcellular compartments can be "eclipsed" by a predominant subset of specific protein localizing in another cell compartment their actual distribution. Therefore, tiny amounts of physiologically relevant proteins could be biologically relevant. Still, their function in some locations can be overlooked (or eclipsed) because of the high expression level of the same protein in another subcellular compartment(s). Although, this concept is not particularly novel. For example, it is already known that many different proteins can localize to distinct cellular locations (e.g., permanent mitochondrial and peroxisomal localization of many proteins or transient localization of particular proteins to separate cell compartments). The authors apply a yeast system and an α-complementation assay to test further the role of such eclipsed proteins in mitochondrial biology. Specifically, they focus on the ubiquitin (Ub, or as abbreviated incorrectly in this manuscript; Ubi) conjugation pathway, components of which have never been convincingly shown to localize inside the mitochondria. This work proposes that certain ubiquitination events can occur inside yeast mitochondria. This work would represent a significant/exceptional discovery if supported by compelling data. However, the major problem with this work is that the conclusions are based on the ectopic expression of distinct proteins. This approach is not failproof in precise protein expression/delivery to the specific subcellular locations and is likely to result in a non-specific localization. Thus, the problem of eclipsed proteins is addressed by the methodology that may lead to the artificial generation of eclipsed overexpressed proteins. A more effective approach would be if the authors found a way to study this issue with endogenous proteins. The need for overexpression of mitochondria-targeted ubiquitin makes it challenging to reconcile the physiological role of these fundings. In addition, some critical technical issues and omissions further reduce the potential impact of this work (see Specific comments above). For example, strong evidence of mitochondria fraction purity and additional evidence that all the essential constructs used in this work are not misdirected to a different compartment are needed.

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

Summary: In this study, the authors detected a set of components of a ubiquitination system in the mitochondrial matrix in budding yeast using the subcellular compartment-dependent α-complementation assay. The authors detected the conjugates of mitochondrial targeting signal sequence-directed HA-Ub (preSu9-HA-Ub) in the mitochondrial matrix. The immunoprecipitates of the preSu9-HA-Ubi conjugates were highly enriched for the mitochondrial matrix proteins. Subsequently, the authors focused on the Rad6 E2 ubiquitin conjugating enzyme in the mitochondrial matrix and evaluated its inactivation-altered ubiquitination pattern in the organelle. The authors conclude that ubiquitination occurs in the mitochondrial matrix because of the eclipsed targeted components of the ubiquitination machinery.

Major comments: The authors argued that the proteins that were modified with preSu9-HA-Ubi, which was forced to be imported into the mitochondria, are present in the mitochondrial matrix, because these species are resistant to trypsin digestion. However, it was possible that they formed severe aggregates on the cytosolic surface of the mitochondria, and hence, were resistant to the proteinase. In other words, a small amount of proteins that were not imported into the mitochondria could be deposited on the cytosolic surface of the mitochondria, where they were modified with preSu9-HA-Ubi by cytosolic Rad6. To confirm if the preSu9-HA-Ubi-modified proteins were really present in the mitochondrial matrix, they should perform the protease protection assay in the presence of an appropriate detergent (Figure 3D). In addition, subcellular fractionation of the organelle by density gradient centrifugation, indirect immunofluorescence microscopic analysis of the preSu9-HA-Ubi conjugates, and/or experiments on the in vitro import of preSu9-HA-Ubi and Rad6 into the mitochondria would strongly support the authors conclusion. Other experiments that might support the authors conclusion would be to test whether the band pattern for the preSu9-HA-Ubi conjugates changes when the mitochondrial import is impaired.

Response: We will attempt to perform 1) Protease protection assay in the presence of a detergent (Figure 3D). 2) Subcellular fractionation of the organelle by density gradient centrifugation. 3) In vitro import of Rad6 into the mitochondria.

Minor comments: In Figure 3B, the molecular weight distributions of the preSu9-HA-Ubi conjugates and those of the preFum-HA-Ubi conjugates are different. Is there any reason for this difference?

In Figure 3E, the position of "-" (MG132) for lane 1 is not correct.

In Figure 6A: The band pattern for preSu9-HA-Ubi (lane 13) in the rad6-delta cells expressing Ubc8-alpha is different from that of the wild-type cells expressing Ubc8-alpha (lane 12) as well as that obtained from the rad6-delta cells harboring empty plasmids (lane 9). Is there any explanation for this observation?

In Figure 7B and S6: The level of preSu9-Rad6-alpha-SL17 in the rad6-delta cells is always lower than that in the wild-type cells (compare lanes 13 and 10 in Figure 7B, and lanes 13 and 12 in Figure S6). Is there any explanation for this observation? The protease protection assay (with detergent control) is needed to fully confirm that preSu9-Rad6-alpha-SL17 is present in the mitochondria.

In Figure S7, the authors presented the matrix proteins, Ilv5 and Aco1, detected in the preSu9-HA-Ubi IPed samples and described this observation in the main text. However, the authors also showed the blots for Idh1 and Fum1, which were also pulled down with preSu9-HA-Ubi from the WT cells more than from the rad6-delta cells. Is this correct? If so, please elucidate this observation in the main text.

Figure 8D and 8E are not cited in the main text. Although there are no explanations for these figures in the main text, it looks like Rad6-deltaN11-alpha resides in the mitochondrial fraction. However, the alpha-complementation assay suggests that it resides in the cytosol. Please explain this discrepancy.

First page of the discussion section, item 6): E2 Rad6, but not E3 Rad6?

Figure S7: HA-Ub (cytosolic form) control is needed in addition to the empty vector control.

Figure S7, left panel: There is an unnecessary line break in "Hsp60" and "Ilv5."

Figure S7, right panel: There is an unnecessary line break in "Hsp60."

CROSS-CONSULTATION COMMENTS I agree with comments of reviewer 1 and 2. -Validation of the complementation assay. -I also think that it is important to address whether intra-mitochondrial ubiquitination can be observed with endogenous level of ubiquitin. If even a small amount of preSu9-HA-Ub is mistargeted to the cytosol, proteins at the cytosolic side of mitochondrial outer membrane could be ubiquitinated and detected in the mitochondrial fraction. -Preparation of mitochondria with more sophisticated purification methods (i.e. high resolution density gradient) would be needed to separate mitochondria from ER and other organelles. -More information is needed in the materials and methods section.

Reviewer #3 (Significance (Required)): Significance Although the results are interesting and very important, as mentioned in the major comments section, additional experiments are needed to support their model. However, researchers working on the mitochondrial biology and ubiquitin systems might be interested in and influenced by the reported findings.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this manuscript, Zhang et al. investigate whether ubiquitination occurs inside mitochondria of the budding yeast S. cerevisiae. They first observe thanks to a sensitive complementation assay that several components of the yeast ubiquitination (and deubiquitination) machinery can localize inside mitochondria. To be able to specifically probe ubiquitin conjugates assembled inside mitochondria they fused HA-tagged ubiquitin to a mitochondrial targeting sequence. Using this construct, they demonstrate that ubiquitin conjugates can be assembled in mitochondria. A series of elegant experiments demonstrates that the pattern of ubiquitin conjugates depends on the mitochondrial localization and the activity of the ubiquitin conjugating enzyme Rad6. Altogether, these results convincingly demonstrate that ubiquitination can occur inside yeast mitochondria when ubiquitin is intentionally targeted inside this organelle. It however remains unclear whether mitochondrial ubiquitination occurs in endogenous conditions (without targeting ubiquitin into this compartment) and whether it affects mitochondrial functions.

Major comments:

1) The materials and methods section is lacking important information (western blot protocol, details of antibodies, strains, plasmids...). It is thus difficult to evaluate how several experiments were performed and how their design (e.g. the promoters chosen to express tagged proteins) could impact the interpretation of the results. This is a major issue that needs to be corrected. The main text should also explicitly indicate whether tagged proteins used in the alpha-complementation assay are overexpressed or not.

2) Despite the previous comment, the data presented in the manuscript convincingly demonstrate that multiple components of the ubiquitination machinery can localize within mitochondria and that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is modified to be intentionally targeted into this compartment. However, little data is shown to support the hypothesis that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is not fused to a mitochondrial targeting sequence. Thus, in my opinion, the evidences presented in the current manuscript are not sufficient to conclude that ubiquitin conjugates are assembled in mitochondria in endogenous conditions (as this is done implicitly). Additional evidences are needed to draw this conclusion (see some experimental suggestions hereafter). Without further evidences, the speculative aspects of the claim that "ubiquitination occurs in the mitochondrial matrix" should be discussed explicitly.

3) The authors used a mass spectrometry approach to identify mitochondrial ubiquitination substrates. However, they have not yet succeeded in identifying a substrate whose modification is specifically regulated by a given component of the mitochondrial ubiquitination machinery. They have also not identified a phenotype or process impacted by mitochondrial ubiquitination. Thus, at this stage, the biological consequences of mitochondrial ubiquitination remain elusive.

4) The authors have not directly investigated whether ubiquitin itself (without a mitochondrial targeting sequence) localizes in mitochondria. I encourage them to address this question since it would provide an important piece of evidence suggesting that mitochondrial ubiquitination can occur in endogenous conditions. This could be done using the alpha-complementation assay and the results could be presented within Figure 1. Ideally this experiment should be performed without overexpressing ubiquitin. Note that if the authors decide to use a C-terminally tagged form of ubiquitin for this experiment, the GG motif of ubiquitin should be mutated to avoid cleavage of the alpha tag by cellular DUBs. This form of ubiquitin will not be conjugatable, but this is not an issue for this experiment since its aim is to determine whether ubiquitin can be targeted to mitochondria, not to probe conjugates.

5) In the top panels of Figure 2 and S1, free ubiquitin is well detectable in the total and cytosolic fractions. It is however not clear to me whether it is also detectable in the concentrated mitochondrial fraction. If yes and if it would be resistant to trypsin digestion, it would provide an additional evidence that endogenous ubiquitin can be targeted to the mitochondrial matrix (see previous comment).

6) The data shown in the top panel of Figure 2 and S1 also suggest that free ubiquitin is less concentrated in mitochondria than in the cytosol (since it is more difficult to detect in the concentrated mitochondrial fraction than in the cytosolic fraction, see previous comment). It is thus possible that the use of preSu9-HA-Ubi (or preFum1-HA-Ubi) lead to an artificially high intra-mitochondrial concentration of free ubiquitin. As the concentration of free ubiquitin is known to impact ubiquitination processes, I encourage the authors to compare the relative levels of free ubiquitin present in the mitochondrial fraction prepared from wt and preSu9-HA-Ubi (or preFum1-HA-Ubi) expressing cells. If free ubiquitin is detectable in mitochondrial fractions and resistant to trypsin (see previous comment), this could be done by repeating the experiment shown in Figure 3B and probing the blot with an antibody that recognizes free ubiquitin.

7) I strongly encourage the authors to provide more data indicating that "ubiquitination occurs in mitochondria" by performing experiments that do not rely on the use of the preSu9-HA-Ubi or other forms of ubiquitin that are intentionally targeted to mitochondria. For instance, they could analyse the pattern of HA-Ubi conjugates of trypsin digested mitochondrial fractions prepared from wt, rad6-delta, and rad6-delta complemented with preSu9-Rad6-alpha-SL17. Note that if trypsin digested mitochondrial fractions are too contaminated by ubiquitinated proteins present outside mitochondria to perform this experiment, the authors may use the unspecific DUB Usp2 as an alternative protease to strip ubiquitinated proteins from the mitochondria periphery.

Minor comments:

1) Overall, the manuscript is well organized and easy to follow. The text is clearly written; the figures are well annotated.

2) The authors should provide full images of all the blots with anti-ubiquitin and anti-HA antibodies so that one can see the bands corresponding to free ubiquitin (or free HA-Ubi). For instance, in Figure 3B, it is not possible to see the presence (or absence) of the band corresponding to free HA-Ubi because the very bottom of the image is cut.

3) The authors should indicate whether the MTS of Su9 (and Fum1) are expected to be cleaved after import of preSu9-HA-Ubi (and preFum1-HA-Ubi) in mitochondria. They should also label on the corresponding immunoblots the presence (or absence) of the band corresponding to the free preSu9-HA-Ubi (and preFum1-HA-Ubi) (or HA-Ubi if the MTS is expected to be cleaved from these constructs).

4) In Figure 3B, the ubiquitin conjugates produced with preSu9-HA-Ubi and preFum1-HA-Ubi have different migration patterns. I think this should be explicitly mentioned and discussed. Could it be due to the presence of lysine residues in the Su9 or Fum1 MTS that could lead to the assembly of artificial ubiquitin chains?

5) The authors indicate that "endogenous Rad6 [...] is expressed at very low levels and can hardly be detected in the mitochondrial fraction by WB (Figure S5)". I did not manage to observe the band corresponding to endogenous Rad6 in the mitochondrial fraction in the pdf. The authors should provide a more contrasted or better quality image.

CROSS-CONSULTATION COMMENTS

• I agree with reviewer 2 that proper validation of the complementation assay is crucial for this manuscript. I was myself wondering whether it uses endogenously tagged proteins or whether it is based on an overexpression system. I imagine this information will be detailed in the manuscript in preparation mentioned by the authors. I am therefore wondering whether it would be possible to ask the authors to provide the draft of this manuscript (or at least the validation part).

• I agree with most comments of reviewer 3. Regarding the hypothesis that preSu9-HA-Ubi could form aggregates on the cytosolic surface of the mitochondria, I think that the results presented on Figure 7B rather argue against it (since they indicate that Rad6 localized inside mitochondria can restore the pattern of ubiquitin conjugates). That's why (in my opinion) the major question the author now need to adress is whether intra-mitochondrial ubiquitination occurs in endogenous conditions (ie without forcing ubiquitin into this compartment and without E2 or E3 overexpression).

Significance

The finding that ubiquitination occurs inside mitochondria would be an important conceptual advance, which would open new perspectives both for ubiquitination and mitochondrial biology research. However, the significance of the current manuscript is limited because the presented evidences heavily rely on the use of artificial conditions (ubiquitin tagged with a mitochondrial-targeting sequence) that may trigger irrelevant ubiquitination events. The significance would be much higher if the authors would provide further evidences indicating that intra-mitochondrial ubiquitination occurs in endogenous conditions and/or if they had identified a mitochondrial process specifically impacted by mitochondrial ubiquitination.

Expertise of the reviewer: Ubiquitination, Yeast biology, protein-protein interactions. No specific expertise in mitochondrial biology

I am very much a dev, but anything but an expert when it comes to the web or SSGs. This didn't seem that unsolvable to me although I'll acknowledge I do more in Liquid than one "should".

(ETA: Wait, no, the author agrees with me that doing a lot in one's templates is good.)

Reviewer #3 (Public Review):

In this work, Chen et al. measured the DNA binding dynamics of HIF transcription factors using single-particle tracking. In particular, they examined the impact of heterodimerization between the alpha and beta subunits, the integrity of the DNA binding domain and the nature of the transactivation domain in DNA binding. As expected, they found that the stoichiometry between the heterodimerization partners directly impacts the bound fraction of the beta subunit which is devoid of a DNA binding domain. More interestingly, using domain swaps between HIF-1alpha and HIF2-alpha they found that the transactivation domain of the alpha subunit plays a major role in determining the bound fraction of the beta subunit (and thus the heterodimer). This work is important because it increases our understanding of how TF search the genome, beyond the traditional conception of the "addressing tag" provided solely by the DNA binding domain. This work is thus of interest to the broad audience of scientists studying gene regulation.

Umfang des Ölmärkte: 100 Mill. Barrel am Tag.

Russischer Anteil: 10%

Verbrauch der USA: Ca ein Drittel

Reviewer #1 (Public Review):

Ahmed et al. examine the changes in the enhancer landscape that may contribute to the transition from Barrett's oesophagus (BO) to oesophageal adenocarcinoma (OAC), building upon their past works looking at the chromatin changes within this transition. They identified a repertoire of eRNA regions that display differential expression between OAC and BO, validating their association to enhancers using H3K27ac levels, CUT&TAG, and KAS-seq. The authors look further into the target genes and regulatory TFs that may define eRNA levels, finding several TFs - AP1, KLF5, CTCF, and HNF1 - that have previously been implicated in OAC and confirming that sets of eRNA target genes were downregulated upon depletion of these TFs. Ahmed et al. also showed that eRNA target genes were relevant to OAC phenotypes, akin to that of DEGs in whole RNA-seq datasets. The authors lastly validate the activity of certain eRNAs targeting JUP, MYBL2, and CCNE1 using functional methods to confirm enhancer activity and effects on cell viability, as well as clinical features such as the age of diagnosis and survival time.

The landscape of eRNA activity seems to be well validated. However, deeper analyses to support the relevance of the function of key eRNAs, their specificity in regulating target genes, and the interaction with other OAC features would further support these findings.

Hypothes.is tools<br /> https://jonudell.info/h/tools.html

facet tools<br /> copy annotations<br /> tag rename<br /> annotation powered survey<br /> pagefit

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

Manuscript number: RC-2022-01481R

Corresponding author(s): Sebastian Voigt. Mirko Trilling, David Schwefel

1. General Statements [optional]

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2. Description of the planned revisions

Reviewer #1: Evidence, reproducibility and clarity

Using proteome profiling of rat CMV infected cells, the authors of this study identify the E27 protein of rat cytomegalovirus as being crucial for proteasomal degradation of STAT2. Since E27 shares 56% sequence identity to the previously characterized STAT2 antagonist M27 of murine CMV the authors investigated association of E27 with the Cullin4-RING UbL CRL4. Using gel filtration chromatography they provide evidence that E27 forms a stable ternary complex with DDB1 and STAT2 suggesting that E27 bridges STAT2 to DDB1 which is further corroborated by data from cross-linking mass spectrometry. A cross-linked DDB1/DDA1/E27/STAT2 complex was then used for cryo-EM imaging experiments. The subsequent single particle analysis yielded a density map at 3.8 A resolution that was further used to generate an E27 molecular model. At this point it should be noted that resolution was not very high and data form AlphaFold2 prediction and CLMS experiments were necessary to build a model which was described as having "sufficient quality", however, no quality parameters are included for this model. In this model, a cryptic zinc-binding motif was identified that turned out to be well conserved in M27. At this point the study switches to a mutational analysis of M27: MCMV mutants either lacking M27 or bearing an AxAxxAA triple mutation were investigated both in cell culture and in animal models. Surprisingly, the M27-AxAxxA mutant while exhibiting attenuated IFN inhibition was still more active than an M27 deletion mutant. Later during the study it is postulated that this may be due to the fact that E27 binding to STAT2 abrogates the interaction with IRF9, however, this is only predicted from modeling and no experimental data are provided for this hypothesis. Furthermore, modeling approaches were used to predict how E27 replaces endogenous CRL4 substrate receptors and how E27 recruits STAT2 to mediate CRL4-catalysed ubiquitin transfer.

Reviewer #1: Significance

__Reviewer #1: __This is an interesting and well written paper describing for the first time in molecular detail how a cytomegalovirus-encoded interferon antagonist degrades STAT2 by mimicking the molecular surface properties of cellular CRL4 substrate receptors.

This study should be of broad interest for both virologists and structural biologists.

Authors Response: We thank the reviewer for the insightful and constructive evaluation. We are very grateful for highlighting the significance of our work.

Reviewer #1: Major points

__Reviewer #1: __To my opinion the authors should perform mutational analysis in the context of E27 and RCMV. I accept that switching to M27 may be easier due to established procedures for MCMV mutagenesis and analysis, however, since all structural work is primarily done on E27 it would be consequent to confirm these structural predictions in the context of E27 before switching to a related protein.

