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Referee #2

Evidence, reproducibility and clarity

• Are the key conclusions convincing?

The following conclusions were not warranted by the findings: Endoderm emergence can occur in the absence of extraembryonic tissues and embryonic architecture: It is unclear if extraembryonic tissues (e.g., primitive endoderm and visceral endoderm-like cells) are absent in the early phase of gastruloid development. While the gastruloid does not replicate the morphological feature of the post-gastrula embryo, it nevertheless has a certain degree of tissue organization. Perhaps the emergence of DE-like cells in 2-D culture would be a more convincing model for "the absence of extraembryonic tissues and embryonic architecture".<br> The FoxA2+/Sox17+ endoderm progenitors never transitioning through the mesenchymal intermediates and never leaving the epithelial compartment that they arise: In view of that the stereotypic morphogenetic activity was not documented during the development of the gastruloid, it is not possible to exclude the possibility of the progenitors undergo a partial EMT (loss of epithelial feature and cellular polarity and display of morphogenetic movement, as in vivo) in the transition from progenitor to the epithelial endoderm cells. The DE-like cells when first discerned in the gastruloid are apparently epithelialized. In the absence of lineage tracing results, It is not clear whether they are still residing in the "epithelial compartment that they arise".

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

The mature endoderm cells are patterned segmentally in the gastruloid. The findings that the molecular phenotype (marker expression) of the mature endoderm cells "aligns with (cellular) identities along the entire length of the embryonic gut tube" are not sufficient evidence of spatial A-P patterning of endoderm cells. Only the spatial regionalization of Pax6-expressing cells (Fig. 8) and Cdx2-expressing cells (Fig 4C) were shown on different gastruloid specimens. The expression pattern of Foxa2/Cdh1 (Fig 5d) was not informative of tissue patterning. It is also unclear if a structure reminiscent of the embryonic gut (closed or partly open) was formed (or self-organised) in the gastruloid. Whether endoderm cells are patterned or not is, however, irrelevant for the understanding of the mode of endoderm formation, unless the timing and the mechanism of allocation of endoderm cells of specific segmental property has been studied in the gastruloid.

• 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.

Specific points:

1. The information regarding the spatial localization of specific germ layer markers in the gastruloids at different timepoints would be important to understand how the morphology progresses and how it is comparable to the developing embryo itself. How is the organisation of the mesoderm and endoderm layers in comparison to embryo in the early timepoints and later timepoints of gastruloids?
2. Clarify if Foxa2 and Sox17 double positive cells exist in the Cdh1 patches (Fig 3a). In Fig 4, authors have demonstrated the development of epithelial primordium with overlaying mesodermal wings, however it is important to show if Foxa2, Sox17, or other definitive endoderm markers co-express in these cells.
3. It was suggested that E-Cadherin is maintained during endoderm differentiation. N-cadherin expression may be examined to determine if N-cad is expressed in the other region of gastruloids.
4. In Fig 6, FACS quantification is not proportional to the expression of the TBra:GFP as shown in the microscopic images at 96 hr, 120hr. Fig 6D does not show the TBra:GFP positive cells on the y -axis in the top-left quadrant, even though it is quite visible in microscopy - at 96, 120 hr. Microscopic images suggest TBra signal is almost completely lost at 128hr whereas FACS does not represent that. Infact, at 120 hours, the plot shows opposite of what microscopy shows.
5. Gastruloids were sampled at 96-168 hours for single cell transcriptome analysis. However, the specimens documented in this study were those only up to 144 hours. How does the gastruloid morphology look at 168 hours? It is essential to show the morphology and characterise the further development from 144 to 168 hours, to compare the single cell RNA seq data with the morphology of the gastruloid.
6. In Fig 7, it is surprising to see that the proportion of cells in the two clusters 13, 4 that mark endoderm are a minor portion of the whole dataset collected, whereas the microscopic images suggest that the majority of the gastruloid structure from 120hr onwards is marked by Foxa2 and shows the epithelial primordium morphology as claimed.
7. The single-cell RNA-seg data should be analysed for the co-expression of multiple segment-specific cell markers to ensure that the mature endoderm cells align with high-confidence with the known cell types in different segments of the embryonic gut, and that the localization of representative cell types can be validated spatially along an endoderm structure in single gastruloids.

• 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 suggested experiments may be accomplished in a few months.