Authors Response: As the Reviewer appreciated, there were multiple reasons for the switch from RCMV-E E27 to MCMV M27. Most importantly, the MCMV in vivo infection model in mice is very well-established. Please also note that MCMV is applied far more often by virologists and immunologist as a standard model. Thus, the extension of our findings from RCMV to MCMV increases the relevance and outreach of the study. By performing the experiments in the MCMV context, we also aimed to emphasise that the function of the zinc-binding motif, which structurally organises the DDB1-binding domain, is functionally conserved among E27/M27-like proteins. Obviously, Reviewer #1 could ask why we do not solve the structure of M27 parallel to E27. With the sole exception of E27, none of the rodent M27 homologues could be produced recombinantly in a soluble form, preventing the purification and structure analysis of M27.

Since we agree with Reviewer #1 that the extension from E27 to M27 may read “a bit rough” without a mutational analysis in the E27 context, we will construct RCMV-E E27 mutants leading to Cys=>Ala exchanges in the Zn-binding motif. An analysis of the interaction between DDB1 and these E27 mutants will be included in the revised manuscript.

__Reviewer #1: __Moreover, data on the replication of the generated E27 deletion RCMV should be included in the manuscript (i.e. growth curves).

Authors Response: RCMV mutants lacking the E27 gene exhibit an impaired replication. According to the suggestion, the growth curves will be part of the revised manuscript.

Reviewer #1: The hypothesis that STAT2/E27 interaction is sterically incompatible with IRF9 binding is only based on structural prediction. It would help if the authors could present experimental evidence for such a mechanism.

Authors Response: The hypothesis is based on three lines of argumentation: (i) structural data regarding the binding interface between STAT2 and E27 covering the known STAT2-IRF9 interface (Fig. 7F) (Rengachari et al., 2018). (ii) The finding that M27 mutants incapable to bind DDB1 and induce STAT2 degradation along the ubiquitin proteasome pathway retain a residual capacity to inhibit ISRE signaling, suggesting that the binding of M27 to STAT2 suffices to elicit some signaling inhibitory functions (Fig. 7G). (iii) To elicit their function, CRL4 substrate receptors such as E27 interact with two partners. As we discussed elsewhere (Le-Trilling and Trilling, 2020), a simultaneous development of two independent traits violates evolutionary and probability theories. Thus, these receptors must acquire their binding interfaces sequentially, and the first interaction must provide an evolutionary advantage allowing the fixation of the allele in the population. Afterwards, the second binding interface evolves. Thus, a hypothesis in which E27/M27 precursors evolved the capacity to bind STAT2, preventing its association with IRF9 thereby establishing relevant but incomplete IFN inhibition (before the DDB1 interface was invented leading to STAT2 degradation by the proteasome), provides a parsimonious explanation for all these findings without violating evolutionary constraints. To corroborate our argumentation, we will analyse if E27 indeed displaces IRF9 from STAT2 by analytical gel filtration and/or co-immunoprecipitation experiments.

Reviewer #2: Evidence, reproducibility and clarity

__Reviewer #2: __The manuscript entitled "Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism" by Dr. Schwefel and colleagues cleverly combines biochemistry, mass-spectrometry, Cryo-EM and cell biology to dissect how RCMV-E hijacks its hosts ubiquitylation machinery to mediate proteasomal degradation of STAT2, a key player driving the antiviral IFN response. They identify E27 as DDB1-binding element, which is able promote CRL4-dependent ubiquitylation of STAT2, and demonstrate its effect on STAT2 levels by knockout RCMV-E strains. These findings are supported by in vitro reconstitution of the DDB1/E27/STAT2 complex and analyses via XL-MS and Cryo-EM. The obtained data are then powerfully validated and analysed in mutational strains via infection of homologue in vivo models. The results collectively explain how E27 mimics endogenous CRL4 substrate receptors, thereby recruiting STAT2 to be targeted by CLR4 for ubiquitylation in a NEDD8-dependent manner.

Overall this is an important study that provides convincing insights on how rodent CMVs antagonize their host interferon response by exploiting its ubiquitin-proteasome system.

The manuscript is well written and its introduction is extraordinarily comprehensive. There are a few minor points for the authors to consider below.

Authors Response: We thank the reviewer for this very positive assessment.

Reviewer #2: Significance

Reviewer #2: The work of Schwefel and colleagues combines several powerful state-of-the art techniques to dissect the mechanism of the viral protein E27 and, for the first time, provides a rational for its ability to act as STAT2 antagonist. They performed outstanding structure-function analyses of the ubiquitin system, including the first global proteomic profiling of RCMV-infected cells, setting the standard for its human counterpart as rodent CMVs are commonly used as infection models. The manuscript is highly suitable for publication in any of the journals associated with the review commons platform.

Authors Response: Again, we thank the reviewer for these kind words and the appreciation of our work.

Reviewer #2: CROSS-CONSULTATION COMMENTS

Reviewer #2: This reviewer agrees that at least testing mutants in the E27 in some assays would be appropriate.

Authors Response: As detailed in the response to Reviewer #1, we will generate RCMV-E E27 mutants targeting the Zn-binding motif by site-directed mutagenesis. An analysis of the interaction between DDB1 and these E27 mutants will be included in the revised manuscript.

Reviewer #3: Evidence, reproducibility and clarity

__Reviewer #3: __Le-Trilling et al. present the first proteomic analysis of RCMV-infected cells, where they identified STAT2 as one of the most heavily downregulated (and degraded) proteins. This analysis showed that RCMV mediated degradation of STAT2 is conserved in closely related species used as animal models (rat and mouse) and human, despite the intra-host adaptation of each CMV. They also identify E27 as the RCMV factor that targets STAT2 for degradation, that exhibits ~50% homology with MCMV pM27. This study also identifies a Zinc binding motif in E27 using Cryo-EM which is conserved in other CMV species and is potentially involved in antagonising Type I and III responses.

Reviewer #3: Significance

__Reviewer #3: __The present work provides the first proteomics analysis of RCMV infection in rat cells, comparing infected vs non-infected rat fibroblasts to access potential RCMV targets. Then, it focuses on the characterisation of RCMV E27 and its role targeting and interacting with STAT2 (plus recruiting the Cul4 complex for STAT2 degradation). Finally, it provides the Cryo-EM structure of E27 and its CMV homologues, and the structure of the complex of E27 with elements of the CUL4 complex and STAT2. This is the first time that E27 function and structure are characterised. These are all novel findings - although the mouse homologue M27 has previously been found to interact with and degrade STAT2 (published by some of the same authors in Plos pathogens in 2011, (https://doi.org/10.1371/journal.ppat.1002069). Therefore the chief novel information is the structural studies.

The manuscript will be of interest to researchers working with human and animal herpesviruses.

My field of expertise is in Virology, Innate Immunity and host-virus interactions from an evolutionary perspective. I do not have expertise in Cryo-EM, so I could not evaluate the methods used in the section.

__Authors Response: __We thank the reviewer for the positive evaluation of our work and its significance.

Reviewer #3: Major points

__Reviewer #3: __1. The authors claim the identification of a Zinc-binding motif in the protein E27 (RCMV) using Cryo-EM, then validation of the phenotype with MCMV WT, delM27 and M27 AxAxxA. To justify the change to MCMV to perform the functional validation, they stated "MCMV M27, the closest E27 homologue, exhibits 56% and 76% amino acid sequence identity and similarity, respectively (Fig. S4B). E27 and M27 AlphaFold2 structure predictions are almost indistinguishable (RMSD of 1.195 Å, 6652 aligned atoms) (Figs. 3B, S4A), and structural alignment of these predictions demonstrated conservation of side chain positions involved in zinc-binding (Fig. 3C). Thus, M27 represents a valid model to study functional consequences of interference with the zinc coordination motif through site-directed mutagenesis, and to test the predictive power of our E27/M27 model". Although they rationalise the change to MCMV to validate the functional outcomes of the newly identified zinc binding motif with alignments and Cryo-EM data, it falls within the DDB1 binding region that is less conserved (Fig S4B). The addition of a mouse model here provides a solid result but given the aim of the paper is to provide a proper characterisation of RCMV and elucidate some inter-species adaptations, I strongly recommend the validation with E27 here given the potential impact of this motif. Rather than having to repeat this in a rat model (which would clearly be a large amount of work), this could simply be achieved by constructing the relevant deletion / mutant viruses and assessing in vitro in a relevant cell line (readout - either virus titre or luciferase assay as shown in Figure 3G/H).

__Authors Response: __Please also see our responses to the other reviewers. Briefly, we will apply side-directed mutagenesis to alter the CxCxxC motif in E27 that binds the zinc ion, and analyse the interaction of these E27 mutants with DDB1. In this context, we would like to add that almost two thirds of E27 residues in direct contact with DDB1 are at least type-conserved in M27, and the zinc-coordinating side chains are totally conserved (Fig. 3C). Together with a predicted similar structural organization of the respective binding regions (Fig. S11), and in light of our MCMV mutagenesis results (Fig. 7), it is highly likely that the DDB1-binding mode is conserved between E27 and M27. As mentioned above, we will put this assumption to the test in the revision process.

__Reviewer #3: __Furthermore, in Figure 2, the GF assay was performed using full-length DDB1, however CLMS was performed using DDB1 delBPB (interchange between these two proteins continues in the remainder of the paper). This should be at least justified, and preferably one or other of wt DDB1 and DDB1 delBPB used in the GF or CLMS assay where this has not yet been performed. Later on in the results section (Fig 5E), the authors use wt DDB1 while in fig 4 they used the delBPB to describe the interaction with E27 - would be relevant to have consistency across the paper and some supplementary data that could support using one or the other in each assay.

__Authors Response: __Protein complex preparations including full length DDB1 did not yield cryo-EM reconstructions at appropriate resolution for model building, almost certainly due to the known flexibility of the DDB1 BPB, impeding proper alignment of the cryo-EM particle images. This is why we switched to DDB1ΔBPB. Importantly, the structure model including full length DDB1 (Fig. S12B) clearly demonstrates that the BPB is located on the opposite side of the E27 binding interface on DDB1 (where it is situated to flexibly connect to the CUL4 scaffold to create the ubiquitination zone around immobilised substrates [Fig. 6]). This rules out an involvement of DDB1 BPB in E27- and/or STAT2-binding processes. Several previous studies have employed DDB1ΔBPB to facilitate structure determination, and have successfully applied the resulting structural models for functional follow-up experiments in the context of complete CRL4 assemblies (Bussiere et al., 2020; Petzold et al., 2016; Slabicki et al., 2020). Nevertheless, we will repeat GF experiments with DDB1ΔBPB for consistency and include these data in the revised manuscript.

Reviewer #3: Minor points

__Reviewer #3: __2. Although they present sufficient detail in the methods, further details in the text should be given as to the number of repeats performed in each case, and whether the data shown is representative or based on an average of repeats (preferably the latter; if representative, the data for other repeats should be shown in supplementary information).

Authors Response: We will add this information in the revised version of the manuscript.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Reviewer #1: Major points

__Reviewer #1: __Resolution of the cryoEM structure is rather low and many predictions of the manuscript are based on modeling using AlphaFold2 prediction. The authors describe their model as of "sufficient quality", however, no quality measures are included in the manuscript. At least the discussion should address limitations of the used approach.

Authors Response: While we apologize for not sufficiently describing our quality measures, we respectfully disagree regarding the conclusion. Our resolution (3.8 Å, map 1) lies well within the 3–4 Å resolution range of the vast majority of structures deposited to the Electron Microscopy Data Bank during the last five years (https://www.emdataresource.org/statistics.html). Nevertheless, de novo modelling in this resolution regime is challenging. This is why we sought additional guidance through cross-linking mass spectrometry (XL-MS) restraints and AlphaFold2. Please also note that modelling of E27 was not based solely on the AlphaFold2 prediction. Instead, a partial model corresponding to the α-domain was manually built in map 1, guided by XL-MS information (see Methods - “Model building and refinement” and Fig. S5B, grey cartoon). This partial model proved to be in very good agreement with AlphaFold2 predictions (RMSD of 1.489 Å, 2764 aligned atoms). Only after this initial sanity check, the computational prediction was used for model completion, adjustment, and refinement.

We now added graphical overviews of model fits in Figs. S5 and S10. Furthermore, we included detailed views of the fit of relevant side chains involved in intermolecular interaction to the experimental density (Fig. S7, S9). We also calculated and listed quality indicators of the model-to-map fit in Table S1 (correlation coefficients and model resolution based upon model-map FSC). To ensure the validity of our atomic model using an alternative method besides cryo-EM and XL-MS, we have performed site-directed mutagenesis of critical binding regions in E27, followed by in vitro reconstitution and analytical GF (Fig. S7B, C, S9B, C). The text was revised accordingly (see p10 [ll22] and p14 [ll26]).

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__Reviewer #1: __The authors identify a cryptic zinc-binding motif in E27 that is conserved in homologous proteins. For this reviewer it is not clear: is there experimental evidence for zinc binding of E27 or can the presence of zinc reliably be detected in their structural data? If not, it would be worth to confirm zinc binding.

Authors Response: Our structural data show a tetragonal metal coordination geometry, involving three cysteine side chains and one histidine side chain, with coordination bond lengths of 2.2 Å between the histidine nitrogen and the metal ion, and of 2.4 Å between the cysteine sulfurs and the metal ion. The density feature cannot be explained by another type of side chain interaction, e.g. a disulfide bond, because this would lead to a steric clash with the remaining adjacent side chains. Based on the knowledge on metal-binding sites in proteins and metal-coordination chemistry, these characteristics indicate the presence of a structural zinc-binding site for the following reasons: (i) after magnesium, zinc is the second most prevalent metal in the Protein Data Bank (https://metalpdb.cerm.unifi.it/getSummary), however, magnesium is coordinated octahedrally by oxygen ligands (Tang and Yang, 2013); (ii) the most abundant zinc ligands are cysteine and histidine; (iii) the most abundant zinc coordination number is four ligands; (iv) the average coordination bond lengths are 2.12±0.19 Å and 2.33±0.12Å for nitrogen-zinc and sulfur-zinc interactions, respectively (Ireland and Martin, 2019; Laitaoja et al., 2013), which is in very good agreement with our structural observations. We included this argumentation in the revised manuscript (see p9 [ll21]), and added Fig. S5C for visualization.

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Reviewer #2: Minor points

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Reviewer #2: Page 2, line 3. "Here," should be inserted before "Global proteome profiling..." to highlight the work of this manuscript.

Authors Response: We changed the text accordingly.

Reviewer #2: Page 3, line 21. "IFNs" instead of "IFN"

Authors Response: We changed the text accordingly.

Reviewer #2: Page 4, lines 9,15,27. "Ubiquitin Ligases (UbL)" is not a common abbreviation and could be mistaken for Ubl (Ubiquitin-like proteins). Possible abbreviation is "E3s" for Ubiquitin E3 ligases

Authors Response: We have amended the respective abbreviations accordingly.

Reviewer #2: Page 4 line 25. "RBX1" is the more common term for "ROC1"

Authors Response: This has been corrected throughout the manuscript.

Reviewer #2: Page 5 lines 1-9. Citing of the first structure of DDB1 in complex with a viral protein is recommended. (Ti Li et al. Cell 2006)

Authors Response: We thank the reviewer for this important suggestions and cited this landmark publication.

Reviewer #2: Figure 1 a) STAT2 dot is cut off in second panel. I recommend highlighting STAT2 in both panels.

We amended the figure accordingly. We furthermore additionally highlighted the “STAT2” text in both panels by increasing the font size and putting it in bold type.

Reviewer #2: Page 7 line 17. "Cross-linking MS (CLMS)" is commonly abbreviated as (XL-MS)

Authors Response: We changed the text accordingly.

Reviewer #2: Figure 2 a-c) These panels could benefit from thinner lines in order to increase visibility of chromatograms and cross-links.

Authors Response: The panels were changed accordingly.

Reviewer #2: Figure 2 a-b) Could the authors elaborate on why STAT2 is stoichiometrically

underrepresented in the SDS-PAGE of the E27/DDB1/STAT2 complex?

Authors Response: We applaud Reviewer #2 for their in-depth examination. Honestly, we were also puzzled by this. Based on the cryo-EM single particle analysis, we found an explanation: We separated a major contamination in silico during 2D classification (~12% of all particles). Out of curiosity, we reconstructed a density map from these particles (now shown in Fig. S3). The map was identical to a previous cryo-EM structure of the E. coli protein ArnA (Yang et al., 2019), a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013). ArnA migrates similar to E27 on the SDS-PAGE, the band runs just a little bit faster (compare fraction 6 [ArnA] and fractions 8/9 [E27] from the SDS-PAGE of the analytical GF run of E27 in isolation, Fig. 2A, green trace). However, in analytical GF, ArnA elutes at higher molecular weight fractions, since it forms a hexamers (Ve~10.2 ml). Incidentally, this elution volume of the ArnA hexamer almost equals the one of DDB1 or DDB1ΔBPB/DDA1/E27/STAT2 complexes. This leads to a superposition of ArnA and E27 bands in the respective SDS-PAGE lanes corresponding to GF fraction 6. Accordingly, we conclude that it is actually not STAT2 that is underrepresented, but rather E27 seems overrepresented due to SDS-PAGE band overlap with the ArnA contaminant. We have now indicated the contaminant in Fig. 2A, amended the legend, and extended Fig. S3 to indicate at which point of the cryo-EM analysis the contaminating ArnA particles were separated, and to show the ArnA model to map fit.

In addition to this, it might be that potential STAT2 degradation products (marked by ** in Fig. 2), which seem to co-migrate with STAT2/E27 complexes, occupy FL STAT2 binding sites on E27.

Reviewer #2: Paragraph "The E27 structure.." page 9. Placing this paragraph after the overall

structure is recommended.

Authors Response: Accordingly, we have now moved this section to the end of the results section.

Reviewer #2: Figure 3 a) The grey mesh being laid over the ribbon structures is not contributing to the overall visibility. Adding a panel of the cryo-EM structure alone in cost of alphafold models is recommended.

Figure 4a) same issue with grey mesh

Authors Response: Thank you very much for the very good suggestions. We have removed the mesh representation, and included panels just showing the segmented cryo-EM map in the new Fig. 3A.

Reviewer #2: c) panels could benefit from fewer amino acids being labeled/shown

Authors Response: We understand the motives of the Reviewer. However, we would prefer to depict all relevant side chain interactions in these panels. The rearrangement of the figure, i.e. showing the overview of the interacting regions before the detailed panels, should make them more accessible (new Fig. 3B).

__Reviewer #2: __d) may want to avoid red-green coloring to improve for colorblindness

Authors Response: We are deeply sorry for our ignorance in this regard. We changed the colors accordingly (see new Fig. 3B, C).

__Reviewer #2: __Figure 6a) s.a grey mesh

Authors Response: We removed the mesh representations and included panels just showing the segmented cryo-EM density in the new Fig. 5C.

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Reviewer #2: CROSS-CONSULTATION COMMENTS

__Reviewer #2: __A 3.8 A overall resolution map and the approach to fitting may be suitable, but it is unclear from the authors' figures whether the side-chains shown in the figures are clearly visible in the map or if they are modeled by some other approach. Side chains should ideally be visible in the maps if shown in figures, and if not, close-ups of the corresponding regions of the maps should be shown with sufficient depthcue to allow the reader to gauge how the map corresponds to the model.

Authors Response: This is a crucial point. As mentioned in the response to Reviewer #1, major point 2, we have now included very detailed views of the fit of relevant side chains involved in intermolecular interaction to the experimental density (Fig. S7, S9).

__Reviewer #2: __Along these lines, the figures with the mesh maps do not clearly show how well the model fits the map. This needs to be clearly visible in figures, and ideally maps and models provided to reviewers in order for the reviewers to gauge the level of accuracy of the fit.

Authors Response: Please see our response to Reviewer #1, major point 2. Briefly, we have now included graphical overviews of model fits in Figs. S5 and S10. We also calculated and listed quality indicators of the model-to-map fit in Table S1 (correlation coefficients and model resolution based upon model-map FSC). To ensure the validity of our atomic model using an alternative method besides cryo-EM and XL-MS, we have performed site-directed mutagenesis of critical binding regions in E27, followed by in vitro reconstitution and analytical GF (Fig. S7B, C, S9B, C). The text was extended accordingly (see p10 [ll22] and p14 [ll26]).

__Reviewer #2: __At minimum, the authors have nicely assembled proteomics and cell biological data indicating that E27 hijacks CRL4 to turn over Stat2 in rat cells in a manner paralagous to M27 hijacking in mouse cells, biophysical/structural data for a model of a CUL4-DDB1-E27-Stat2 complex, and mutagenesis of a putative zinc binding site in M27.

I feel most of the issues raised by all 3 reviewers could be addressed in the text, with more clarity about the structural models, and better explanation for why the construct with proteins from various organisms were used for structural studies (the authors had made human DDB1 before, and it expressed well, and perhaps didn't consider to make from rat? Or this mixture expressed, purified best? Gave best quality EM data?).

Authors Response: We thank Reviewer #2 for her/his overall assessment. As mentioned in the two cross-consultation comments before, and in the response to Reviewer #1, major point 2, we strived to provide adequate measures allowing to judge the quality of our structural models in the present updated version of the manuscript. In addition, as indicated in the response to reviewer #3, major point 2, we have now added Fig. S12 and extended the Discussion to explain and justify the use of different protein constructs.

__Reviewer #2: __Also, the presentation of the zinc binding site should come after the overall structure. As for the use of MCMV to assess the role of the zinc binding site, placing this last in the text might allow this to flow better.

Authors Response: Thank you very much for this suggestion. The manuscript has been restructured as recommended: details of the zinc-binding motif and the MCMV assays are now shown in Fig. 7 and described in the text just before the Discussion.

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Reviewer #3: Major points

__Reviewer #3: __2. Given that previous data in mice showed that the E27 homologue pM27 binds a component of host Cullin4-RING UbLs (CRL4), to induce the poly-ubiquitination of STAT2, the current study also addressed if this mechanism was preserved in RCMV. Yet, they seemed to do this with E27, rnSTAT2 and hsDDB1 - Page 7 lines 1 to 3: "These results prompted us to explore the association of E27 with Rattus norvegicus (rn) STAT2 and Homo sapiens (hs) DDB1 in vitro. Importantly, 1128 of 1140 amino acids are identical between hsDDB1 and rnDDB1 (...)". They identify the residues and regions where the DDB1 is different between both species, but should provide a structure/alignment with this highlighted. In addition, DDB1 is a DNA damage protein that is annotated in the Rattus norvegicus genome. The authors should justify the assays between rnSTAT2-hsDDB1 instead of using the both proteins from rn, and present the equivalent data for rnDDB1 in the paper.

Authors Response: Among the 12 alterations between human and rat DDB1, 4 are type-conserved (Fig. S12A). Thus, >99% of amino acids are identical or similar. We mapped all exchanges on a model of full length human DDB1 bound to E27 and the rat STAT2 CCD. None are involved in intermolecular interactions (Fig. S12B, C). Please note that due to the high conservation of DDB1 across eukaryotes, this inter-species approach has been used by us and others to study DDB1-containing complexes (e.g., the SV5V, WHX, SIV Vpx and Vpr, zebrafish DDB2, and chicken CRBN proteins have been in vitro reconstituted with human DDB1 for structural characterisation) and valid biological conclusions have been drawn from these studies (Angers et al., 2006; Banchenko et al., 2021; Fischer et al., 2014; Fischer et al., 2011; Li et al., 2006; Li et al., 2010; Schwefel et al., 2015; Schwefel et al., 2014; Wu et al., 2015).

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Reviewer #3: Minor points

__Reviewer #3: __1. In fig 5D, the authors present the H-box alignment, where it is clear that this motif is not conserved. The lack of H-box conservation should be discussed in the results and discussion, to provide an explanation for the competition/binding observed.

Authors Response: We respectfully disagree. There is conservation of amino acid side chains, regarding their physicochemical properties, observable in the H-box motif. Furthermore, the secondary structure is conserved. Please note, that the H-box is not our invention but rather represented a well-accepted motif known in the field, see e.g., (Li et al., 2010). We extended the discussion to cover this point (p21 [ll15]).

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__Reviewer #3: __3. The authors commence their abstract justifying the study on the grounds of the usefulness of rodent HCMV counterparts as common infection models for HCMV. They should return to this theme in the discussion - what is the usefulness of their findings with regards to HCMV (particularly given the relatively low homology between E27 and HCMV pUL27, and the alternative mechanism for STAT2 antagonism encoded by HCMV UL145)?

Authors Response: We extended the discussion in this regard. Briefly, our data, to our knowledge for the first time, reveal that RCMV (like MCMV) exploits CRL4 to induce proteasomal degradation of STAT2. With pUL145, HCMV relies on an analogous protein. In clear contrast to HCMV, RMCV and MCMV are both amenable to in vivo experiments in small animal models. Over 40 years ago, HCMV has been called the troll of transplantation due to its grim impact on immunosuppressed individuals after transplantation surgery (Balfour, 1979). Despite tremendous efforts, HCMV still harms and kills graft recipients. While MCMV allows various experiments regarding general principles of cytomegaloviral pathogenesis and antiviral immunity, one shortcoming is that the mouse obviously is a rather small animal, preventing various chirurgical and solid organ transplantation (SOT) procedures. In clear contrast, SOT procedures that are indispensable for human medicine can be recapitulated in rat models. Thus, according to our opinion, our work lays the molecular foundation for future studies addressing the relevance of STAT2 and CMV-induced STAT2 degradation in rat SOT models.

4. Description of analyses that authors prefer not to carry out

-

• *

References

Andersen, K.R., Leksa, N.C., and Schwartz, T.U. (2013). Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81, 1857-1861.

Angers, S., Li, T., Yi, X., MacCoss, M.J., Moon, R.T., and Zheng, N. (2006). Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590-593.

Balfour, H.H., Jr. (1979). Cytomegalovirus: the troll of transplantation. Arch Intern Med 139, 279-280.

Banchenko, S., Krupp, F., Gotthold, C., Burger, J., Graziadei, A., O'Reilly, F.J., Sinn, L., Ruda, O., Rappsilber, J., Spahn, C.M.T., et al. (2021). Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses. PLoS pathogens 17, e1009775.

Bussiere, D.E., Xie, L., Srinivas, H., Shu, W., Burke, A., Be, C., Zhao, J., Godbole, A., King, D., Karki, R.G., et al. (2020). Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex. Nat Chem Biol 16, 15-23.

Fischer, E.S., Bohm, K., Lydeard, J.R., Yang, H., Stadler, M.B., Cavadini, S., Nagel, J., Serluca, F., Acker, V., Lingaraju, G.M., et al. (2014). Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49-53.

Fischer, E.S., Scrima, A., Bohm, K., Matsumoto, S., Lingaraju, G.M., Faty, M., Yasuda, T., Cavadini, S., Wakasugi, M., Hanaoka, F., et al. (2011). The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation. Cell 147, 1024-1039.

Ireland, S.M., and Martin, A.C.R. (2019). ZincBind-the database of zinc binding sites. Database (Oxford) 2019.

Laitaoja, M., Valjakka, J., and Janis, J. (2013). Zinc coordination spheres in protein structures. Inorg Chem 52, 10983-10991.

Le-Trilling, V.T.K., and Trilling, M. (2020). Ub to no good: How cytomegaloviruses exploit the ubiquitin proteasome system. Virus Res 281, 197938.

Li, T., Chen, X., Garbutt, K.C., Zhou, P., and Zheng, N. (2006). Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase. Cell 124, 105-117.

Li, T., Robert, E.I., van Breugel, P.C., Strubin, M., and Zheng, N. (2010). A promiscuous alpha-helical motif anchors viral hijackers and substrate receptors to the CUL4-DDB1 ubiquitin ligase machinery. Nature structural & molecular biology 17, 105-111.

Petzold, G., Fischer, E.S., and Thoma, N.H. (2016). Structural basis of lenalidomide-induced CK1alpha degradation by the CRL4(CRBN) ubiquitin ligase. Nature 532, 127-130.

Rengachari, S., Groiss, S., Devos, J.M., Caron, E., Grandvaux, N., and Panne, D. (2018). Structural basis of STAT2 recognition by IRF9 reveals molecular insights into ISGF3 function. Proceedings of the National Academy of Sciences of the United States of America 115, E601-E609.

Schwefel, D., Boucherit, V.C., Christodoulou, E., Walker, P.A., Stoye, J.P., Bishop, K.N., and Taylor, I.A. (2015). Molecular Determinants for Recognition of Divergent SAMHD1 Proteins by the Lentiviral Accessory Protein Vpx. Cell host & microbe 17, 489-499.

Schwefel, D., Groom, H.C., Boucherit, V.C., Christodoulou, E., Walker, P.A., Stoye, J.P., Bishop, K.N., and Taylor, I.A. (2014). Structural basis of lentiviral subversion of a cellular protein degradation pathway. Nature 505, 234-238.

Slabicki, M., Kozicka, Z., Petzold, G., Li, Y.D., Manojkumar, M., Bunker, R.D., Donovan, K.A., Sievers, Q.L., Koeppel, J., Suchyta, D., et al. (2020). The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K. Nature 585, 293-297.

Tang, S., and Yang, J.J. (2013). Magnesium Binding Sites in Proteins. In Encyclopedia of Metalloproteins, R.H. Kretsinger, V.N. Uversky, and E.A. Permyakov, eds. (New York, NY: Springer New York), pp. 1243-1250.

Wu, Y., Koharudin, L.M., Mehrens, J., DeLucia, M., Byeon, C.H., Byeon, I.J., Calero, G., Ahn, J., and Gronenborn, A.M. (2015). Structural Basis of Clade-specific Engagement of SAMHD1 (Sterile alpha Motif and Histidine/Aspartate-containing Protein 1) Restriction Factors by Lentiviral Viral Protein X (Vpx) Virulence Factors. The Journal of biological chemistry 290, 17935-17945.

Yang, M., Chen, Y.S., Ichikawa, M., Calles-Garcia, D., Basu, K., Fakih, R., Bui, K.H., and Gehring, K. (2019). Cryo-electron microscopy structures of ArnA, a key enzyme for polymyxin resistance, revealed unexpected oligomerizations and domain movements. J Struct Biol 208, 43-50.

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Reply to the reviewers

Manuscript number: RC-2022-01528

Corresponding author(s): Elena Taverna and Tanja Vogel

1. General Statements [optional]

We thank the reviewers for the comments and points they raised. We think what we have been asked is a doable task for us and we are confident we will manage to address all points in a satisfactory manner.

2. Description of the planned revisions

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

Reviewer’s comment: The manuscript investigated the role of DOT1L during neurogenesis especially focusing on the earlier commitment from APs. Using tissue culture method with single-cell tracing, they found that the inhibition of DOT1L results in delamination of APs, and promotes neuronal differentiation. Furthermore, using single cell RNA-seq, they seek possible mechanisms and changes in cellular state, and found a new cellular state as a transient state. Among differentially expressed genes, they focused on microcephaly-related genes, and found possible links between epigenetic changes led by DOT1L inhibition and epigenetic inhibition by PRC2. Based on these findings, they suggested that DOT1L could regulate neural fate commitment through epigenetic regulation. Overall, it is well written and possible links from epigenetic to metabolic regulation are interesting. However, there are several issues across the manuscript.

Response to Reviewer and planned revision:

We thank the reviewer’s 1 for her/his comments and constructive criticism.

We hope the revision plan will address the points raised by the reviewer in a satisfactory manner.

Major issues:

* *Reviewer’s comment: 1) It is not clear whether the degree of H3K79 methylation (or other histones) changes during development, and whether DOT1L is responsible for those changes. It is necessary to show the changes in histone modifications as well as the levels of DOT1L from APs to BPs and neurons, and to what extent the treatment of EPZ change the degree of histone methylation.

Response to Reviewer and planned revision:

• As for the level of DOT1L protein We tried several commercially available antibodies, but they do not work in the mouse, even after multiple attempts and optimization. So, unfortunately we will not be able to provide this piece of information.

• As for the level of DOT1L mRNA We will provide info regarding the DOT1L mRNA level in APs, BPs and neurons by using scRNAseq data from E12, E14, E16 WT cerebral cortex.

• As for the levels of H3K79methylation, we did not intend to claim that the histone methylation is responsible for the reported fate transition. We will edit the text to avoid any possible confusion. If it is deemed to be necessary to address the point raised by the reviewer, we do have 3 options, that we here in order of priority and ease of execution from our side.

• immunofluorescence with an Ab against H3K79me2 using CON and EPZ-treated hemispheres.

• FACS sort APs, BPs and neurons from CON and EPZ-treated hemispheres, followed by immunoblot for H3K79me2 to assess the H3K79me2 levels. As for the FACS sorting, we will use a combinatorial sorting in the lab on either a TUBB3-GFP or a GFP-reporter line using EOMES-driven mouse lines. This strategy has already been employed in the lab by Florio et al., 2015 and we will use it with minor modifications.
• scCut&Tag for H3K79me2 from CON and EPZ-treated hemispheres. This option entails a collaboration with the Gonzalo Castelo-Branco lab in Sweden and might therefore require additional time to be established and carried out. Reviewer’s comment:

Furthermore, the study mainly used pharmacological bath application. DOT1L has anti-mitotic effect, thus it is not clear whether the effect is coming from the inhibition of transmethylation activity.

Response to Reviewer and planned revision:

In a previous work we used a genetic model (DOT1L KO mouse) that showed microcephaly (Franz et al. 2019). For this study, we wanted to fill a gap in knowledge by understating if the DOT1L effect was mediated by its enzymatic activity. For this reason, we choose to use the pharmacological inhibition with EPZ, whose effect on DOT1L activity has been extensively reported and documented in literature (EPZ is a drug currently in phase clinical 3 studies).

The stringent focus of this study on the pharmacological inhibition is thus a step toward understanding what specific roles DOT1L can play, both as scaffold or as enzyme.

Here, we concentrate on the enzymatic function and the scaffolding function is beyond the scope of this specific study. We can further discuss and elaborate on the rationale behind this in the revised manuscript.

Reviewer’s comment:

In addition, the study assumed that the effect of EPZ is cell autonomous. However, if EPZ treatment can change the metabolic state in a cell, it would be possible that observed effects was non-cell autonomous. It would be important to address if this effect is coming in a cell-autonomous manner by other means using focal shRNA-KD by IUE.

Response to Reviewer and planned revision:

We did not claim that the effect of EPZ is cell autonomous, we are actually open on this point, as we consider both explanations to be potentially valid. We will edit the text to avoid any possible confusion on what we assume and what not.

As a general consideration, it is entirely possible that the effects are non-cell autonomous. We will comment and elaborate on that in the revised manuscript.

If the reviewer/journal considers this a point that must be addressed experimentally, then we will proceed as follows:

• DOT1L shRNA-KD via in utero electroporation, followed by either
• in situ hybridization for ASNS to check if ASNS transcript is increased upon DOT1L shRNA-KD compared to CON
• FACS sorting of the positive electroporated cells (CON and DOT1L shRNA-KD), followed by qPCR to assess the levels of ASNS
• If the reviewer wants us to check for a more downstream effect on fate, then we will immuno-stain the DOT1L shRNA-KD and CON with TUBB3 AB and/or TBR1 AB (as already done in the present version of the manuscript). Reviewer’s comment: 2) The possible changes in cell division and differentiation were found by very nice single-cell tracing system. However, changes in division modes occurring in targeted APs such as angles of mitotic division and the expression of mitotic markers were not addressed. These information is critical information to understand mechanisms underlying observed phenotype, delamination, differentiation and fate commitment.

Response to Reviewer and planned revision:

Previous effects of DOT1L manipulation on the mitotic spindle were observed in a previous paper, using DOT1L KO mouse (Franz et al. 2019). Considering that in our experiments we do use a pharmacological inhibition, we will address this point by quantifying the spindle angle in CON and EPZ-treated cortical hemispheres.

We will co-stain for DAPI to visualize the DNA/chromosomes, and for phalloidin (filamentous actin counterstain) that allows for a precise visualization of the apical surface and of the cell contour, as it stains the cell cortex.

Of note, the protocols we are referring to are already established in the lab, based on published work from the Huttner lab (Taverna et al, 2012; Kosodo et al, 2005).

Reviewer’s comment: 3) The scRNA-seq analysis indicated interesting results, but was not fully clear to explain the observed results in histology. In fact, in single cell RNA-seq, the author claimed that cells in TTS are increased after EPZ treatment, which are more similar to APs. However, in histological data, they found that EPZ treatment increased neuronal differentiation. These data conflicts, thus I wonder whether "neurons" from histology data are actually neurons? Using several other markers simultaneously, it would be important to check the cellular state in histology upon the inhibition/KD of DOT1L.

Response to Reviewer and planned revision:

The reviewer’s comment is valid, and we indeed found that TTS cells are an intermediate state between APs and neurons in term of transcriptional profile. This is the reason why we called this cell cluster transient transcriptional state.

We plan to address this point by staining for TBR1 and/or CTIP2 in CON and EPZ-treated hemispheres and to expand with this EOMES and SOX2 co-staining.

Minor issues:

Reviewer’s comment: Figure 1 - It is not clear delaminated cells are APs, BPs or some transient cells (Sox2+ Tubb3+??). It is important to use several cell type-specific and cell cycle markers simulnaneously to characterize cell-type specific identity of the analysed cells by staining. These applied to Fig1B,D,E,F,G,as well as Fig2,3.

Response to Reviewer and planned revision:

We will address this point by using a combinatorial staining scheme for several fate markers such as TUBB3, EOMES and SOX2, as suggested by the reviewer.

Reviewer’s comment: - Please provide higher magnification images of labelled cells (Fig 1H)

Response to Reviewer and planned revision:

In the revised manuscript, we will provide higher magnification for the staining.

Reviewer’s comment: - Please provide clarification on the criteria of Tis21-GFP+ signal thresholding.

Response to Reviewer and planned revision:

In the revised manuscript, we will provide a clarification on the criteria of Tis21-GFP+ signal thresholding.

Reviewer’s comment: - Splitting the GFP signal between ventricular and abventricular does not convincingly support the "more basal and/or differentiated" states after EPZ treatment.

Response to Reviewer and planned revision:

We will provide a clarification regarding this point.

Reviewer’s comment: - Please explain the presence of Tis21-GFP+ cells at the apical VZ.