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

Yes for the methods. It is not clearly indicated how many replicates were performed to assure consistency/reproductivity of the gastruloid results.

• Are the experiments adequately replicated and statistical analysis adequate?

Statistical results were not provided for most of the immunostaining experiments, either in the main text or in the figure legends.

Minor comments:

• Specific experimental issues that are easily addressable.

Majority of the images presented in the manuscript are shown as Maximum Intensity Projections, and it is not clearly stated if the localisation of the cells expressing specific protein markers are present on the surface or in the internal layers of the gastruloids. Optical slices of the gastruloid images may be presented as supplementary information.

• Are prior studies referenced appropriately?

Yes, except for the study on endoderm formation by lineage tracing in vivo, high-resolution single-cell analytics and functional analysis of genetic mutant embryos.

• Are the text and figures clear and accurate?

Results may be presented with reference to the data figures in the appropriate sequential order, or the figures may be re-organised to match the presentation in the Results.

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

While taking into consideration the limitation of this embryo model for studying morphogenesis, highlight the interesting/unique findings of the gastruloid study, albeit they may have been discovered in the study of embryos in vivo.

Reduce the verbosity throughout the manuscript, especially the Results and Discussion.

Significance

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

This study demonstrated that definitive endoderm (DE)-like cells can be generated in the stem cell-derived embryo-like structure, the gastruloid. This observation is significant for that gastruloids can serve as an amenable experimental model, in comparison to other 2D invitro differentiation models, for elucidating the requisite cellular process in endoderm differentiation and the acquisition of cell identity. It was inferred that, in the gastruloid, epithelial-mesenchyme transition may not be a requisite cellular process for the formation of the DE-like cells and that endoderm formation may not involve progression through an intermediate mesenchymal state. The progenitor cells may have acquired and retained the attribute of epithelization during differentiation and organization the DE-like cells into an endoderm layer.<br> This study on endoderm development, however, is confounded by the inherent limitation of the experimental model: lack of extraembryonic tissue components, the atypical morphological structure and the deviation from the in vivo schedule of development and morphogenesis. This may raise doubt of the relevance of the findings to the understanding of the morphogenetic activity and molecular control of endoderm formation during gastrulation in the embryo.

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

Knowledge gleaned from the present study on the gastruloid study added little to that of a recent study of the morphogenetic program of endoderm formation in the mouse embryo and the ESC differentiation model (https://doi.org/10.1038/s41556-021-00694-x ) . This recent study has advanced the understanding of the functional attributes that segregate the endoderm progenitors and mesoderm progenitors in the primitive streak and the posterior epiblast, and has characterised the role of epithelial plasticity and the modulation of EMT activity under the control of Forkhead box transcription factor A2 and modulation of WNT signalling in the formation of the definitive endoderm.

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

Developmental biologists, stem cell scientists and embryo modellers

• 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.

Mouse embryogenesis, gastrulation, in vitro stem cell differentiation, advanced microscopy, single cell transcriptomics.

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Referee #1

Evidence, reproducibility and clarity

The study adresses a long-standing question in mouse gastrulation: the existence of a mesendodermal progenitor, similar to other species. Recent data in the mouse point towards a direct transition from epiblast to endoderm without going through a bipotent progenitor, and without loss of epithelial characteristics (notably Probst 2021). The authors take advantage of the gastruloid system to follow the emergence of endoderm cells. Through co-staining with markers of various germ layers at different timepoints, live imaging, and reanalysis of single cell RNASeq data, they propose a model in which endoderm cells differentiate from E-cadherin positive cells without going through a bipotent stage. Interestingly, they then organise a rod like structure along the anterior-posterior axis of the gastruloid, that display some polarity illustrated by a marker of anterior gut.

The data present show very high reproducibility, and authors fully exploit the organoid system by analysing a large amount of samples and showing very similar results. The results are qualitatively very convincing. For the sake of clarity, it may help to provide some table illustrating the proportion of gastruloids behaving precisely as the best example shown. It would also strengthen the message even more to provide some quantitation of co-expression for the main markers. As the behaviour seems very consistent, it is likely that such quantification would not be very arduous, and it would show the strength of the model.

The manuscript is pleasantly written and all statements are clearly explained. It is a bit long though, in particular the introduction, some of which might read more like a review of the field. It is less striking in the Results part, but there is some level of repetition that is a bit distracting.

Significance

The question is important and timely, and the data are clear and convincing. There have been a number of publications addressing it in the last years, either in the embryo or in gastruloids (notably Hashmi 2021). All data appear to point in the same direction, and this study is certainly an important contribution. An original aspect to my knowledge is the self organisation of the central rod. The system is simple, reproducible, and opens novel possibilities to dispose of a large number of cells to explore the emergence of endoderm subpopulations.

The fact that several studies converge is rather an advantage as they all have specificities, and I believe they are adequately cited here.

In summary this is an important and well conducted study that may just benefit from some additional quantification to prove robustness. In terms of writing, it is pleasant and quite literary, but perhaps a little bit too much so. The introduction could be shortened, and the results a bit more to the point.

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Reply to the reviewers

Reply to the Reviewers

We would like to thank the reviewers for their thoughtful comments and efforts towards improving our manuscript. Based on the reports, we have revised the manuscript entitled “Bi-phasic effect of gelatin in myogenesis and skeletal muscle r__egeneration__ (RC-2021-00854 )”. We have addressed all the concerns, revised the text and figures. Modified parts are in red, and line numbers are tracked in this response letter. Our detailed point-by-point responses to the reviewers’ comments are listed below. We believe that these changes strengthen the manuscript and are grateful to the reviewers for all of their suggestions.

Point-by-point description of the revisions

R__eviewer #2 (Evidence, reproducibility and clarity (Required)):__

\*Summary:***

The manuscript by Xiao Ling Liu and colleagues titled "Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration" deals with the effect of gelatin on differentiation of myoblast cell line, in vitro, and on skeletal muscle regeneration upon muscle injury, in vivo. In vivo, the gelatin is a product of collagen breakdown associated with skeletal muscle regeneration upon acute or chronic muscle damage.

Specifically, the authors define a dose-dependent effect of gelatin, beneficial at low dose and detrimental at high dose. This effect is mediated by the level of ROS accumulation leading to the induction of different cytokines with opposite effects on skeletal muscle regeneration.

**Major comments:**

The experimental purpose is well tackled from both biochemical and functional point of view, and the proposed experiments are quite exhaustive.

Response: We are grateful for the encouraging comments from this reviewer.

However, I would suggest some additional experimental analyses to improve the robustness and quality of the study, as well as text and figure editing, as reported below.

Regarding the additional experiments/analyses/images:

1. • Figure 5: I would suggest to add an image of C2C12 cells in GM (growth medium), as representative images of proliferation analysis upon LCG/HCG/NAC treatment.* Response: We appreciate this suggestion by the reviewer. New images of C2C12 cells upon LCG/HCG/NAC treatment have now been added as in Supplementary Figure 4B. The new results are described in main text Lines 237-239.

• Figure 5: I would suggest to repeat the main si-NOX2 experiments with an alternative siRNA to rule out off target effects.*

Response: We thank the reviewer for this suggestion. We have now added a new siRNA targeting NOX2 with an independent sequence and shown the results in Figure 5F-J and Supplementary Figure 4H-J. The sequences of si-NOX2 and si-NC are shown in Materials and Methods Lines 681-687. The newly added siRNAs confirmed results with the previous siRNA, suggesting unlikely off-target effects. This result is described in the main text Lines 246-256.

• In vivo experiments could be improved by adding DHE or DCFH staining on muscle TA cryosections to quantify the level of oxidative stress.*

Response: We appreciate this suggestion by the reviewer. We have now stained DCFH-DA in TA cryosections to quantify oxidative status in situ and show the results in Supplementary Figure 8A-B. Indeed, low- and high-dose gelatin injections both triggered ROS production, and high-dose injection resulted in a high accumulation of ROS. The new result is described in the main text Lines 318-321.

• The proposed model could be better tackled by additional in vivo treatment with Ab anti IL-6 or anti TNFalpha in combination with CTX and LCG or HCG, followed by H/E staining at 14 dpi.*

Response: We appreciate this suggestion by the reviewer. We have now added in vivo treatment with IL-6 or TNFα neutralizing antibody (Ab) in combination with CTX and LCG or HCG. The procedure is illustrated in Figure 8J and described in main text Lines 522-525. H&E staining showed that anti-IL-6 Ab injection significantly reduced the beneficial effect of LCG, but had no effect on HCG-treated mice. By contrast, anti-TNFα Ab injection significantly suppressed infiltration of macrophages into the injury site upon HCG, reversed the deleterious effect of HCG on muscle repair, resulting in myofibers with higher CSA and more myofibers with central nuclei. The new results are described in Figure 8J-K and main text Lines 331-346.