Response to Reviewer and planned revision:

Tis21-GFP+ cells at the apical VZ has been extensively reported in the literature, since the first paper by Haubensak et al. regarding the generation of the Tis21-GFP+ line. In a nutshell, T Tis21-GFP+ cells are present throughout the VZ (therefore also in the apical portion) as neurogenic, Tis21-GFP positive cells are undergoing mitosis at the apical surface. Indeed, the presence of Tis-21 GFP signal have been extensively used by the Huttner lab and collaborators to score apical neurogenic mitosis. In addition, since AP undergo interkinetic nuclear migration, it follows that Tis21-GFP+ nuclei are going to be present throughout the entire VZ.

In the revised manuscript, we will explain this point and cite additional literature.

Reviewer’s comment: - Order the legends in same order as the bars.

Response to Reviewer and planned revision:

We will follow reviewers’ recommendation and order the legends accordingly.

Reviewer’s comment: Figure 2 -Fig 2B) The difference between CON and EPZ apical contacts is not clear and does not match with the graph in Fig 2E.

Response to Reviewer and planned revision:

We will explain Fig. 2B in more detail and provide additional images in the revised manuscript.

Reviewer’s comment: -Supp Fig 2 - are these injected slices cultured in control conditions? Please include this in the text and figure/figure legend

Response to Reviewer and planned revision:

In the revised manuscript, the text will be changed to address this point and provide clearer info.

Reviewer’s comment: Fig 2C) The EPZ-treated DxA555+ cells exhibit morphological change of cell shape. Is this phenotype? please comment on the image shown for EPZ treatment panel.

Response to Reviewer and planned revision:

We thank the reviewer for having raised this point.

The change in morphology might be a consequence of delamination and or of cell fate. In the revised manuscript, we will certainly better comment on this very relevant point and expand the discussion accordingly.

Reviewer’s comment: Fig 2F - 2G) Data presented on EOMES+ and TUBB3+ % are counterintuitive. The authors claimed that TUBB3+ cells are increased and neuronal differentiation is promoted. However, no changes in EOMES+ are observed. What is the explanation? Did the author check the double positive cells? These could be TSS cells?

Response to Reviewer and planned revision:

We thank the reviewer to have raised this point.

As envisioned by the reviewer, we suspect that the counterintuitive data might be due to TSS cell, which based on our scRNAseq data are expressing at the same time several cell type specific markers. It is possible that, since the treatment with EPZ is 24h long, cells (like the TTS cluster) have no time to completely eliminate the EOMES protein. If that were to be the case, then we would expect to still detect (as we indeed do) EOMES immunoreactivity.

To address this point, we will:

• analyze scRNA-seq data and check which is the extent of co-expression of Eomes and Tubb3 mRNAs in the TTS population.
• Check for EOMES and TUBB3 double positive cells in the microinjection experiment. Reviewer’s comment: Figure 2 and Figure 3) the number of pairs analyzed for EPZ is twice as that of Con for comparison of the parameters taken into account. Please include n of each graph in the figure legend of the specific panel if not the same for all panels in that figure (i.e. for figure 3)

Response to Reviewer and planned revision:

We will revise the text accordingly.

Reviewer’s comment:

Figure 3) The data indicated that the number of daughter cell pairs in EPZ samples is almost double than Control. Is this the phenotype? More numbers of daughter cells in EPZ treated samples were observed from the same number of injections? or the number of injected cells were different?

Response to Reviewer and planned revision:

Due to technical reasons, we indeed performed a higher number of injections in EPZ-treated slices. We think this is the main reason behind the difference in number.

If the reason were to be biological, one would expect to see the same trend in IUE experiments, but this is actually not the case. This does suggest/corroborate the idea that the reason behind the difference is mainly technical.

Reviewer’s comment: Figure 4)

• Please clarify if the single cell transcriptomic analysis has been performed only once, and if yes, how statistical testing to compare the cell proportion is carried out with only one batch. Fig 4G)

Response to Reviewer and planned revision:

As for the scRNAseq on microinjected cells:

the scRNA-seq analysis was done once using cells pooled from 3 different microinjection experiments performed in 3 different days.

As for the scRNAseq on IUE cells:

The scRNA-seq analysis was done once using cells pooled from 2-3 different IUE experiments performed in 3 different days.

For all scRNAseq experiments the statistical testing is achieved by intrasample comparisons according to established bioinformatics pipelines. We will better explain this point in the revised manuscript.

Reviewer’s comment: Figure 4 and 5) - Figures are not supportive of the statement regarding APs' neurogenic potential upon DOT1L inhibition. TSS transcriptomic profile resembles more progenitors than neurons. Please comment on TSS neurogenic capacity taking into account the provided GO and RNAseq.

Response to Reviewer and planned revision:

We thank Reviewer 1 for raising this point, It is indeed true that TTS resemble more AP than neurons (as indicated in the Fig. S5B, C). We took that to indicate the fact that these cells are transient and therefore still maintain some AP features. Interestingly, TTS downregulate cell division markers, suggesting a restriction of proliferative potential, as one would expect for cells with an increased neurogenic potential. We will discuss this point in the revised manuscript.

Reviewer’s comment: - Please provide GO analysis for APs and BPs.

Response to Reviewer and planned revision:

Following the reviewer’s suggestion, we will incorporate a more careful and in-depth analysis in the revised version of the manuscript.

Reviewer’s comment: - Reconstruct figure 5A by listing genes in the same order in both Con and EPZ and prioritize EPZ-Con differences instead of cell-cell differences.

Response to Reviewer and planned revision:

We will revise Figure 5A based on the reviewer’s comment.

Reviewer’s comment:

Moreover, the presented genes in the heatmap is not the same in two conditions (i.e. NEUROG1 is present in EPZ but absent in Con). Please justify.

Response to Reviewer and planned revision:

This observation is based on different activities of transcription factor networks in the control and EPZ condition. They are not supposed to be the same as the cell states are altered and different TF are expressed and active upon the treatment in the diverse cell types. In a revised manuscript we will justify this point.

Reviewer’s comment: Fig 5D)

• Please explain why binding of EZH2 on the promoter of Asns is strongly reduced in comparison to a mild significant reduction of H3K79me/H3K27me3 in EPZ compared to Control.

Response to Reviewer and planned revision:

Several explanations are possible

First, the variation can be due to batch effects.

Second, the acute reduction of EZH2 might not be directly accompanied by a reduced histone mark, which is reduced either by cell division or by demethylases. The two processes of getting rid of the mark might be slower than the reduction of EZH2 presence at the respective site.

Based on the reviewer’s comment, we will explain this point in the revised manuscript.

• *

Reviewer’s comment:

Also is the changed directly medicated by DOT1L?

Please test whether DOT1L can bind the promoter of Asns.

Response to Reviewer and planned revision:

To address this relevant issue we will proceed with the following protocol:

• electroporate a tagged version of DOT1L into ESCs
• select ESCs and differentiate them into NPC_48h.
• treat NPC with DMSO (Con) or EPZ
• harvest CON and EPZ-treated NPC
• perform ChIP-qPCR DOT1L at the Asns promoter Reviewer’s comment: Please provide the expression patterns of DOT1L and Asns during neuronal differentiation.

Response to Reviewer and planned revision:

As for Dot1l

Dot1l expression was shown in Franz et al 2019, by ISH from E12.5 to E18.5.

As for Asns

We will provide E14.5 in situ staining of Asns in the developing mouse brain using the Gene Paint database (see Figure below).

We will also show immunostainings for ASNS at mid-neurogenesis, provided that Ab against ASNS works in the mouse.

Other General comments:

Reviewer’s comment: Please Indicate VZ, SVZ and CP on the side of the pictures/ with dot lines in the pictures both for primary figures and supplementary.

Response to Reviewer and planned revision:

We will revise the figures accordingly.

Reviewer’s comment: - The Results and figures sometimes do not support the statement made by the authors

Response to Reviewer and planned revision:

We will carefully check on this and eliminate any overinterpretation or non-supported statements from the text.

• Schemes are not informative/explanatory enough, i.e. time windows of treatment and sample collection, culture conditions details.

Response to Reviewer and planned revision:

We will revise the schemes to include more details. In particular, we plan to add a supplementary figure with a detailed visual description of the protocol, to match the detailed description presented in the materials and methods.

Reviewer’s comment: - A more extensive characterization of TTS cells in terms of differentiation progression and integration would be enlightening

Response to Reviewer and planned revision:

In general, we are facing two main challenges while studying the TTS population: one is the lack of a specific marker gene for TTS, the other is the relatively small size of the TTS subpopulation.

For these reasons, our ability to carry on an in-depth analysis of this cell state is limited.

Considering the reviewer’s comment, in the revised manuscript we will expand the analysis ad characterization of the differentiation potential of TTS using RNA velocity trajectory.

We can also expand the discussion on this point.

Reviewer’s comment: - Picture quality can be improved, provide high magnification images.

Response to Reviewer and planned revision:

We will revise the figures to include higher magnification images.

Reviewer #1 (Significance (Required)):

Reviewer’s comment: The study could be important for the specific field in neural development. It aims to understand mutations in respective genes and brain malformation. If the link between epigenetic and metabolic changes is clearly shown, it will be interesting. However, the current manuscript is still rather descriptive, and clear mechanistic insights were not provided. The study have potentials and additional data will strength the value of study.

Response to Reviewer and planned revision:

We will address the direct impact of DOT1L and H3K79me2 on the Asns gene locus during the revision (see the rationale of the experimental strategy also in the revision plan above). We hope we will thus provide a mechanistic link between epigenetics and altered metabolome.

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

Reviewer’s comment: Appiah et al. present a concise manuscript that provides details and possible mechanisms of their previous work (Franz et al., 2019; Ferrari et al., 2020). The study uses diverse lines of investigation to arrive at most conclusions. However, as interesting as the data is, we find that at the present state, it is not sufficient to prove that, indeed, the asparagine metabolism is regulated by DOTL1/PRC2 crosstalk. The neurogenic shift presented in the first part of the paper is not comprehensive and, therefore, not very convincing. The quality of images provided in the main and supplementary data is less than ideal. Additional data analysis and interpretation of the scRNA seq data may be needed. The authors finally conclude with rescue experiments done in culture and in-vivo, which we believe is the stand-out part of this study. Overall the manuscript has some interesting observations that are often over-interpreted with less supporting data. The manuscript reads well but requires additional data and changes in the claims/interpretation to be suited for publication.

Response to Reviewer and planned revision:

In the revised manuscript, we hope we will address the comments and concerns raised by the reviewer in a satisfactory manner. Comments

Reviewer’s comment: 1) Abstract: Is this statement correct: "DOT1L inhibition led to increased neurogenesis driven by a shift from asymmetric self-renewing to symmetric neurogenic divisions of APs. AP undergoes symmetric division for self-renewal and asymmetric neurogenic divisions.

Response to Reviewer and planned revision:

Based on the current literature (cit. Huttner and Kriegstein), AP undergo:

• symmetric division for proliferative division at early stages of neurogenesis
• asymmetric self-renewing division, generating an AP and a BP at mid neurogenesis. This division is also described as neurogenic, as it produces a BP, that is a step further than AP in term of neurogenic potential.
• symmetric consumptive division at late neurogenesis To avoid any possible confusion, we will re-phrase the sentence to include the adjective “consumptive” and specify the composition of the progeny.

In the revised manuscript, the sentence will read as follow:

"DOT1L inhibition led to increased neurogenesis driven by a shift of APs from asymmetric self-renewing (generating one AP and one BP) to symmetric consumptive divisions (generating two neurons)"

Reviewer’s comment: All the data is based on treatments with EPZ (DOTL1 inhibitor), yet no information is shown to support its targeted activity in this system. A proof of principle in the chosen experimental system is missing; for instance, examining the activity or protein level of DOTL1 and decreased methylation of the target(s) is essential.

Response to Reviewer and planned revision:

EPZ is a well characterized drug, that has been used previously in our lab and by others as well.

As for our lab, the information regarding the inhibitor, its activity and efficiency in inhibiting DOT1L towards H3K79me2 was shown in Franz et al. Supplementary Fig. S6 D, E.

In the present manuscript, an additional confirmation that EPZ targets DOT1L in regard to its H3K79me2 activity is shown in Fig. 5D.

We would refer to this information more explicitly in a revised manuscript.

Reviewer’s comment: 2) Figure 1: The scoring of centrosomes and cilia is insufficient to conclude delamination and increase in basal fates. The effect could be on ciliogenesis or centrosome tethering to the apical end-feet of the AP, and other possible explanations for this observation also exist. The images are too small; larger images or graphic representations could be helpful in addition to the data.

Response to Reviewer and planned revision:

We did not intend to claim that the change in centrosome location demonstrate delamination, but only that it suggests delamination. This criterion has been extensively used as a proxy for delamination by several labs working on the cell biology of neurogenesis, such Huttner and Gotz labs. If the issue persists, we can re-phrase in a more cautious way the text referring to Figure 1 to highlight that the data only suggest delamination.

Response to Reviewer and planned revision:

To make a statement regarding delamination, I would like to see either the dynamics of delamination (organotypic slices images), staining with BP markers, or morphological changes of AP (staining that will reveal loss of adherence) or comparable data to support the observation. In my opinion Supp. Figure 1 is insufficient; the single image is not convincing; I would like to see 3D reconstruction and better-quality images.

Response to Reviewer and planned revision:

We can certainly provide better images and co-stain with relevant markers.

We think it is beyond the scope of the manuscript embarking in live imaging as we are not studying the dynamics of delamination per se.

Reviewer’s comment: Tis21 data (1H), again of low quality, is only a single piece of evidence and the conclusion "suggesting that the acquisition of a basal fate was paralleled by a switch to neurogenesis" is premature. I think other cell cycle exit reporters, Fucci markers, pHis, BrdU, NeuroD, or Tbr2 reporters (Li et al., 2020, (Haydar and Sestan labs)) to name a few, are necessary to establish the conclusions. The authors should show other markers such as PAX6, EOMES, or other upper-layer markers upon cell cycle exit in the SVZ/CP. These additional experiments will assist in cell fate analysis.

Response to Reviewer and planned revision:

We completely understand the points raised by the reviewer, and we plan to address them by co-staining with PAX6/SOX2, PH3 and/or EOMES.

We think establishing the Fucci or EOMES mouse system is beyond the scope of the manuscript. In addition, given the present setting of all labs involved, it would be logistically unattainable (see also comments in the section below).

We think the co-staining scheme and plan will be informative enough to satisfactory address the concerns raised by the reviewer.

Reviewer’s comment: 2) Figure 2: The microinjection experiments are elegant; the images, however, do not complement the experiment. The images of the microinjected cells seem not to be reconstructed from z-stacked optical slices, so often, processes are not continuous (panel B, for example); therefore, it is not clear if an apical process is indeed missing or just not seen.

Response to Reviewer and planned revision:

The mentioned images are reconstructed from continuous Z-stacks, as we always do given the type of data. We can provide better reconstructions and/or additional images.

Reviewer’s comment:

The data analysis should include other parameters; BrdU staining could have given information on cell cycle exit, PAX6, SOX2, and EOMES on the location of the cells in the VZ/sVZ. The quality of images showing EOMES and TUBB3 staining is so low that it makes the reader doubt the validity of the quantifications. "Taken together, these data suggest that the inhibition of DOT1L might favor the acquisition of a neuronal over BP cell fate" This interpretation should be subjected to more investigations. It is possible that this treatment just accelerates the AP-> BP -> Neuronal fate. The author's claim needs to be backed by additional experiments or be changed.

Response to Reviewer and planned revision:

To address this point, we will include in the revised manuscript staining and co-staining with PAX6, SOX2 (see also response above) and provide a BrdU labeling experiment.

Reviewer’s comment: 3) Figure 3: The experiment concept and its performance are impressive, yet the data is insufficient. The images in A that are supposed to be representative show two cells; their location is not clear, and the expression of GFP is not clear; in fact, both pairs seem to be GFP negative (not clear what is the threshold for background). Staining with anti-GFP and a second method to follow neurogenesis is necessary.

Response to Reviewer and planned revision:

We did use different staining methods and schemes to follow neurogenesis. As specified above, we will deepen our analysis by using additional markers, such as TBR1.

Reviewer’s comment: 4) On page 9, lines 8-10, the authors claim that their number of cells was "sufficient" for single-cell analysis; the numbers are Response to Reviewer and planned revision:

In the revised manuscript, we will include the analysis of how many cells are needed to identify cluster of 6 cell types in this paradigm, based for example on the algorithms developed in Treppner et al. 2021.

Reviewer’s comment: 5) The authors use Seurat and RaceID without their appropriate citations in the first mention during the results. The authors also stop immediately after DEG analysis along with clustering. The authors could analyze their RNA-seq data with a trajectory; to say the least, the identification/characterization of TTS and neurons as Neurons I, II, and III are insufficient. There could be multiple ways to show the "fate" of cells in the isolated FACS, which the authors have missed.

Response to Reviewer and planned revision:

We will include the respective citations in a revised manuscript. We provide already differentiation trajectories but will include other methods, including scVelo of FateID to extend the trajectory analyses. We kindly ask the reviewer to also refer to the comments above regarding the TTs cluster characterization as part of our effort to provide a better picture of the different clusters.

Reviewer’s comment: 6) The authors detected candidates like Fgfr3, Nr2f1, Ofd1, and Mme as part of their treated (different approaches) datasets (from their DEG analysis). They correctly cite Huang et al., 2020 but fail to give us a sense of the consequences of these gene dysregulations. The authors can also validate if these proteins are expressed in their treated cells.

Response to Reviewer and planned revision:

In the revised manuscript we will comment on the function of the four genes mentioned.

In addition, we will validate the expression of these genes on protein and transcriptional level through immunostainings -provided that antibodies are working in our system- or smFISH, respectively.

Reviewer’s comment: 7) The authors list a few GO terms (page 10, lines 1-10) and associate them with reduced proliferation; they must cite relevant studies. The authors can also add supplementary data showing which genes in their data correspond to these GO terms.

Response to Reviewer and planned revision:

We thank the reviewer for pointing out the missing citations.

We of course agree on the need to add them, and we will do so in the revised manuscript.

Reviewer’s comment: 8) On Page 11, lines 3-7, the authors describe their method to arrive at the 17 targets with TF activity from the previous analysis. Can the authors describe the method used to correlate the two? The reviewer understands this could be MEME analysis or analysis of earlier datasets of Ferrari et al. 2020. But it must be explicitly stated, and a few examples in supplementary need to be exemplified as this analysis is key to discovering the three metabolic genes.

Response to Reviewer and planned revision:

In the revised manuscript, we will clarify the exact analysis that resulted in the identification of the 17 target genes, using the specific tool for gene network analysis, that is based on our scRNA-seq data alone, but not on the Ferrari et al 2020 data set.

3. Description of the revisions that have already been incorporated in the transferred manuscript

n/a

4. Description of analyses that authors prefer not to carry out

Reviewer’s comment: Tis21 data (1H), again of low quality, is only a single piece of evidence and the conclusion "suggesting that the acquisition of a basal fate was paralleled by a switch to neurogenesis" is premature. I think other cell cycle exit reporters, Fucci markers, pHis, BrdU, NeuroD, or Tbr2 reporters (Li et al., 2020, (Haydar and Sestan labs)) to name a few, are necessary to establish the conclusions. The authors should show other markers such as PAX6, EOMES, or other upper-layer markers upon cell cycle exit in the SVZ/CP. These additional experiments will assist in cell fate analysis.