**Minor comments:**

1. Each acronym should be indicated in full in the main text at the first mention (for instance BHP, NAC and others). Moreover, I would suggest to add an acronym list for reagents and factors Response: We thank the reviewer for this suggestion and have now added an acronym list in Material and Methods, Lines 466-504.

Experimental methods should be better detailed; for instance I would suggest:

1. • Add a detailed description of the quantification of differentiation indexes* Response: We thank the reviewer for this suggestion. We have now added a detailed description on the quantification method of myogenesis and differentiation to Material and Methods, Lines 577-582.

• Explain how cell growth (OD 450nm) and optical density (570nm) assays have been performed*.

Response: We thank the reviewer for pointing this out. The cell growth was examined by measuring dehydrogenase activities that generate a soluble formazan dye, whose OD 450nm value was measured as a proportional value to the number of viable cells in the sample (CCK-8 kits according to manufacturer’s instructions).

The transwell cell migration ratio was determined by measuring the optical density of crystal violet staining (570 nm) of migrated cells on the bottom of the transwell filters (transwell filters have 8 μm pores to allow cells to pass through). The non-migrated cells on the top of the transwell filter were scraped away.

Detailed descriptions have now been added to Material and Methods, Lines 593-609.

• Explain how ROS species and antioxidant enzymes have been measured (Fig 4C and 4D)*

Response: We thank the reviewer for pointing this out. The levels of ROS species (O2.- , OH· and H2O2) and antioxidant enzymes in Figure 4C and 4D were examined using commercial kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, cells were lysed in RIPA lysis buffer and protein concentrations were determined using BCA method. The O2.- level and SOD activity were measured by adding electron transfer substances that reduce azo blue tetrazole to blue methionine. The activity of SOD was evaluated by the absorption of methionine. GSH-PX facilitates the reaction between H2O2 and GSH to produce H2O and oxidized glutathione (GSSG). The activity of GSH-PX can thus be obtained by measuring the consumption of GSH in this enzymatic reaction. OH· was measured using Fenton reaction. The level of H2O2 was determined according to the reaction with molybdic acid. We have now added the corresponding information to Figure. 4 legend and main text Lines 1066-1067. Detailed protocols can be found in Material and Methods, Lines 704-714.

Figures and figure legends:

1. Please add in the figures the figure number in order to facilitate the reading of the pdf file Response: We thank the reviewer for this suggestion and have added figure numbers to the PDF file.

The sequence of the panels should be coherent with the alphabet and reading left to right and up to down

Response: Yes, we have rearranged the figure layouts to be coherent.

• In the Figure 1, I would suggest to add the whole TA sections for H/E staining in order to appreciate the overall beneficial or detrimental effect of LCG and HCG, respectively.*

Response: We thank the reviewer for this suggestion and have added the whole-section view of TA with H&E staining as in Supplementary Figure 1C. The results are described in the main text Lines 132-136.

• I would suggest to show Supplementary figures 1C and 1D in the Figure 1*.

Response: We thank the reviewer for this suggestion and have now moved Supplementary Figures 1C and 1D to Figure 1F and 1G.

In the Supplementary Fig 2A, I would suggest to the authors to show and comment only the data about proliferation: the earlier orientation, fusion and differentiation of C2C12 exposed to LCG are a consequence of the positive effect of LCG on proliferation

Response: We thank the reviewer for this suggestion and have removed data and comments except for proliferation of C2C12 in Supplementary Figure 2A.

• The quality of representative images of western blot is not always high: the bands are fuse and, consequently, the quantification is not reliable. For instance Fig S4 B, S4 G; in Fig 2 the representative image does not really represent the reported quantification.*

Response: We thank the reviewer for this suggestion and have replaced western blot images in Supplementary Figure 4B, 4G and Figure 2.