Response to Reviewer and planned revision:

As pointed out above, we think establishing the Fucci or EOMES mice system is beyond the scope of the manuscript as it will not provide more information than the ones we will obtain from systematic and extensive co-staining experiments. In addition, all labs involved are facing a logistic issue (animal house not ready yet, construction works etc) that made the importing and setting up of the colony unattainable for the next 6-10months. If the reviewer and/or the editorial board think this is a major point compromising the entire revision, we kindly ask to contact us again so that we can discuss the issue and arrive to a shared conclusion.

There's an interesting dichotomy between streaming information and batched data here, but it isn't well delineated and doesn't add much to the discussion as a result. Perhaps distilling it down may help? There's a kernel of something useful here, but it isn't immediately apparent.

Relation to stock and flow or the idea of the garden and the stream?

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Reply to the reviewers

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

In recent years, the field has investigated crosstalk between cGMP and cAMP signaling (PMID: 29030485), lipid and cGMP signaling (PMID: 30742070), and calcium and cGMP signaling (PMID: 26933036, 26933037). In contrast to the Plasmodium field, which has benefited from proteomic experiments (ex: PMID 24594931, 26149123, 31075098, 30794532), second messenger crosstalk in T. gondii has been probed predominantly through genetic and pharmacological perturbations. The present manuscript compares the features of A23187- and BIPPO-stimulated phosphoproteomes at a snapshot in time. This is similar to a dataset generated by two of the authors in 2014 (PMID: 24945436), except that it now includes one BIPPO timepoint. The sub-min​​ute phosphoproteomic timecourse following A23187 treatment in WT and ∆cdpk3 parasites is novel and would seem like a useful resource.

CDPK3-dependent sites were detected on adenylate cyclase, PI-PLC, guanylate cyclase, PDE1, and DGK1. This motivated study of lipid and cNMP levels following A23187 treatment. The four PDEs determined to have A23187-dependent phosphosites were characterized, including the two PDEs with CDPK3-dependent phosphorylation, which were found to be cGMP-specific. However, cGMP levels do not seem to differ in a CDPK3- or A23187-dependent manner. Instead, cAMP levels are elevated in ∆cdpk3 parasites. This would seem to implicate a feedback loop between CDPK3, the adenylyl cyclase, and PKA/PKG: CDPK3 activity reduces adenylyl cyclase activity, which reduces PKA activity, which increases PKG activity. The authors don't pursue this direction, and instead characterize PDE2, which does not have CDPK3-dependent phosphosites, and seems out of place in the study

Response:

We agree with reviewer 1 that a feedback loop between CDPK3, the adenylyl cyclase and PKA/PKG is certainly one of several possibilities (and we acknowledge this in the manuscript).

We felt, however, that given the observation that A23187 and BIPPO treatment leads to phosphorylation of numerous PDEs (hinting at the presence of an Ca2+-regulated feedback loop), it was entirely relevant to study these in greater detail. Coupled with the A23187 egress assay on ΔPDE2 parasites - our findings suggest that PDE2 plays an important role in this signalling loop (an entirely novel finding). While PDE2 appears to exert its effects in a CDPK3-independent manner (indeed suggesting that CDPK3 might exert its effects on cAMP levels in a different fashion), this does not detract from the important finding that PDE2 is one of the (likely numerous) components that is regulated in a Ca2+-dependent feedback loop to regulate egress.

We have modified our writing to better reflect the fact that our decision to pursue study of the PDEs was not solely CDPK3-centric.

While we feel that our reasoning for studying the PDEs is solid, we appreciate that further clarification on the putative CDPK3-Adenylate cyclase link would make it easier for the reader to follow the rationale.

We have not studied the direct link between CDPK3 and the Adenylate Cyclase β in more detail, as ACβ alone was shown to not play a major role in regulating lytic growth (Jia et al., 2017).

**MAJOR COMMENTS**

1.Some of the key conclusions are not convincing.

The data presented in Figure 6E, F, and G and discussed in lines 647-679 are incongruent. In Figure 6E, the plaques in the PDE2+RAP image are hardly visible; how can it be that the plaques were accurately counted and determined not to differ from vehicle-treated parasites?

Are the images in 6E truly representative? Was the order of PDE1 and PDE2 switched? The cited publication by Moss et al. 2021 (preprint) is not in agreement with this study, as stated. That preprint determined that parasites depleted of PDE2 had significantly reduced plaque number and plaque size (>95% reduction); and parasites depleted of PDE1 had a substantially reduced plaque size but a less substantial reduction in plaque number.

Response:

The plaques for PDE2+RAP were counted using a microscope since they are difficult to see by eye. We thank the reviewer for detecting our incorrect reference to Moss et al. (2021). This has been corrected in the text. We confirm, however, that the images in 6E are representative of what we observed and do indeed differ from what was seen by Moss et al.. We have acknowledged this clearly in the text.

The differences cannot easily be explained other than by the different genetic systems used. Further studies of the individual PDEs will likely illuminate their role in invasion/ growth, but we feel this would be beyond the scope of this study.

Unfortunately, the length of time required for PDE depletion (72h) is incompatible with most T. gondii cellular assays (typically performed within one lytic cycle, 40-48h). Although the authors performed the assays 3 days after initial RAP treatment, is there evidence that non-excised parasites don't grow out of the population. This should be straightforward to test: treat, wait 3 days, infect onto monolayers, wait 24-48h fix, and stain with anti-YFP and an anti-Toxoplasma counterstain. The proportion of the parasite population that had excised the PDE at the time of the cellular assays will then be known, and the reader will have a sense of how complete the observed phenotypes are. As a reader, I will regard the phenotypes with some level of skepticism due to the long depletion time, especially since a panel of PDE rapid knockdown strains (depletion in __Response:

1. Cellular assays using KO parasites are commonly performed at the point at which protein depletion is detected. Both our western blots and plaque assay results demonstrate that, at the point of assay, there is no substantial outgrowth of non-excised parasites. The original manuscript also includes PCRs performed at the 72 hr time point (See Fig. 6B) to support this.
2. We appreciate the reviewer’s comment re the panel of PDE KD strains. The reviewer notes that there are substantial limitations to conditional KO systems, which similarly applies to KD systems - there are notable pros and cons to each approach. When designing our strategy (pre-publication of the Moss et al., 2022), we made a deliberate decision to use conditional KO strains in light of the fact that residual protein levels in KD systems can cause significant problems, particularly for membrane proteins (all of the investigated PDEs have a transmembrane domain). Tagging of proteins with the degradation domain can have further issues, leading to protein mis-localisation, which we have experienced with several unrelated proteins in the lab.

   The authors should qualify some of their claims as preliminary or speculative, or remove them altogether.

The claims in lines 240-260 are confusing. It seems likely that the two drug treatments have at least topological distinctions in the signaling modules, given that cGMP-triggered calcium release is thought to occur at internal stores, whereas A23187-mediated calcium influx likely occurs first at the parasite plasma membrane.The authors' proposed alternative, that treatment-specific phosphosite behavior arises from experimental limitations and "mis-alignment", is unsatisfying for the following reasons: (1) From the outset, the authors chose different time frames to compare the two treatments (15s for BIPPO vs. 50s for A23187); (2) the experiment comprises a single time point, so it does not seem appropriate to compare the kinetics of phosphoregulation. There is still value in pointing out which phosphosites appear treatment-specific under the chosen thresholds, but further claims on the basis of this single-timepoint experiment are too speculative. Lines 264-267 and 281-284 should also be tempered.

Relatedly, graphing of the data in Figure 1G (accompanying the main text mentioned above) was confusing. Why is one axis a ratio, and the other log10 intensity? What does log10 intensity tell you without reference to the DMSO intensity? Wouldn't you want the L2FC(A23187) vs. L2FC(BIPPO) comparisons? Could you use different point colors to highlight these cases on plot 1E? Additionally, could you use a pseudocount to include peptides only identified in one treatment condition on the plot in 1E? (Especially since these sites are mentioned in lines 272-278 but are not on the plot)

Response:

1. The kinetics of the responses to A23187 and BIPPO are very different. This is why treatment timings are purposely different as they were selected to align pathways to a point where calcium levels peak just prior to calcium re-uptake. We make no mention of kinetic comparisons, and merely demonstrate that at the chosen timepoints, overall signalling correlation is very high. The observation that most of the sites that behave differently between conditions sit remarkably close to the threshold for differential regulation (in the treatment condition where they are not DR - see Fig. 1G) led us to speculate that many of these sites are likely on the cusp of differential regulation. While it is entirely possible that some of these differences are, in fact, treatment specific (and we clearly acknowledge this in the text), we simply state that we cannot confidently discern clear signalling features that allow us to distinguish between the two treatments. We feel that this is an entirely relevant observation given the observed preponderance of both A23187 and BIPPO-dependent DR phosphosites on proteins in the PKG signalling pathway (as current models place this upstream of Ca2+release).
2. Log10 intensity only serves to spread the data for easier visualisation. The only comparison being made relates to the LFCs. Fig. 1Gi shows the LFC scores (x axis) for all sites regulated following A23187 treatment (for which peptides were also identified in BIPPO treatment). On this plot we have highlighted the sites that are differentially regulated following BIPPO but not A23187 treatment (with red showing the DRup and blue showing the DRdown sites). This demonstrates that many of the sites that are regulated following BIPPO but not A23187 treatment cluster close to the threshold for differential regulation in the A23187 dataset - suggesting that many of these sites are likely on the cusp of differential regulation. Fig. 1Gii shows the reverse. While we could highlight the above-mentioned sites on the plot in Fig. 1E, we do not feel that it would demonstrate our point as clearly.

We feel that including a pseudocount on Fig. 1E for peptides lacking quantification in one treatment condition would be visually misleading as the direct correlation being made in Fig. 1E is BIPPO vs A23187 treatment. The sites mentioned in lines 272-278 in the original manuscript (now lines 268-276) are available in the supplement tables.

3.Additional experiments would be essential to support the main claims of the paper.

Genetic validation is necessary for the experiments performed with the PKA inhibitor H89. H89 is nonspecific even in mammalian systems (PMID: 18523239) and in this manuscript it was used at a high concentration (50 µM) The heterodimeric architecture of PKA in apicomplexans dramatically differs from the heterotetrameric enzymes characterized in metazoans (PMID: 29263246), so we don't know what the IC50 of the inhibitor is, or whether it inhibits competitively. Two inducible knockdown strains exist for PKA C1 (PMID: 29030485, 30208022). The authors could request one of these strains and construct a ∆cdpk3 in that genetic background, as was done for the PDE2 cKO strain. Estimated time: 3-4 weeks to generate strain, 2 weeks to repeat assays.

Response:

1. While we appreciate that H89 is not 100% specific for PKA, this is not our only line of evidence that cAMP levels are altered. We demonstrate that cAMP levels are elevated in CDPK3 KO parasites – further substantiating our finding.

The H89 concentration used in our experiment is in keeping with/lower than the concentrations used in other Toxoplasma publications (Jia et al., 2017), and both the Toxoplasma and Plasmodium fields have shown convincingly that H89 treatment phenocopies cKD/cKO of PKA (see Jia et al., 2017; Flueck et al., 2019).

While we agree that the genetic validation suggested by reviewer 1 would serve to further support our findings (though it would not provide further novel insights), the suggested time frame for experimental execution was not realistic. Line shipment, strain generation, subcloning and genetic validation would take substantially longer than 3-4 weeks.

cGMP levels are found to not increase with A23187 treatment, which is at odds with a previous study (lines 524-560). The text proposes that the differences could arise from the choice of buffer: this study used an intracellular-like Endo buffer (no added calcium, high potassium), whereas Stewart et al. 2017 used an extracellular-like buffer (DMEM, which also contains mM calcium and low potassium). An alternative explanation is that 60 s of A23187 treatment does not achieve a comparable amount of calcium flux as 15 s of BIPPO treatment, and a calcium-dependent effect on cGMP levels, were it to exist, could not be observed at the final timepoint in the assay. The experiments used to determine the kinetics of calcium flux following BIPPO and A23187 treatments (Fig. 1B, C) were calibrated using Ringer's buffer, which is more similar to an extracellular buffer (mM calcium, low potassium). In this buffer, A23187 treatment would likely stimulate calcium entry from across the parasite plasma membrane, as well as across the membranes of parasite intracellular calcium stores. By contrast, A23187 treatment in Endo buffer (low calcium) would likely only stimulate calcium release from intracellular stores, not calcium entry, since the calcium concentration outside of the parasite is low. Because calcium entry no longer contributes to calcium flux arising from A23187 treatment, it is possible that the calcium fluxes of A23187-treated parasites at 60 s are "behind" BIPPO-treated parasites at 15 s. The researchers could control these experiments by *either* (i) performing the cNMP measurements on parasites resuspended in the same buffer used in Figure 1B, C (Ringer's) or (ii) measuring calcium flux of extracellular parasites in Endo buffer with BIPPO and A23187 to determine the "alignment" of calcium levels, as was done with intracellular parasites in Figure 1C. No new strains would have to be generated and the assays have already been established in the manuscript. Estimated time to perform control experiments with replicates: 2 weeks. This seems like an important control, because the interpretation of this experiment shifts the focus of the paper from feedback between calcium and cGMP signaling, which had motivated the initial phosphoproteomics comparisons, to calcium and cAMP signaling. Further, the lipidomics experiments were performed in an extracellular-like buffer, DMEM, so it's unclear why dramatically different buffers were used for the lipidomics and cNMP measurements.

Response:

While the initial calibration experiments to measure calcium flux were indeed performed in Ringer’s buffer, the parasites were intracellular. We therefore chose to measure cNMP concentrations of extracellular parasites syringe lysed in Endo buffer, which is better at mimicking intracellular conditions than any other described buffer.

As the reviewer suggested, we measured the calcium flux of extracellular parasites in Endo buffer upon stimulation with either A23187 or BIPPO.

We found that peak calcium response to BIPPO in Endo buffer was similar to that of intracellular parasites (~15 seconds post treatment) (See Supp Fig. 6A). Upon treatment with A23187, extracellular parasites in Endo buffer had a much faster response compared to their intracellular counterparts, with peak flux measured at ~25 seconds post treatment (see Supp Fig. 6B). This indeed does suggest that extracellular parasites in Endo buffer behave differently to A23187 compared to their intracellular counterparts. However, peak calcium response is still occuring within the experimental time course and is not being missed, as the reviewer worries. Moreover, since we are able to detect increased cAMP levels in A23187 treated parasites, Ca2+ flux appears sufficient to alter cNMP signalling.

We did notice however that the intensity of the calcium flux was much weaker in Endo buffer compared to intracellular parasites (see Supp Fig. 6B). We found that this was due to the lack of host-derived Ca2+, since supplementation of Endo buffer with 1 uM CaCl2 restored the intensity of the calcium response to match that of intracellular parasites (see Supp Fig. 6C). We therefore decided to repeat our cGMP measurements, this time using extracellular parasites in Endo buffer supplemented with 1 uM CaCl2. However, we found no differences in cGMP levels in the response to ionophore under these conditions (now Supp Fig. 6D) compared to the previous experiments, so the conclusions from the previous data do not change.

As for the lipidomics experiments, we chose to use DMEM so that our dataset could be compared with other published lipidomic datasets (Katris et al., 2020; Dass et al., 2021) where DMEM was also used as a buffer when measuring global lipid profiles of parasites.

We now acknowledge in the paper that Endo buffer has its shortcomings, and that this could be the reason why we do not detect changes in cGMP concentrations. We do, however, believe that Endo buffer is the best alternative to intracellular parasites and is supported by its consistent use in numerous publications studying Toxoplasma signalling (McCoy et al., 2012; Stewart et al., 2017).

Additional information is required to support the claim that PDE2 has a moderate egress defect (lines 681-687). T. gondii egress is MOI-dependent (PMID: 29030485). Although the parasite strains were used at the same MOI, there is no guarantee that the parasites successfully invaded and replicated. If parasites lacking PDE2 are defective in invasion or replication, the MOI is effectively decreased, which could explain the egress delay. Could the authors compare the MOIs (number of vacuoles per host cell nuclei) of the vehicle and RAP-treated parasites at t = 0 treatment duration to give the reader a sense of whether the MOIs are comparable?

Response:

Since PDE2 KO parasites have a substantial growth defect, we did notice that starting MOIs were consistently lower for the RAP-treated samples compared to the DMSO-treated samples. However, this was also the case for PDE1 KO parasites where we did not see an egress delay. We also found that the egress delay was still evident for ∆CDPK3 parasites, despite having higher starting MOIs than WT parasites in our experiments. Therefore there does not appear to be a link between starting MOIs and the egress delay.

To be sure of our results, we also performed egress assays where we co-infected HFFs with mCherry-expressing WT parasites (WT ∆UPRT) and GFP-expressing PDE2 cKO parasites that were treated with either DMSO or RAP or ∆CDPK3 parasites. This recapitulated our previous findings, confirming the deletion of PDE2 leads to delay in A23187-mediated egress.

4.A few references are missing to ensure reproducibility.

The manuscript states that the kinetic lipidomics experiments were performed with established methods, but the cited publication (line 497) is a preprint. These are therefore not peer reviewed and should be described in greater detail in this manuscript, including any relevant validation.

Response:

We thank the reviewer for pointing this out. We have included a greater description of the methods used in the materials and methods section such that the experiment is reproducible, as per the reviewer’s suggestion. We decided to still make mention of the BioRxiv preprint since we thought it was appropriate for the reader to be informed of ongoing developments in the field.

Please cite the release of the T. gondii proteomes used for spectrum matching (lines 972-973).

Response:

We have included this as per the reviewer’s suggestion.

Please include the TMT labeling scheme so the analysis may be reproduced from the raw files.

Response:

We have included this as per the reviewer’s suggestion in Supp Fig. 3A.

5.Statistical analyses should be reviewed as follows:

Have the authors examined the possibility that some changes in phosphopeptide abundance reflect changes in protein abundance? This may be particularly relevant for comparisons involving the ∆cdpk3 strain. Did the authors collect paired unenriched proteomes from the experiments performed? Alternatively, there may be enriched peptides that did not change in abundance for many of the proteins that appear dynamically phosphorylated.

Response:

We did not collect unenriched proteomes from the experiments performed (although we did perform unenriched mixing checks to ensure equal loading between samples), and believe that this wasn’t a necessity for the following reasons:

1. For within-line treatment analyses, treatment timings are so short (a maximum of 15-50s in the single timepoint experiment) that it would be unlikely to detect substantial changes in protein abundance. Moreover, these unlikely events would affect all phosphosites across a protein, and therefore be detectable.

In our CDPK3 dependency timecourse experiments, we normalise both the WT and ∆CDPK3 strain to 0s, and measure signalling progression over time. Therefore, any difference at timepoints that are not “0” are not originating from basal differences. We also see a consistent increase/decrease in phosphosite detection across the sub-minute timecourse, further confirming that the observed changes are truly down to dynamic changes in phosphorylation and not protein levels.

In the single timepoint CDPK3 dependency analyses (44 regulated sites identified, Data S2), we acknowledge that there could be some risk of altered starting protein abundance between lines. However, if protein abundance were responsible for the changes in phosphosite detection, we would expect all phosphosites across the protein to shift, and we do not observe this. Moreover, when we look at these CDPK3 dependent proteins and compare their phosphosite abundance in untreated WT and ∆CDPK3 lines, we find that for each protein, either all or the majority of phosphosites detected are unchanged (highlighting that there is no substantial difference in this protein’s abundance between lines). Where there are phosphosite differences between lines, these are only ever on single sites on a protein while most other sites are unchanged - implying that these are changes to basal phosphorylation states and not protein levels.

It seems like for Figs. 3B and S5 the maximum number of clusters modeled was selected. Could the authors provide a rationale for the number of clusters selected, since it appears many of the clusters have similar profiles.