• Please specify in the figure legends the meaning of the acronym in the axis title (for instance RFU, MFI or DCF) and in the axis title the unit of measure (for instance Count (?)).*

Response: We thank the reviewer for this suggestion and have spelt out “RFU” as “Relative fluorescence intensity”, changed “MFI” to “Relative fluorescence intensity of NOX2” in Figure 4H and Figure 8H. The unit of measurement is the fluorescence intensity. The related modifications have been described in the legend of Figure 4H and Figure 8H, Lines 1071, 1125-1126. We have now included “DCF: 2',7'-dichlorofluorescein” in the acronym list in Material and Methods, Line 473. DCF positive ratio is now used to reflect ROS level in the population. We have explained “Count” in the legend as “Cell counts”.

The authors always wrote "filed" in place of "field"**.

Response: We apologize for this typo and have corrected it with others throughout the text.

• In the figures 7 and 8 the letters for densitometry panel are missed.*

Response: We could not identify those missing labels and letters in the densitometry panel. We suspect this issue could be from the soft wares opening the documents.

• In the figure legend 8 the panel letters do not match the panels in the figure*.

Response: We thank the reviewer for pointing this mistake out and have corrected Figure legend 8.

• Figure 3I: replace "nucleis" with "nuclei"*.

Response: We thank the reviewer for pointing out this mistake and have modified Figure 3I.

Text editing:

1. • Line 64: Satellite cells (SCs) would be more appropriate than myoblasts* Response: We thank the reviewer’s suggestion and have replaced myoblasts with satellite cells in the main text Line 60.

• Line 64: Please define more carefully the location of SCs*

Response: We thank the reviewer for this suggestion. SCs are underneath the basal laminin and myofiber plasma membrane in the resting skeletal muscle. The results are described in the main text Lines 60-65.

• Line 67: MyoD+/Myog+ would be more appropriate than Pax7+/Myog+*

Response: We thank the reviewer’s suggestion and have changed pax7+/MyoG+ into MyoD+/MyoG+ in the main text Line 64.

• Line 67: (Pax7+)/Myog+ "myocytes" in place of myotubes ... and fuse with each other to generate myotubes*

Response: We thank the reviewer for pointing this out and have modified the sentence in Lines 64-65.

• Line 69: Please add Myf6/MRF4 to MRFs list*

Response: We thank the reviewer for pointing out and have added Myf6/MRF4 to MRFs list in the main text Line 66.

• Line 112: replace "its" with "their"*

Response: We thank the reviewer for pointing out this mistake and have replaced “its” with “their”.

• Lines 330-331: "promoted IL-6" "enhanced TNF", please insert secretion/production*

Response: We thank the reviewer’s suggestion and have inserted “production and secretion” behind IL-6 and TNFa in the main text Lines 310-312. .

• Line 352-353: this sentence is not necessary*

Response: We have deleted this sentence.

Reviewer #2 (Significance (Required)):

The study reports robust and interesting data applicable to both basic research and translational research, such as tissue engineering applications.

Response: We thank the reviewer for sharing this positive and important opinion on our work. We are delineating the biological pathways of gelatin treatments, motivated by the application of this biocompatible and industrial material for treating disease and aging-related skeletal muscular dystrophies

Keywords for field of expertise of this reviewer:

Skeletal muscle regeneration

Duchenne Muscular Dystrophy

Inflammation

Macrophages

Oxidative stress

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

**Summary**

The Review Commons submission by Liu and colleagues entitled "Biphasic effect of gelatin in myogenesis and skeletal muscle regeneration" provide a systematic in vitro and in vivo evaluation of the effects of myogenic cell exposure to low or high dose gelatin. Through these analyses they uncover pro- and anti-regenerative effects of gelatin that are dose dependent and through a series of cell and molecular studies, they attribute these dual effects to a ROS-IL6/TNFa signaling axis. The study is well designed and executed. For the most part the figures and experimental details are clear and transparent, and most of the conclusions are supported by the data. Specific comments follow.

Response: We are grateful for the reviewer’s encouraging comments.

**Major Comments**

1. • Study framing in the Introduction is mis-aligned with the research conducted. Currently the Introduction sets the stage for an in vivo exploration of the effects of endogenous gelatin produced in the course of muscle regeneration. However, there are no experiments in this paper investigating the presence or effects of endogenous gelatin.* Response: We thank the reviewer for pointing this out and apologize for the misleading parts in our abstract and introduction. The reported phenomenon of a temporary breakdown of collagen after skeletal muscle injury has inspired us to apply gelatin for achieving a pro-regenerative effect. Although it will be a very interesting biological pathway to study, no adequate research tools are available for measuring endogenous gelatin in vivo. We have re-written parts in abstract and introduction to avoid confusion, see Lines 34-35, 78-93.