The number of clusters is chosen automatically by the Mclust algorithm as the value that maximizes the Bayes Information Criterion (BIC). BIC in effect balances gains in model fit (increasing log-likelihood) against increasing the number of parameters (i.e. number of clusters).

Please include figure panel(s) relating to gene ontology. Relevant information for readers to make conclusions includes p-value, fold-enrichment or gene ratio, and some sort of metric of the frequency of the GO term in the surveyed data set. See PMID: 33053376 Fig. 7 and PMID: 29724925 Fig. 6 for examples or enrichment summaries. Additionally, in the methods, specify (i) the background set, (ii) the method used for multiple test correction, (iii) the criteria constituting "enrichment", (iv) how the T. gondii genome was integrated into the analysis, (v) the class of GO terms (molecular function, biological process, or cellular component), (vi) any additional information required to reproduce the results (for example, settings modified from default).

Response:

We have included the additional information requested in the materials and methods.

We purposely did not include GO figure panels as our analyses are being done across many clusters, making it very difficult to display this information cohesively. We have included all data in Tables S2-S5. These tables included all the relevant information on p-value, enrichment status, ratio in study/ratio in population, class of GO terms etc.

The presentation of the lipidomics experiments in Figure 4A-C is confusing. First, the ∆cdpk3/WT ratio removes information about the process in WT parasites, and it's unclear why the scale centers on 100 and not 1. Second, the data in Figure S6 suggests a more modest effect than that represented in Fig. 4; is this due to day to day variability? How do the authors justify pairing WT and mutant samples as they did to generate the ratios?

Response:

This is a common strategy used by many metabolomics experts (Bailey et al., 2015; Dass et al., 2021; Lunghi et al., 2022). We had originally chosen to represent the data as a ratio since this form of representation helps get rid of the variability that arises between experiments and allows us to see very clear patterns which would otherwise go unnoticed. This variability arises from the amount of lipids in each sample which varies between parasites in a dish, the batch of FBS and DMEM used, and the solutions and even room temperature used to extract lipids on a given day.

However, we agree with the reviewer that depicting the data in Figure 4A-C as a ratio of ∆CDPK3/WT parasites can be confusing, so we have now changed the graphs, plotting WT and ∆CDPK3 levels instead, and have moved the ratio of ∆CDPK3/WT to the Supplementary Figure 5.

The significance test seems to be performed on the difference between the WT and ∆cdpk3 strains, but not relative to the DMSO treatment? Wouldn't you want to perform a repeated measures ANOVA to determine (i) if lipid levels change over time and (ii) if this trend differs in WT vs. mutant strain?

Response:

The reviewer correctly points out that ANOVA is often used for time courses, but we must point out that it is not always strictly appropriate since it can overlook the purpose of the individual experiment design, which in this case is, 1) to investigate the role of CDPK3 compared to the WT parental strain, and 2) specifically to find the exact point at which the DAG begins to change after stimulus to match the proteomics time course.

Our data is clearly biassed towards earlier time points where we have 0, 5, 10, 30, 45 seconds where DAG levels are mostly unchanged compared to the single timepoint 60 seconds which shows a significant difference in DAG using our method of statistical comparison by paired two tailed t-test. Therefore, it would be unwise to use ANOVA when we really want to see when the A23187 stimulus takes effect, which appears to be after the 45 second mark. Therefore, analysing the data by ANOVA would likely provide a false negative result, where the result is non-significant but there is clearly more DAG in WT than CDPK3 after 60 seconds. T-tests are commonly used when comparing the same cell lines grown in the same conditions with a test/treatment, and in this case the test/treatment is CPDK3 present or absent (Lentini et al., 2020).

In the main text, it would be preferable to see the data presented as the proteomics experiments were in Figure 4B and 4C, with fold changes relative to the DMSO (t = 0) treatment, separately for WT and ∆cdpk3 parasites.

Response:

We have now changed the way that we represent the data, plotting %mol instead of the ratio.

Signaling lipids constitute small percentages of the overall pool (e.g. PMID: 26962945), so one might not necessarily expect to observe large changes in lipid abundance when signaling pathways are modulated. Is there any positive control that the authors could include to give readers a sense of the dynamic range? Maybe the DGK1 mutant (PMID: 26962945)?

Response:

DGK1 is maybe not a good example because the DGK1 KO parasites effectively “melt” from a lack of plasma membrane integrity ((Bullen et al., 2016), so this would likely be technically challenging. We don’t see the added value in including an additional mutant control since we can already see the dynamic change over time from no difference (0 seconds) to significant difference (60 seconds) between WT and CDPK3 for DAG and most other lipids. We already see a significant difference between WT and CDPK3 after 60 seconds for DAG, and we can clearly see in sub-minute timecourses the changes or not at the specific points where the A23187 is added (0-5 seconds), the parasites acclimatise, for the A23187 to take effect (10-30 seconds) and for the parasite lipid response to be visible by lipidomics (45-60 +seconds).

Figure 4E: are the differences in [cAMP] with DMSO treatment and A23187 treatment different at any of the timepoints in the WT strain? The comparison seems to be WT/∆cdpk3 at each timepoint. Does the text (lines 562-568) need to be modified accordingly?

Response:

In WT (and ∆CDPK3) parasites, [cAMP] is significantly changed at 5s of A23187 treatment (relative to DMSO). We have modified our figures to include this analysis. The existing text accurately reflects this.

Figure 6I: is the difference between PDE2 cKO/∆cdpk3 + DMSO or RAP significant?

Response

In our original manuscript, there was no statistical difference in [cAMP] between PDE2cKO/∆CDPK3+DMSO and PDE2cKO/∆CDPK3+DMSO+RAP, likely due to the variation between biological replicates. To overcome the issues in variability between replicates, we have now included more biological replicates (n=7). This has led to a significant difference in [cAMP] between PDE2cKO/∆CDPK3 DMSO- and RAP-treated parasites and between PDE2cKO DMSO- and RAP-treated parasites (now Fig. 6I).

**MINOR COMMENTS**

1.The following references should be added or amended:

Lines 83-85: in the cited publication, relative phosphopeptide abundances of an overexpressed dominant-negative, constitutively inactive PKA mutant were compared to an overexpressed wild-type mutant. In this experimental setup, one would hypothesize that targets of PKA should be down-regulated (inactive/WT ratios). However, the mentioned phosphopeptide of PDE2 was found to be up-regulated, suggesting that it is not a direct target of PKA.

Response:

We thank the reviewer for spotting this error, we have now modified our wording.

Cite TGGT1_305050, referenced as calmodulin in line 458, as TgELC2 (PMID: 26374117).

Response:

We have included this as per the reviewer’s suggestion.

Cite TGGT1_295850 as apical annuli protein 2 (AAP2, PMID: 31470470).

Response:

We have included this as per the reviewer’s suggestion.

Cite TGGT1_270865 (adenylyl cyclase beta, Acβ) as PMID: 29030485, 30449726.

Response:

We have included this as per the reviewer’s suggestion.

Cite TGGT1_254370 (guanylyl cyclase, GC) as PMID: 30449726, 30742070.

Response:

We have included this as per the reviewer’s suggestion.

Note that Lourido, Tang and David Sibley, 2012 observed that treatment with zaprinast (a PDE inhibitor) could overcome CDPK3 inhibition. The target(s) of zaprinast have not been determined and may differ from those of BIPPO (in identity and IC50). The cited study also used modified CDPK3 and CDPK1 alleles, rather than ∆cdpk3 and intact cdpk1 as used in this manuscript. That is to say, the signaling backgrounds of the parasite strains deviate in ways that are not controlled.

Response:

While it is true that zaprinast targets have not been unequivocally identified, zaprinast-induced egress is widely thought to be the result of PKG activation, a conclusion that is further supported by the finding that Compound 1 completely blocks zaprinast-induced egress (Lourido, Tang and David Sibley, 2012). Similarly, BIPPO-induced egress is inhibited by chemical inhibition of PKG by Compound 1 and Compound 2 (Jia et al., 2017). Moreover, like zaprinast, BIPPO has been clearly shown to partially overcome the ∆CDPK3 egress delay (Stewart et al., 2017).

2.The following comments refer to the figures and legends:

Part of the legend text for 1G is included under 1H.

Response:

This has been corrected

Figure 1H: The legend mentions that some dots are blue, but they appear green. Please ensure that color choices conform to journal accessibility guidelines. See the following article about visualization for colorblind readers: https://www.ascb.org/science-news/how-to-make-scientific-figures-accessible-to-readers-with-color-blindness____/ . Avoid using red and green false-colored images; replace red with a magenta lookup table. Multi-colored images are only helpful for the merged image; otherwise, we discern grayscale better. Applies to Figures 1B, 5C, 6D. (Aside: anti-CAP seems an odd choice of counterstain; the variation in the staining, esp. at the apical cap, is distracting.)

Response:

We thank reviewer #1 for bringing this to our attention, and have modified our colour usage for all IFAs and Figures 1H and 3E.

We chose CAP staining as the antibody is available in the laboratory and stains both the apical end (which has been shown to contain several proteins important for signalling as well as PDE9) and the parasite periphery, the location of CDPK3.

Figure 1B: When showing a single fluorophore, please use grayscale and include an intensity scale bar, since relative values are being compared.

Response:

We have modified this as per the reviewer’s suggestion

Figure 1C: it is difficult to compare the kinetics of the calcium response when the curves are plotted separately. Since the scales are the same, could the two treatments be plotted on the same axes, with different colors? Additionally, according to the legend, a red line seems to be missing in this panel.

Response:

Fig1C is not intended to compare kinetics, merely to show peak calcium release in each separate treatment condition. We have removed mention of a red line in the figure legend.

Figure 2A: Either Figure S4 can be moved to accompany Figure 2A, or Figure 2A could be moved to the supplemental.

Figure S4 has now been incorporated into Figure 2.

Reviewer #1 (Significance (Required)):

This manuscript would interest researchers studying signaling pathways in protozoan parasites, especially apicomplexans, as CDPK3 and PKG orthologs exist across the phylum. To my knowledge, it is the first study that has proposed a mechanism by which a calcium effector regulates cAMP levels in T. gondii. Unfortunately, the experiments fall short of testing this mechanism.

Response:

We thank reviewer #1 for their comments, but disagree with their assessment that the key points of the manuscript “fall short of experimental testing”.

1. We demonstrate that, following both BIPPO and A23187 treatment, there is differential phosphorylation of numerous components traditionally believed to sit upstream of PKG activation (as well as several components within the PKG signalling pathway itself).
2. We show that some of these sites are CDPK3 dependent, and that deletion of CDPK3 leads to changes in lipid signalling and an elevation in levels of cAMP (dysregulation of which is known to alter PKG signalling).
3. We show that pre-treatment with a PKA inhibitor is able to largely rescue this phenotype.
4. We demonstrate that a cAMP-specific PDE is phosphorylated following A23187 treatment (i.e. Ca2+ flux)
5. We show that this cAMP specific PDE plays a role in A23187-mediated egress.
6. While the latter PDE may not be directly regulated by CDPK3, these findings suggest that there are likely several Ca2+-dependent kinases that contribute to this feedback loop.

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

**Summary:**

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

In this manuscript, Dominicus et al investigate the elusive role of calcium-dependent kinase 3 during the egress of Toxoplasma gondii. Multiple functions have already been proposed for this kinase by this group including the regulation of basal calcium levels (24945436) or of a tyrosine transporter (30402958). However, one of the most puzzling phenotypes of CDPK3 deficient tachyzoites is a marked delay in egress when parasites are stimulated with a calcium ionophore that is rescued with phosphodiesterase (PDE) inhibitors. Crosstalk between, cAMP, cGMP, lipid and calcium signalling has been previously described to be important in regulating egress (26933036, 23149386, 29030485) but the role of CDPK3 in Toxoplasma is still poorly understood.

Here the authors first take an elegant phosphoproteomic approach to identify pathways differentially regulated upon treatment with either a PDE inhibitor (BIPPO) and a calcium ionophore (A23187) in WT and CDPK3-KO parasites. Not much difference is observed between BIPPO or A23187 stimulation which is interpreted by the authors as a regulation through a feed-back loop.

The authors then investigate the effect of CDPK3 deletion on lipid, cGMP and cAMP levels. The identify major changes in DAG, phospholipid, FFAs, and TAG levels as well as differences in cAMP levels but not for cGMP. Chemical inhibition of PKA leads to a similar egress timing in CDPK3-KO and WT parasites upon A23187 stimulation.

As four PDEs appeared differentially regulated in the CDPK3-KO line upon A23187, the authors investigate the requirement of the 4 PDEs in cAMP levels. They show diverse localisation of the PDEs with specificities of PDE1, 7 and 9 for cGMP and of PDE2 for cAMP. They further show that PDE1, 7 and 9 are sensitive to BIPPO. Finally, using a conditional deletion system, they show that PDE1 and 2 are important for the lytic cycle of Toxoplasma and that PDE2 shows a slightly delayed egress following A23187 stimulation.

**Major comments:**

-Are the key conclusions convincing?

The title is supported by the findings presented in this study. However I am not sure to understand why the authors imply a positive feed back loop. This should be clarified in the discussion of the results.

Response:

We believe in a positive feedback loop as, upon A23187 treatment (resulting in a calcium flux), ΔCDPK3 parasites are able to egress, albeit in a delayed manner. This egress delay is substantially, but not completely, alleviated upon treatment with BIPPO (a PDE inhibitor known to activate the PKG signalling pathway). In conjunction with our phosphoproteomic data (where we see phosphorylation of numerous pathway components upstream of PKG upon BIPPO and A23187 treatment - both in a CDPK3 dependent and independent manner), these observations suggest that calcium-regulated proteins (CDPK3 among them) feed into the PKG pathway. As deletion of CDPK3 delays egress, it is reasonable to postulate that this feedback is one that amplifies egress signalling (i.e. is positive).

The phosphoproteome analysis seems very strong and will be of interest for many groups working on egress. However, the key conclusion, i.e. that a substrate overlaps between PKG and CDPK3 is unlikely to explain the CDPK3 phenotype, seems premature to me in the absence of robustly identified substrates for both kinases.

Response:

We certainly do not fully exclude the possibility of a substrate overlap but do lean more heavily towards a feedback loop given (a) the inability to clearly detect treatment-specific signalling profiles and (b) the phospho targets observed in the A23187 and BIPPO phosphoproteomes. We have further clarified our reasoning, and overall tempered our language in the manuscript as per the reviewer’s suggestion.

I am not sure there is a clear key conclusion from the lipidomic analysis and how it is used by the authors to build their model up. Major changes are observed but how could this be linked with CDPK3, particularly if cGMP levels are not affected?

Response:

Our phosphoproteomic analyses identify several CDPK3-dependent phospho sites on phospholipid signalling components (DGK1 & PI-PLC), suggesting that there is indeed altered signalling downstream of PKG. To test whether these lead to a measurable phenotype, we performed the lipidomics analysis. We did not pursue this arm of the signalling pathway any further as we postulated that the changes in the lipid signalling pathway were less likely to play a role in the feedback loop. Nevertheless, we felt that it was worthwhile to include these findings in our manuscript as they support the conclusions drawn from the phosphoproteomics - namely that lipid signalling is perturbed in CDPK3 mutants. We, or others, may follow up on this in future.

We agree with the reviewer that it is surprising that cGMP levels remain unchanged in our experiments when we treat with A23187. Given the measurable difference in cAMP levels between WT and ΔCDPK3 parasites, we postulate that CDPK3 directly or indirectly downregulates levels of cAMP. This would, in turn, alter activity of the cAMP-dependent protein kinase PKAc. Jia et al. (2017) have shown a clear dependency on PKG for parasites to egress upon PKAc depletion, but were also unable to reliably demonstrate cGMP accumulation in intracellular parasites. Similarly, their hypothesis that dysregulated cGMP-specific PDE activity results in altered cGMP levels has not been proven (the PDE hypothesised to be involved has since been shown to be cAMP-specific).

While it is possible that our collective inability to observe elevated cGMP levels is explained by the sensitivity limits of the assay, it is similarly possible that cAMP-mediated signalling is exerting its effects on the PKG signalling pathway in a cGMP-independent manner.

The evidence that CDPK3 is involved in cAMP homeostasis seems strong. However, the analysis of PKA inhibition is a bit less clear. The way the data is presented makes it difficult to see whether the treatment is accelerating egress of CDPK3-KO parasites or affecting both WT and CDPK3-KO lines, including both the speed and extent of egress. This is important for the interpretation of the experiment.

Response:

Fig. 4F shows that there is a significant amount of premature egress in both WT and ∆CDPK3 parasites following 2 hrs of H89 pre-treatment (consistent with previous reports that downregulation of cAMP signalling stimulates premature egress). When we subsequently investigated A23187-induced egress rates of the remaining intracellular H89 pre-treated parasites (Fig. 4Gi-ii) we found that the ∆CDPK3 egress delay was largely rescued. We have moved Fig. 4F to the supplement (now Supp Fig. 5E) in order to avoid confusion between the distinct analyses shown in 4F (pre-treatment analyses) and 4G (egress experiment). These experiments provided a hint that cAMP signalling is affected, which we then validate by measuring elevated cAMP levels in CDPK3 mutant parasites.

The biochemical characterisation of the four PDE is interesting and seems well performed. However, PDE1 was previously shown to hydrolyse both cAMP and cGMP (____https://doi.org/10.1101/2021.09.21.461320____) which raises some questions about the experimental set up. Could the authors possibly discuss why they do not observe similar selectivity? Could other PDEs in the immunoprecipitate mask PDE activity? In line with this question, it is not clear what % of "hydrolytic activity (%)" means and how it was calculated.

The experiments describing the selectivity of BIPPO for PDE1, 7 and 9 as well as the biological requirement of the four tested PDEs are convincing.

Response:

We believe that the disagreement between our findings and those published by Moss and colleagues are due to the differences in experimental conditions. We performed our assays at room temperature for 1 hour with higher starting cAMP concentrations (1 uM) compared to them. They performed their assays at 37ºC for 2 hours with 10-fold lower starting cAMP concentrations (0.1 uM). We have now repeated this set of experiments using the Moss et al. conditions, and find that PDEs 1, 7 and 9 can be dual specific, while PDE2 is cAMP-specific, thereby recapitulating their findings (Now included in the revised manuscript under Supp Fig. 7B). However, we also now performed a timecourse PDE assay using our original conditions and show that the cAMP hydrolytic activity for PDE1 can only be detected following 4 hours of incubation, compared to cGMP activity that can be detected as early as 30 minutes, suggesting that it possesses predominantly cGMP activity (See Supp Fig. 7C). We therefore believe that our experimental setup is more stringent, because if one starts with a lower level of substrate and incubates for longer and at a higher temperature, even minor dual activity could make a substantial difference in cAMP levels. Our data suggests that the cAMP hydrolytic activity of PDEs 1, 7 and 9 is substantially lower than the cGMP hydrolytic activity that they display.

We have also included a clear description of how % hydrolytic activity was calculated in the methods section.

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

The claim that CDPK3 affects cAMP levels seems strong however the exact links between CDPK3 activity, lipid, cGMP and cAMP signalling remain unclear and it may be important to clearly state this.

Response:

We have modified our wording in the text to more clearly describe our current hypothesis and reasoning.