1.a. The impact of the study would indeed be increased by including a systematic characterization of endogenous gelatin levels during muscle regeneration in healthy mice as compared to in those where fibrosis is prevalent. A demonstration that ROS-IL6/TNFa levels align with patterns seen in the in vitro studies, and pharmacological manipulations to 'rescue' would all provide a demonstration of a hormesis gelatin response in vivo. Meaning, is this process something that naturally occurs in the physiological context, or is it one that is possible, but only be supraphyiological gelatin injections?

Response: We resonate with the reviewer and would have loved to investigate whether what we have observed is a fundamental mechanism of the natural healing process. But we are currently limited by the lacking of adequate tools for endogenous gelatin quantification. We appreciate the reviewer’s suggestion to compare normal and aberrant repairing processes such as fibrosis and would like to explore the possibility in a separate future study.

1.b. Alternatively to 1.a., the authors should reframe the Introduction to focus on understanding the effects of gelatin as a biomaterial that is being used in regenerative medicine applications. In this case, the authors should delete/edit/reframe lines 74-102 and instead use lines 103 on to motivate the study so as to be consistent.

Response: We have rephrased sections of Abstract and Introduction to focus on gelatin as a biomaterial. Please find the changes in the main text Lines 34-35, 78-93.

• Satellite cell conclusions in Figures 2C-D that are based upon representative images provided in 2A, are questionable. Pax7 staining in mouse tissue sections is notoriously difficult and the antibodies can have dramatic lot to lot variability. The immunostaining provided in the representative images is not convincing, and hence, draws into question the conclusions based upon them.*

Response: We thank the reviewer for the suggestion and have optimized Pax7 staining based on the published protocol by Feng et al., 2018 in JoVE. The new images can be found in Figure 2A.

2.a. If the authors wish to leave the satellite cell conclusions in their study, they will need to optimize their Pax7 staining and repeat this study. They should focus on Pax7+ objects that contain a nucleus and are located below the basal lamina. Also, the word 'activation' in line 180 should be edited to 'expansion' as the histological analysis and study design preclude an evaluation of satellite cell activation.

Response: Our new results after optimizing staining protocol support that low-dose gelatin injection causes more Pax7+ cells (green) underneath the basal lamina (red) in injured TA muscle and high-dose gelatin injection suppressed the number of Pax7+ SCs at 7 D.P.I. The new results were shown in Figure 2A and described in the main text Lines 152-155.

We have changed the word “activation” into “expansion” according to reviewer’s suggestion in the main text Line 161.

2.b. Alternatively to 2.a., it would not diminish the impact of this study to remove the 'satellite cell' findings in their entirety from the manuscript.

Response: Please see above. We hope the new images are convincing to this reviewer.

• It is surprising that the molecular hallmarks of low vs high gelatin injection shown in Fig. 8 would still be present at a time point 2-weeks after the initial injection.*

Response: Please see below 3.b.

3.a. It would increase the impact of the study to better understand the basis of this surprising observation. This point links to point 1.a. as one would ideally need to quantify baseline gelatin levels pre-injury and post-injury. For example, is the injected gelatin still present 14-days after injection? Or is the MMP profile altered in a way that sustains these levels one direction or the other? Etc.

Response: Please see below 3.b.

3.b. Alternatively to 3.a., the authors should use the Discussion to note this point and speculate on the significance.

Response: We thank the reviewer for making this important point. The sustained effect of gelatin materials has been reported in several previous studies, and we now have discussed possible mechanisms such as MMP expression profiles and lasting interplays between SCs, macrophages, ECM, and myoblasts. See the main text Lines 437-459.

**Minor Comments**

1. • The authors should conduct a careful review of the manuscript to address minor typos and grammatical errors.* Response: We thank the reviewer for pointing this out and have carefully reviewed the manuscript and corrected minor typos and grammatical errors.

• It is unclear from the Figure Legends, Results, or Methods what the 'PBS' condition in Figure 1 refers to. Is this the uninjured control? If so, consider using 'Cntrl' as the label and then defining it in the figure legend for clarity.*

Response: We thank the reviewer for pointing this out. Yes, PBS/phosphate-buffered saline is the vehicle for delivering CTX thus representing the uninjured model. In the revised manuscript, we have replaced “PBS: phosphate-buffered saline” with “Ctrl” in Figures, Figure Legends, Results and Methods.

• It is unclear from the Figure Legends, Results, or Methods what is quantified to read-out the 'myogenesis index' and 'fusion index' that is reported in Fig. 5D & E. Please reconcile.*

Response: This point has been addressed in the previous section. Myotube formation was quantified using myogenesis and fusion index. Myogenesis index (% nuclei within MyHC-stained myocytes/total nuclei) and fusion index (% nuclei in myotubes with >5 nuclei/total nuclei in MyHC-stained cells) are now explained both in legends and in Materials and Methods. Please see the main text Lines 577-582.

Reviewer #3 (Significance (Required)):

The manuscript constitutes a technical advance, and offers a molecular mechanism, in support of the notion that intramuscular injection of low dose gelatin serves to expedite the process of skeletal muscle regeneration. This study has translational implications for a regenerative medicine application of this knowledge. It is my opinion that this aspect of the study is well supported by the results and requires Response: We thank the reviewer for sharing this positive and important comment. Indeed, the motivation of this work was to delineate biological pathways of gelatin treatments in myogenesis and muscle regeneration for potential therapeutic applications.

The manuscript would constitute both a technical and a conceptual advance by addressing Major Point 1a as the authors would show, for the first time, that low and high dose gelatin levels naturally exist in vivo to mediate the process of muscle endogenous repair. This would be highly significant, because as the authors rightly point out in Lines 74-76 of their manuscript "...by what mechanisms ECM regulates the functional, morphological, and molecular events of skeletal muscle regeneration remain poorly understood". It is my opinion that making this latter point would require 1-3 months of additional studies.

Response: We thank the reviewer for pointing to this important scientific direction. But regrettably, missing adequate tools for quantifying endogenous gelatin in vivo is currently prohibitive.

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Referee #2

Evidence, reproducibility and clarity

Summary

The Review Commons submission by Liu and colleagues entitled "Biphasic effect of gelatin in myogenesis and skeletal muscle regeneration" provide a systematic in vitro and in vivo evaluation of the effects of myogenic cell exposure to low or high dose gelatin. Through these analyses they uncover pro- and anti-regenerative effects of gelatin that are dose dependent and through a series of cell and molecular studies, they attribute these dual effects to a ROS-IL6/TNFa signaling axis. The study is well designed and executed. For the most part the figures and experimental details are clear and transparent, and most of the conclusions are supported by the data. Specific comments follow.

Major Comments

1. Study framing in the Introduction is mis-aligned with the research conducted. Currently the Introduction sets the stage for an in vivo exploration of the effects of endogenous gelatin produced in the course of muscle regeneration. However, there are no experiments in this paper investigating the presence or effects of endogenous gelatin.

1.a. The impact of the study would indeed be increased by including a systematic characterization of endogenous gelatin levels during muscle regeneration in healthy mice as compared to in those where fibrosis is prevalent. A demonstration that ROS-IL6/TNFa levels align with patterns seen in the in vitro studies, and pharmacological manipulations to 'rescue' would all provide a demonstration of a hormesis gelatin response in vivo. Meaning, is this process something that naturally occurs in the physiological context, or is it one that is possible, but only be supraphyiological gelatin injections?

1.b. Alternatively to 1.a., the authors should reframe the Introduction to focus on understanding the effects of gelatin as a biomaterial that is being used in regenerative medicine applications. In this case, the authors should delete/edit/reframe lines 74-102 and instead use lines 103 on to motivate the study so as to be consistent.

1. Satellite cell conclusions in Figures 2C-D that are based upon representative images provided in 2A, are questionable. Pax7 staining in mouse tissue sections is notoriously difficult and the antibodies can have dramatic lot to lot variability. The immunostaining provided in the representative images is not convincing, and hence, draws into question the conclusions based upon them.

2.a. If the authors wish to leave the satellite cell conclusions in their study, they will need to optimize their Pax7 staining and repeat this study. They should focus on Pax7+ objects that contain a nucleus and are located below the basal lamina. Also, the word 'activation' in line 180 should be edited to 'expansion' as the histological analysis and study design preclude an evaluation of satellite cell activation.

2.b. Alternatively to 2.a., it would not diminish the impact of this study to remove the 'satellite cell' findings in their entirety from the manuscript.

1. It is surprising that the molecular hallmarks of low vs high gelatin injection shown in Fig. 8 would still be present at a time point 2-weeks after the initial injection.