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

I think that the manuscript contains a significant amount of experiments that are of interest to scientists working on Toxoplasma egress. Requesting experiments to identify the functional link between above-mentioned pathways would be out of the scope for this work although it would considerably increase the impact of this manuscript. For example, would it be possible to test whether the CDPK3-KO line is more or less sensitive to PKG specific inhibition upon A23187 induced?

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

The above-mentioned experiment is not trivial as no specific inhibitors of PKG are available. Ensuring for specificity of the investigated phenotype would require the generation of a resistant line which would require significant work.

__Response: __We agree that this would be an interesting experiment to further substantiate our findings. As indicated by the reviewer, however, the lack of specific inhibitors of PKG means a resistant line would likely be required to ensure specificity.

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

It is not clear how the % of hydrolytic activity of the PDE has been calculated.

Response: We have included a clearer description of how % hydrolytic activity was calculated in the methods section.

-Are the experiments adequately replicated and statistical analysis adequate?

This seems to be performed to high standards.

**Minor comments:**

-Specific experimental issues that are easily addressable.

I do not have any comments related to minor experimental issues.

-Are prior studies referenced appropriately?

Most of the studies relevant for this work are cited. It is however not clear to me why some important players of the "PKG pathway" are not indicated in Fig 1H and Fig 3E, including for example UGO or SPARK.

Response:

We have modified Fig 1H and 3E to include all key players involved in the PKG pathway.

-Are the text and figures clear and accurate?

While all the data shown here is impressive and well analysed, I find it difficult to read the manuscript and establish links between sections of the papers. The phosphoproteome analysis is interesting and is used to orientate the reader towards a feedback mechanism rather than a substrate overlap. But why do the authors later focus on PDEs and not on AC or CNBD, as in the end, if I understand well, there is no evidence showing a link between CDPK3-dependent phosphorylation and PDE activity upon A23187 stimulation?

Response:

We thank reviewer#2 and appreciate their constructive feedback re the flow of the manuscript.

Our key findings from the phosphoproteomics study were that 1) BIPPO and A23187 treatment trigger near identical signalling pathways, 2) that both A23187 and BIPPO treatment leads to phosphorylation of numerous components both upstream and downstream of PKG signalling (hinting at the presence of an Ca2+-regulated feedback loop) and 3) several of the abovementioned components are phosphorylated in a CDPK3 dependent manner.

While several avenues of study could have been pursued from this point onwards, we chose to focus on the feedback loop in a broader sense as its existence has important implications for our general understanding of the signalling pathways that govern egress.

We reasoned that, given the differential phosphorylation of 4 PDEs following A23187 and BIPPO treatment (none of which had been studied in detail previously), it was relevant to study these in greater detail.

Coupled with the A23187 egress assay on PDE2 knockout parasites - our findings suggest that PDE2 plays a role in the abovementioned Ca2+ signalling loop. While PDE2 may not exert its effects in a CDPK3-dependent manner (and CDPK3 may, therefore, alter cAMP levels in a different fashion), this does not detract from the important finding that PDE2 is one of the (likely numerous) components that is regulated in a Ca2+-dependent feedback loop to facilitate rapid egress.

We have modified our wording to better reflect our rationale for studying the PDEs irrespective of their CDPK3 phosphorylation status.

While we feel that our reasoning for studying the PDEs is solid, we do appreciate that further clarification on the putative CDPK3-Adenylate cyclase link would elevate the manuscript substantially. However, given the data that the ACb is not playing a sole role in the control of egress, this is likely a non-trivial task and requires substantial work.

It is also unclear how the authors link CDPK3-dependent elevated cAMP levels with the elevated basal calcium levels they previously described. This is particularly difficult to reconcile particularly in a PKG independent manner.

Response:

We previously postulated that elevated Ca2+ levels allowed ΔCDPK3 mutants to overcome a complete egress defect, potentially by activating other CDPKs (e.g. CDPK1). It is similarly plausible that elevated Ca2+ levels in ΔCDPK3 parasites may lead to elevated cAMP levels in order to prevent premature egress.

As noted in our previous responses, we acknowledge that our inability to detect cGMP is surprising. However, given the clarity of our cAMP findings, and the phosphoproteomic evidence to suggest that various components in the PKG signalling pathway are affected, we postulate that we are either unable to reliably detect cGMP due to sensitivity issues, or that cAMP is exerting its regulation on the PKG pathway in a cGMP-independent manner. As noted previously, while the link between cAMP and PKG signalling has been demonstrated by Jia et al., it is not entirely clear how this is mediated.

The presentation of the lipidomic analysis is also not really clear to me. Why do the authors show the global changes in phospholipids and not a more detailed analysis?

Response:

We performed a detailed phospholipid profile of WT and ∆CDPK3 parasites under normal culture conditions. However, due to the sheer quantity of parasites required for this detailed analysis, we were unable to measure individual phospholipid species in our A23187 timecourse. We therefore opted to measure global changes following A23187 stimulation.

As the authors focus on the PI-PLC pathway, could they detail the dynamics of phosphoinositides? I understand that lipid levels are affected in the mutant but I am not sure to understand how the authors interpret these massive changes in relationship with the function of CDPK3 and the observed phenotypes.

Response:

Our phosphoproteomic analyses identified several CDPK3-dependent phospho sites on phospholipid signalling components (DGK1 & PI-PLC), suggesting that (in keeping with all of our other data), there is altered signalling downstream of PKG. To test whether these changes lead to a measurable phenotype, we performed the lipidomics analysis. Following stimulation with A23187, we found a delayed production of DAG in ∆CDPK3 parasites compared to WT parasites. Since DAG is required for the production of PA, which in turn is required for microneme secretion, our finding can explain why microneme secretion is delayed in ∆CDPK3 parasites, as previously reported (Lourido, Tang and David Sibley, 2012; McCoy et al., 2012).

We did not follow this arm of the signalling pathway any further as we postulated that the changes in the lipid signalling pathway were less likely to play a role in the feedback loop. Nevertheless, we felt that it was worthwhile to include these findings in our manuscript as they support the conclusions drawn from the phosphoproteomics - namely that lipid signalling is perturbed in CDPK3 mutants. We, or others, may follow up on this in future.

Finally, the characterisation of the PDEs is an impressive piece of work but the functional link with CDPK3 is relatively unclear. It would also be important to clearly discuss the differences with previous results presented in this this preprint: https://doi.org/10.1101/2021.09.21.461320____.

My understanding is while the authors aim at investigating the role of CDPK3 in A23187 induced egress, the main finding related to CDPK3 is a defect in cAMP homeostasis that is not linked to A23187. Similarly, the requirements of PDE2 in cAMP homeostasis and egress is indirectly linked to CDPK3. Altogether I think that important results are presented here but divided into three main and distinct sections: the phosphoproteomic survey, the lipidomic and cAMP level investigation, and the characterisation of the four PDEs. However, the link between each section is relatively weak and the way the results are presented is somehow misleading or confusing.

Response:

As mentioned in a previous response, we chose to study PDEs in greater detail because of our observation that both A23187 and BIPPO treatments lead to their phosphorylation (hinting at the presence of a Ca2+regulated feedback loop). We were particularly intrigued to study the cAMP specific PDE, as CDPK3 KO parasites suggested that cAMP may play a role in the Ca2+ feedback mechanism. As PDE2 may not be directly regulated by CDPK3, Ca2+ appears to exert its feedback effects in numerous ways. We have modified our wording to better reflect our rationale for studying the PDEs irrespective of their CDPK3 phosphorylation status.

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

This is a very long manuscript written for specialists of this signalling pathway and I would suggest the authors to emphasise more the important results and also clearly state where links are still missing. This is obviously a complex pathway and one cannot elucidate it easily in a single manuscript.

Response:

We have included an additional summary in our conclusions to better illustrate our findings and clarify any missing links.

Reviewer #2 (Significance (Required)):

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

This is a technically remarkable paper using a broad range of analyses performed to a high standard.

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

The cross-talk between cAMP, cGMP and calcium signalling is well described in Toxoplasma and related parasites. Here the authors show that, in Toxoplasma, CDPK3 is part of this complex signalling network. One of the most important finding within this context is the role of CDPK3 in cAMP homeostasis. With this in mind, I would change the last sentence of the abstract to "In summary we uncover a feedback loop that enhances signalling during egress and links CDPK3 with several signalling pathways together."

Response:

In light of feedback received from several reviewers, we have made our wording less CDPK3 centric - as our findings relate in part to CDPK3 and, in a broader sense, to a Ca2+ driven feedback loop.

The genetic and biochemical analyses of the four PDEs are remarkable and highlight consistencies and inconsistencies with recently published work that would be important to discuss and will be of interest for the field.

__Response: __We thank reviewer#2 and agree that the PDE findings are of significant importance to the field.

While I understand the studied signalling pathway is complex, I think it would be important to better describe the current model of the authors. In the discussion, the authors indicate that "the published data is not currently supported by a model that fits most experimental results." I would suggest to clarify this statement and discuss whether their work helps to reunite, correct or improve previous models.

__Response: __We have expanded on the abovementioned statement to clarify that the presence of a feedback loop is a major pillar of knowledge required for the complete interpretation of existing signalling data.

Could the authors also speculate about a potential role of PDE/CDPK3 in host cell invasion as cAMP signalling has be shown to be important for this process (30208022 and 29030485)?

__Response: __Existing literature (Jia et al., 2017) suggests that perturbations to cAMP signalling play a very minor role in invasion since parasites where either ACα or ACβ are deleted show no impairment in invasion levels. We currently do not have substantial data on invasion, and are not sure that pursuing this is valuable given the minor phenotypes observed in other studies.

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

This paper is of great interest to groups working on the regulation of egress in Toxoplasma gondii and other related apicomplexan pathogens.

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

I am working on the cell biology of apicomplexan parasites.

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

**Summary:**

Dominicus et al aimed to identify the intersecting components of calcium, cyclic nucleotides (cAMP, cGMP) and lipid signaling through phosphoproteomic, knockout and biochemical assays in an intracellular parasite, Toxoplasma gondii, particularly when its acutely-infectious tachyzoite stage exits the host cells. A series of experimental strategies were applied to identify potential substrates of calcium-dependent protein kinase 3 (CDPK3), which has previously been reported to control the tachyzoite egress. According to earlier studies (PMID: 23226109, 24945436, 5418062, 26544049, 30402958), CDPK3 regulated the parasite exit through multiple phosphorylation events. Here, authors identified differentially-regulated (DR) phosphorylation sites by comparing the parasite samples after treatment with a calcium ionophore (A23178) and a PDE inhibitor (BIPPO), both of which are known to induce artificial egress (induced egress as opposed to natural egress). When the DCDPK3 mutant was treated with A23187, its delayed egress phenotype did not change, whereas BIPPO restored the egress to the level of the parental (termed as WT) strain, probably by activating PKG.

The gene ontology enrichment of the up-regulated clusters revealed many probable CDPK3-dependent DR sites involved in cyclic nucleotide signaling (PDE1, PDE2, PDE7, PDE9, guanylate and adenylate cyclases, cyclic nucleotide-binding protein or CNBP) as well as lipid signaling (PI-PLC, DGK1). Authors suggest lipid signaling as one of the factors altered in the CDPK3 mutant, albeit lipidomics (PC, PI, PS, PT, PA, PE, SM) showed no significant change in phospholipids. To reveal how the four PDEs indicated above contribute to the cAMP and cGMP-mediated egress, they examined their biological significance by knockout/knockdown and enzyme activity assays. Authors claim that PDE1,7,9 proteins are cGMP-specific while PDE2 is cAMP-specific, and BIPPO treatment can inhibit PDE1-cGMP and PDE7-cGMP, but not PDE9-cGMP. Given the complexity, the manuscript is well structured, and most experiments were carefully designed. Undoubtedly, there is a significant amount of work that underlies this manuscript; however, from a conceptual viewpoint, the manuscript does not offer significant advancement over the current knowledge without functional validation of phosphoproteomics data (see below). A large body of work preceding this manuscript has indicated the crosstalk of cAMP, cGMP, calcium and lipid signaling cascades. This work provides a further refinement of the existing model In a methodical sense, the work uses established assays, some of which require revisiting to reach robust conclusions and avoid misinterpretation. The article is quite interesting from a throughput screening point of view, but it clearly lacks the appropriate endorsement of the hits.The authors accept that identifying the phosphorylation of a protein does not imply a functional role, which is a major drawback as there is no experimental support for any phosphorylation site of the protein identified through phosphoproteomics. In terms of the mechanism, it is not clear whether and how lipid turnover and cAMP-PKA signaling control the egress phenotype (lack of a validated model at the end of this study).

Response:

We thank reviewer #3 for their comments, but respectfully disagree with their assessment that the work presented does not advance current knowledge.

1. We demonstrate that, following both BIPPO and A23187 treatment, there is differential phosphorylation of numerous components traditionally believed to sit upstream of PKG activation (as well as numerous components within the PKG signalling pathway itself). While it may have been inferred from previous studies that A23187 and BIPPO signalling intersect, this has never been unequivocally demonstrated - nor has a feedback loop ever been shown.

We provide a novel A23187-driven phosphoproteome timecourse that further bolsters the model of a Ca2+-driven feedback loop.

We show that deletion of CDPK3 leads to a delay in DAG production upon stimulation with A23187.

We show that some of the abovementioned sites are CDPK3 dependent, and that deletion of CDPK3 leads to elevated levels of cAMP (dysregulation of which is known to alter PKG signalling).

We show that pre-treatment with a PKA inhibitor is able to largely rescue this phenotype.

We demonstrate that a cAMP-specific PDE is phosphorylated following A23187 treatment (i.e. Ca2+ flux)

We show that this cAMP specific PDE plays a role in egress.

While the latter PDE may not be directly regulated by CDPK3, these findings suggest that there are likely several Ca2+-dependent kinases that contribute to this feedback loop.

We also firmly disagree with the reviewer’s assertion that without phosphosite characterisation, we have no support for our model. Following treatment with A23187 (and BIPPO), we clearly show broad, systemic changes (both CDPK3 dependent and independent) across signalling pathways previously deemed to sit upstream of calcium flux. Given the vast number of proteins involved in these signalling pathways, and the multitude of differentially regulated phosphosites identified on each of them, it is highly likely that the signalling effects we observe are combinatorial. Accordingly, we believe that mutating individual sites on individual proteins would be a very costly endeavour which is unlikely to substantially advance our understanding of signalling during egress. Moreover, introducing multiple point mutations in a given protein to ablate phosphorylation may lead to protein misfolding and would therefore not be informative. One of the key aims of this study was to assess how egress signalling pathways are interconnected, and we believe we have been able to show strong support for a Ca2+-driven feedback mechanism in which both CDPK3 and PDE2 play a role through the regulation of cAMP.

While we agree with the reviewer’s statement that a large body of work preceding this manuscript has indicated the crosstalk of cAMP, cGMP, calcium and lipid signalling cascades, a feedback loop has not previously been shown. We believe that this finding is absolutely central to facilitate the complete interpretation of existing signalling data. Furthermore, no previous studies have gone to this level of detail in either proteomics or lipidomics to analyse the calcium signal pathway in any apicomplexan parasite. We argue that the novelty in our manuscript is that it is a carefully orchestrated study that advances our understanding of the signalling network over time with subcellular precision. The kinetics of signalling is not well understood and we believe that our study is likely the first to include both proteomic and lipidomic analyses over a timecourse during the acute lytic cycle stage of the disease. In doing so, we found evidence for a feedback loop that controls the signalling network spatiotemporally, and we characterise elements of this feedback in the same study.

**Major Comments:**

Based on the findings reported here there is little doubt that BIPPO and A23187-induced signaling intersect with each other, as very much expected from previous studies. The authors selected the 50s and 15s post-treatment timing of A23187 and BIPPO, respectively for collecting phosphoproteomics samples. At these time points, which were shown to peak cytosolic Ca2+, parasites were still intracellular (Line #171). How did authors make sure to stimulate the entire signaling cascade adequately, particularly when parasites do not egress within the selected time window? There is significant variability between phosphosite intensities of replicates (Line #186), which may also be attributed to insufficient triggers for the egress across independent experiments. This work must be supported by in vitro egress assays with the chosen incubation periods of BIPPO and ionophore treatment (show the induced % egress of tachyzoites in the 50s and 15s).

Response:

1. We appreciate that the reviewer acknowledges that our data clearly shows that BIPPO and A23187-induced signalling intersect. While this may have been expected from previous studies, this has not previously been shown - and is therefore valuable to the field. Specifically, the fact that A23187-treatment leads to phosphorylation of targets normally deemed to sit upstream of calcium release is entirely novel and adds a substantial layer of information to our understanding of how these signalling pathways work together.

Treatments were purposely selected to align pathways to a point where calcium levels peak just prior to calcium reuptake. At these chosen timepoints, we clearly show that overall signalling correlation is very high. We know from our egress assays using identical treatment concentrations (Fig. 2C), that the stimulations used are sufficient to result in complete egress. We are simply comparing signalling pathways at points prior to egress.

As mentioned in point 2, we show convincingly that the treatments used are sufficient to trigger complete egress. As detailed clearly in the text, we believe that these variations in intensities between replicates are due to slight differences in timing between experiments (this is inevitable given the very rapid progression of signalling, and the difficulty of replicating exact sub-minute treatment timings). We demonstrate that the reporter intensities associated with DR sites correlate well across replicates (Supp Fig. 3C), suggesting that despite some replicate variability, the overall trends across replicates is very much consistent. This allows us to confidently average scores to provide values that are representative of a site’s phosphorylation state at the timepoint of interest.

The reviewer’s suggestion that we should demonstrate % egress at the 50s and 15s treatment timepoints is obsolete - we state clearly in the text that parasites have not egressed at these timepoints. Our egress assays (Fig. 2C) further support this.

The authors discuss that CDPK3 controls the cAMP level and PKA through activation of one or more yet-to-be-identified PDEs(s). cAMP could probably also be regulated by an adenylate cyclase, ACbeta that was found to have CDPK3-dependent phosphorylation sites. If CDPK3 is indeed a regulator of cAMP through the activation of PDEs or ACbeta, it would be expected that the deletion of CDPK3 would perturb the cAMP level, resulting in dysregulation of PKAc1 subunit, which in turn would dysregulate cGMP-specific PDEs (PMID: 29030485) and thereby PKG. All these connections need to explain in a more clear manner with experimental support (what is positive and what is negatively regulated by C____DPK3).

Response:

1. We do not firmly state that CDPK3 regulates cAMP by phosphorylation of a PDE - this is one of the possibilities addressed. We acknowledge the possibility that this could also be via the adenylate cyclase (see line 792).

PMID: 29030485 demonstrates clearly a link between cAMP signalling and PKG signalling, but does not demonstrate how this is mediated. The authors postulate that a cGMP-specific PDE is dysregulated given their observation that PDE2 is differentially phosphorylated in a constitutively inactive PKA mutant, however this was not validated experimentally. We and others (Moss et al., 2022), however, demonstrate that PDE2 is cAMP-specific. This suggests that the model built by PMID: 29030485 requires revisiting. We acknowledge clearly in the text that Jia et al. have shown a link between cAMP and PKG signalling, and hypothesise that CDPK3’s modulation of cAMP levels may affect this (this is in keeping with our phosphoproteomic data).

Moreover, the egress defect is not due to a low influx of calcium in the cytosol because when the ionophore A23187 was added to the CDPK3 mutant, its phenotype was not recovered. Rather, the defect may be due to the low or null activity of PKG that would activate PI4K to generate IP3 and DAG. The latter would be used as a substrate by DGK to generate PA that is involved in the secretion of micronemes and Toxoplasma egress. In this context, authors should evaluate the role of CDPK3 in the secretion of micronemes that is directly related to the egress of the parasite.

1. We agree with the reviewer on their point about calcium influx, and have already acknowledged in the text that the feedback loop does not control release of Ca2+ from internal stores as disruption of CDPK3 does not lead to a delay in Ca2+

We agree, and clearly address in the text, that the egress defect could be due to altered PKG/phospholipid pathway signalling.

(Lourido, Tang and David Sibley, 2012; McCoy et al., 2012) have both previously shown that microneme secretion is regulated by CDPK3. We therefore do not deem it necessary to repeat this experiment, but have made clearer mention of their findings in our writing.

When the Dcdpk3 mutant with BIPPO treatment was evaluated, it was observed that the parasite recovered the egress phenotype. It is concluded that CDPK3 could probably regulate the activity of cGMP-specific PDEs. CDPK3 could (in)activate them, or it could act on other proteins indirectly regulating the activity of these PDEs. Upon inactivation of PDEs, an increase in the cGMP level would activate PKG, which will, in turn, promote egress. From the data, it is not clear whether any phosphorylation by CDPK3 would activate or inactivate PDEs, and if so, then how (directly or indirectly). To reach unambiguous interpretation, authors should perform additional assays.

Response:

As mentioned previously, given the abundance of differentially regulated phosphosites, we do not believe that mutating individual sites on individual proteins is a worthwhile or realistic pursuit.

We clearly show systematic A23187-mediated phosphorylation of key signalling components in the PKA/PKG/PI-PLC/phospholipid signalling cascade, and demonstrate that several of these are CDPK3-dependent. We demonstrate that CDPK3 alters cAMP levels (and that the ∆CDPK3 egress delay in A23187 treated parasites is largely rescued following pre-treatment with a PKA inhibitor). We similarly demonstrate that A23187 treatment leads to phosphorylation of numerous PDEs, including the cAMP specific PDE2, and show that PDE2 knockout parasites show an egress delay following A23187 treatment. While PDE2 may not be directly regulated by CDPK3 (suggesting other Ca2+ kinases are also involved), these findings collectively demonstrate the existence of a calcium-regulated feedback loop, in which CDPK3 and PDE2 play a role (by regulating cAMP).

We acknowledge that we have not untangled every element of this feedback loop, and do not believe that it would be realistic to do so in a single study given the number of sites phosphorylated and pathways involved. We do believe, however, that we have shown clearly that the feedback loop exists - this in itself is entirely novel, and of significant importance to the field.

On a similar note, a possible experiment that can be done to improve the work would be to treat the CDPK3 mutant with BIPPO in conjunction with a calcium chelator (BAPTA-AM) to reveal, which proteins are phosphorylated prior to activation of the calcium-mediated cascades?

Response:

We agree that this would be an interesting experiment to carry out but would involve significant work. This could be pursued in another paper or project but is beyond the scope of this work.

The manuscript claims that PDE1, PDE7, PDE9 are cGMP specific, and BIPPO inhibits only cGMP-specific PDEs. All assays are performed with 1-10 micromolar cAMP and cGMP for 1h. There is no data showing the time, protein and substrate dependence. Given the suboptimal enzyme assays, authors should re-do them as suggested here. (1) Repeat the pulldown assay with a higher number of parasites (50-100 million) and measure the protein concentration. (2) Set up the PDE assay with saturating amount of cAMP and cGMP, which is critical if the PDE1,7,9 have a higher Km Value for cAMP (means lower affinity) compared to cGMP. An adequate amount of substrate and protein allows the reaction to reach the Vmax. Once you have re-determined the substrate specificity (revise Fig 5D), you should retest BIPPO (Fig 5E) in the presence of cAMP and cGMP. It is very likely that you would find the same result as PDE9 and PfPDEβ (BIPPO can inhibit both cAMP and cGMP-specific PDE), as described previously

We have repeated our assay using the exact same conditions outlined by Moss et al. This involved using a similar number of parasites, a longer incubation time of 2 hours at a higher temperature (37ºC) and with a lower starting concentration of cAMP (0.1 uM). We demonstrate that we are able to recapitulate both the Moss et al. and Vo et al. (see Supp Fig. 7B). However, we noticed that these reactions were not carried out with saturating cAMP/cGMP concentrations, since all reactions had reached 100% completion at the end of the assay whereby all substrate was hydrolysed. We therefore believe that based on our original assay, as well as the new PDE1 timecourse that we have performed (Supp Fig. 7C), that PDEs 1, 7 and 9 display predominantly cGMP hydrolysing activity, with moderate cAMP hydrolysing activity.

We also repeated the BIPPO inhibition assay using the Moss et al. conditions, and still observe that the cGMP activity of PDE1 is the most potently inhibited of all 4 PDEs. We also see moderate inhibition of the cAMP activities of PDE1 and PDE9, suggesting that cAMP hydrolytic activity can also be inhibited. Interestingly, the cGMP hydrolytic activities of PDEs 7 & 9, which were previously inhibited using our original assay conditions, no longer appear to be inhibited. This is likely due to the longer incubation time, which masks the reduced activities of these two PDEs following treatment with BIPPO.

The authors did not identify any PKG substrate, which is quite surprising as cAMP signaling itself could impact cGMP. Authors should show if they were able to observe enhanced cGMP levels in BIPPO-treated sample (which is expected to stimulate cGMP-specific PDEs). The author mention their inability to measure cGMP level but have they analyzed cGMP in the positive control (BIPPO-treated parasite line)? Why have they focused only on CDPK3 mutant, whereas in their phosphoproteomic data they could see other CDPKs too? It could be that other CDPK-mediated signaling differs and need PKA/PKG for activation.

In the title, the authors have mentioned that there is a positive feedback loop between calcium release, cyclic nucleotide and lipid signaling, which is quite an extrapolation as there is no clear experimental data supporting such a positive feedback loop so the author should change the title of the paper.

Response:

1. As addressed in our previous response to the reviewer, PMID: 29030485 demonstrates clearly a link between cAMP signalling and PKG signalling, but does not confirm how this is mediated. The authors surmise that a cGMP-specific PDE is dysregulated (although the PDE hypothesised to be involved has since been shown to be cAMP-specific), but are similarly unable to detect changes in cGMP levels. This suggests that their model may be incomplete.

The BIPPO treatment experiment suggested by the reviewer was already included in the original manuscript (see Fig. 4D in original manuscript, now Fig. 4E). With BIPPO treatment we are able to detect changes in cGMP levels.

We did not deem it to be within the scope of this study to study every single other CDPK. We chose to study CDPK3, as its egress phenotype was of particular interest given its partial rescue following BIPPO treatment. We reasoned that its study may lead us to identify the signalling pathway that links BIPPO and A23187 induced signalling.

As addressed in greater detail in our response to reviewer #2, the fact that the feedback loop appears to stimulate egress implies that it is positive.

**Minor Comments:**

Materials & Methods

Explanation of parameters is not clear (Line #360-367). Phosphoproteomics with A23187 (8 micromolar) treatment in CDPK3-KO and WT, for 15, 30 and 60s at 37{degree sign}C incubation with DMSO control. Simultaneously passing the DR and CDPK3 dependency thresholds: CDPK3-dependent phosphorylation

__Response: __We have modified the wording to make this clearer as per the reviewer’s suggestion.

Line #368: At which WT-A23187 timepoint did the authors identify 2408 DR-up phosphosites (15s, 30s or 60s)? Or consistently in all? It should be clarified?

__Response: __As already stated in the manuscript (see line 366 in original manuscript, now line 1047), phosphorylation sites were considered differentially regulated if at any given timepoint their log2FC surpassed the DR threshold.

A23187 treatment of the CDPK3-KO mutant significantly increased the cAMP levels at 5 sec post-treatment, but BIPPO did not show any change. The authors concluded that BIPPO presumably does not inhibit cAMP-specific PDEs. However, the dual-specific PDEs are known to be inhibited by BIPPO, as shown recently (____https://www.biorxiv.org/content/10.1101/2021.09.21.461320v1____). Authors do confirm that BIPPO-treatment can inhibit hydrolytic activity of PfPDEbeta for cAMP as well as cGMP (Line #612). Besides, it was shown in Fig 5E that BIPPO can partially though not significantly block cAMP-specific PDE2. The statements and data conflict each other under different subtitles and need to be reconciled. Elevation of basal cAMP level in the CDPK3 mutant indicates the perturbation of cAMP signaling, however BIPPO data requires additional supportive experiments to conclude its relation with cAMP or dual-specific PDE.

Response:

1. The manuscript to which the reviewer refers does not use BIPPO in any of their experiments. They show that continuous treatment with zaprinast blocks parasite growth in a plaque assay, but do not test whether zaprinast specifically blocks the activity of any of the PDEs.

Having repeated the PDE assay using the Moss et al. conditions (as outlined above), we are now able to recapitulate their findings, showing that PDEs 1, 7 and 9 can display dual hydrolytic activity while PDE2 is cAMP specific. As explained further above, we believe that our original set of experiments are more stringent than the Moss *et al. * To confirm this, we also performed an additional experiment, incubating PDE1 for varying amounts of time using our original conditions (1 uM cAMP or 10 uM cGMP, at room temperature). This revealed that PDE1 is much more efficient at hydrolysing cGMP, and only begins to display cAMP hydrolysing capacity after 4 hours of incubation.

We also measured the inhibitory capacity of BIPPO on the PDEs using the Moss *et al. * During the longer incubation time, it seems that BIPPO is unable to inhibit PDEs 7 and 9, while with the more stringent conditions it was able to inhibit both PDEs. We reasoned that since BIPPO is unable to inhibit these PDEs fully, the residual activity over the longer incubation period would compensate for the inhibition, eventually leading to 100% hydrolysis of the cNMPs. We also see that while the cGMP hydrolysing capacity of PDE1 is completely inhibited, its cAMP hydrolysing capacity is only partially inhibited. These findings and the fact that PDE2 is not inhibited by BIPPO are in line with our experiments where we measured [cAMP] and showed that treatment with BIPPO did not lead to alterations in [cAMP].

The method used to determine the substrate specificity of PDE 1,2,7 and 9 resulted in the hydrolytic activity of PDE2 towards cAMP, while the remaining 3 were determined as cGMP-specific. However, PDE1 and PDE9 have been reported as being dual-specific (Moss et al, 2021; Vo et al, 2020), which questions the reliability of the preferred method to characterize substrate specificity by the authors. It is also suggested to use another ELISA-based kit to double check the results.

Response:

As outlined above, we have repeated the assay using the conditions described by Moss et al. (lower starting concentrations of cAMP, 2 hour incubation period at 37ºC) and find that we are able to recapitulate the results of both Moss et al. and Vo et al.. However, using the Moss et al. conditions, the PDEs have hydrolysed 100% of the cyclic nucleotide, suggesting that these conditions are less stringent than the ones we used originally using higher starting concentrations of cAMP and incubating for 1 hour only at room temperature. With enzymatic assays it is always important to perform them at saturating conditions (as already suggested by the reviewer) and therefore we believe that our original conditions are more stringent than the results using the Moss et al. conditions.

Line #607-608: Authors found PDE9 less sensitive to BIPPO-treatment and concluded PDE2 as refractory to BIPPO inhibition; however, the reduction level of activity seems similar as seen in PDE9-BIPPO treated sample? This strong statement should be replaced with a mild explanation.

__Response: __We have tempered our wording as per the reviewer’s suggestion

Figures and legends:

The introductory model in Fig S1 is difficult to understand and ambiguous despite having it discussed in the text. For example, CDPK1 is placed, but only mentioned at the beginning, and the role of other CDPKs is not clear. In addition, the arrows in IP3 and PKG are confusing. The location of guanylate and adenylate cyclase is wrong, and so on... The figure should include only the egress-related signaling components to curate it. The illustration of host cell in orange color must be at the right side of the figure in connection with the apical pole of the parasite (not on the top). Figure legend should also be rearranged accordingly and citations of the underlying components should be included (see below).

__Response: __We have modified Supp Fig. 1 as per the suggestions of reviewer#2 and #3. We have now modified the localisations of the proteins and have also removed the lines showing the cross talk between pathways. We have also highlighted to the reader that this is only a model and may not represent the true localisations of the proteins, despite our best efforts.

In Figure 5D, would you please provide the western blot analysis of samples before and after pulling down to demonstrate the success of your immunoprecipitation assay. Mention the protein concentration in your PDE enzyme assay. Please refer to the M&M comments above to re-do the enzyme assays.

Response:

We have now included western blots for the pull downs of PDEs 1, 2, 7 and 9 (Supp Fig. 7A). We chose not to measure protein concentrations of samples since all experiments were performed using the same starting parasite numbers, and we do not see large differences in activities between biological replicates of the PDEs.

Figure legend 1C: Line #194: There is no red-dotted line shown in graph! Correct it!

__Response: __We have modified this.

Figure 4Gi-ii: Shouldn't it be labelled i: H89-treatment and ii: A23178, respectively instead of DMSO and H89? (based on the text Line #579).

__Response: __Our labelling of Fig. 4Gi-ii is correct as panel i parasites were pre-treated with DMSO, while panel ii parasites were pre-treated with H89. Subsequent egress assays on both parasites were then performed using A23187.

We have modified the figures to include mention of A23187 on the X axis, and modified the figure legend to clarify pre-treatment was performed with DMSO and H89 respectively.

Bibliography:

Line #57 and 58: Citations must be selected properly! Carruthers and Sibley 1999 revealed the impact of Ca2+ on the microneme secretion within the context of host cell attachment and invasion, not egress as indicated in the manuscript! Similar case is also valid for the reference Wiersma et al 2004; since the roles of cyclic nucleotides were suggested for motility and invasion. Also notable in the fact that several citations describing the localization, regulation and physiological importance of cAMP and cGMP signaling mediators (PMID: 30449726 , 31235476 , 30992368 , 32191852 , 25555060 , 29030485 ) are either completely omitted or not appropriately cited in the introduction and discussion sections.

Response:

We have modified the citations as per the reviewer’s suggestions. We now cite Endo et al., 1987 for the first use of A23187 as an egress trigger, and Lourido, Tang and David Sibley, 2012 for the role of cGMP signalling in egress. We also cite all the GC papers when we make first mention of the GC. We have also removed the Howard et al., 2015 citation (PMID: 25555060) when referring to the fact that BIPPO/zaprinast can rescue the egress delay of ∆CDPK3 parasites.

Grammar/Language

Line #31: After "cAMP levels" use comma

Response:

We have modified this.

36: Sentence is not clear. Does conditional deletion of all four PDEs support their important roles? If so, the role in egress of the parasite?

Response:

We have clarified our wording as per the reviewer’s suggestion. We state that PDEs 1 and 2 display an important role in growth since deletion of either these PDEs leads to reduced plaque growth. We have not investigated exactly what stage of the lytic cycle this is.

40: "is a group involving" instead of "are"

Response:

We found no mention of “a group involving” in our original manuscript at line 40 or anywhere else in the manuscript, so we are unsure what the reviewer is referring to.

108: isn't it "discharge of Ca++ from organelle stores to cytosol"?

__Response: __We thank the reviewer for spotting this error. We have now modified this sentence.

120: "was" instead of "were"

__Response: __Since the situation we are referencing is hypothetical, then ‘were’ is the correct tense.

Reviewer #3 (Significance (Required)):

There is a significant amount of work that underlies this manuscript; however, from a conceptual viewpoint, the manuscript does not offer significant advancement over the current knowledge without functional validation of phosphoproteomics data. In terms of the mechanism, it is not clear whether and how lipid turnover and cAMP-PKA signaling control the egress phenotype (lack of a validated model at the end of this study).In a methodical sense, the work uses established assays, some of which require revisiting to reach robust conclusions and avoid misinterpretation.

Compare to existing published knowledge

A large body of work preceding this manuscript has indicated the crosstalk of cAMP, cGMP, calcium and lipid signaling cascades. This work provides a further refinement of the existing model. The article is quite interesting from a throughput screening point of view, but it clearly lacks the appropriate endorsement of the hits.

Response:

Please refer to our first response to reviewer #3 for our full rebuttal to these points. We respectfully disagree with the assessment that the work presented does not advance current knowledge.

Audience

Field specific (Apicomplexan Parasitology)

Expertise

Molecular Parasitology

References

Bailey, A. P. et al. (2015) ‘Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila’, Cell. The Authors, 163(2), pp. 340–353. doi: 10.1016/j.cell.2015.09.020.

Bullen, H. E. et al. (2016) ‘Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma Article Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma’, Cell Host & Microbe, pp. 349–360. doi: 10.1016/j.chom.2016.02.006.

Dass, S. et al. (2021) ‘Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage’, Nature Communications. Springer US, 12(1). doi: 10.1038/s41467-021-22956-w.

Endo, T. et al. (1987) ‘Effects of Extracellular Potassium on Acid Release and Motility Initiation in Toxoplasma gondii’, The Journal of Protozoology, 34(3), pp. 291–295. doi: 10.1111/j.1550-7408.1987.tb03177.x.

Flueck, C. et al. (2019) Phosphodiesterase beta is the master regulator of camp signalling during malaria parasite invasion, PLoS Biology. doi: 10.1371/journal.pbio.3000154.

Howard, B. L. et al. (2015) ‘Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites’, ACS Chemical Biology, 10(4), pp. 1145–1154. doi: 10.1021/cb501004q.

Jia, Y. et al. (2017) ‘ Crosstalk between PKA and PKG controls pH ‐dependent host cell egress of Toxoplasma gondii ’, The EMBO Journal, 36(21), pp. 3250–3267. doi: 10.15252/embj.201796794.

Katris, N. J. et al. (2020) ‘Rapid kinetics of lipid second messengers controlled by a cGMP signalling network coordinates apical complex functions in Toxoplasma tachyzoites’, bioRxiv. doi: 10.1101/2020.06.19.160341.

Lentini, J. M. et al. (2020) ‘DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification’, Nature Communications. Springer US, 11(1). doi: 10.1038/s41467-020-16321-6.

Lourido, S., Tang, K. and David Sibley, L. (2012) ‘Distinct signalling pathways control Toxoplasma egress and host-cell invasion’, EMBO Journal. Nature Publishing Group, 31(24), pp. 4524–4534. doi: 10.1038/emboj.2012.299.

Lunghi, M. et al. (2022) ‘Pantothenate biosynthesis is critical for chronic infection by the neurotropic parasite Toxoplasma gondii’, Nature Communications. Springer US, 13(1). doi: 10.1038/s41467-022-27996-4.

McCoy, J. M. et al. (2012) ‘TgCDPK3 Regulates Calcium-Dependent Egress of Toxoplasma gondii from Host Cells’, PLoS Pathogens, 8(12). doi: 10.1371/journal.ppat.1003066.

Moss, W. J. et al. (2022) ‘Functional Analysis of the Expanded Phosphodiesterase Gene Family in Toxoplasma gondii Tachyzoites’, mSphere. American Society for Microbiology, 7(1). doi: 10.1128/msphere.00793-21.

Stewart, R. J. et al. (2017) ‘Analysis of Ca2+ mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules’, Cellular Microbiology, 19(4). doi: 10.1111/cmi.12685.

Vo, K. C. et al. (2020) ‘The protozoan parasite Toxoplasma gondii encodes a gamut of phosphodiesterases during its lytic cycle in human cells’, Computational and Structural Biotechnology Journal. The Author(s), 18, pp. 3861–3876. doi: 10.1016/j.csbj.2020.11.024.