3.a. It would increase the impact of the study to better understand the basis of this surprising observation. This point links to point 1.a. as one would ideally need to quantify baseline gelatin levels pre-injury and post-injury. For example, is the injected gelatin still present 14-days after injection? Or is the MMP profile altered in a way that sustains these levels one direction or the other? Etc.

3.b. Alternatively to 3.a., the authors should use the Discussion to note this point and speculate on the significance.

Minor Comments

1. The authors should conduct a careful review of the manuscript to address minor typos and grammatical errors.
2. It is unclear from the Figure Legends, Results, or Methods what the 'PBS' condition in Figure 1 refers to. Is this the uninjured control? If so, consider using 'Cntrl' as the label and then defining it in the figure legend for clarity.
3. It is unclear from the Figure Legends, Results, or Methods what is quantified to read-out the 'myogenesis index' and 'fusion index' that is reported in Fig. 5D & E. Please reconcile.

Significance

The manuscript constitutes a technical advance, and offers a molecular mechanism, in support of the notion that intramuscular injection of low dose gelatin serves to expedite the process of skeletal muscle regeneration. This study has translational implications for a regenerative medicine application of this knowledge. It is my opinion that this aspect of the study is well supported by the results and requires <1month of additional edits to finalize the manuscript.

The manuscript would constitute both a technical and a conceptual advance by addressing Major Point 1a as the authors would show, for the first time, that low and high dose gelatin levels naturally exist in vivo to mediate the process of muscle endogenous repair. This would be highly significant, because as the authors rightly point out in Lines 74-76 of their manuscript "...by what mechanisms ECM regulates the functional, morphological, and molecular events of skeletal muscle regeneration remain poorly understood". It is my opinion that making this latter point would require 1-3 months of additional studies.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The manuscript by Xiao Ling Liu and colleagues titled "Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration" deals with the effect of gelatin on differentiation of myoblast cell line, in vitro, and on skeletal muscle regeneration upon muscle injury, in vivo. In vivo, the gelatin is a product of collagen breakdown associated with skeletal muscle regeneration upon acute or chronic muscle damage.

Specifically, the authors define a dose-dependent effect of gelatin, beneficial at low dose and detrimental at high dose. This effect is mediated by the level of ROS accumulation leading to the induction of different cytokines with opposite effects on skeletal muscle regeneration.

Major comments:

The experimental purpose is well tackled from both biochemical and functional point of view, and the proposed experiments are quite exhaustive. However, I would suggest some additional experimental analyses to improve the robustness and quality of the study, as well as text and figure editing, as reported below.

Regarding the additional experiments/analyses/images:

• Figure 5: I would suggest to add an image of C2C12 cells in GM (growth medium), as representative images of proliferation analysis upon LCG/HCG/NAC treatment.
• Figure 5: I would suggest to repeat the main si-NOX2 experiments with an alternative siRNA to rule out off target effects.
• In vivo experiments could be improved by adding DHE or DCFH staining on muscle TA cryosections to quantify the level of oxidative stress.
• The proposed model could be better tackled by additional in vivo treatment with Ab anti IL-6 or anti TNFalpha in combination with CTX and LCG or HCG, followed by H/E staining at 14 dpi.

Minor comments:

Each acronym should be indicated in full in the main text at the first mention (for instance BHP, NAC and others). Moreover, I would suggest to add an acronym list for reagents and factors

Experimental methods should be better detailed; for instance I would suggest:

• Add a detailed description of the quantification of differentiation indexes
• Explain how cell growth (OD 450nm) and optical density (570nm) assays have been performed
• Explain how ROS species and antioxidant enzymes have been measured (Fig 4C and 4D)

Figures and figure legends:

• Please add in the figures the figure number in order to facilitate the reading of the pdf file
• The sequence of the panels should be coherent with the alphabet and reading left to right and up to down
• In the Figure 1, I would suggest to add the whole TA sections for H/E staining in order to appreciate the overall beneficial or detrimental effect of LCG and HCG, respectively.
• I would suggest to show Supplementary figures 1C and 1D in the Figure 1
• In the Supplementary Fig 2A, I would suggest to the authors to show and comment only the data about proliferation: the earlier orientation, fusion and differentiation of C2C12 exposed to LCG are a consequence of the positive effect of LCG on proliferation
• The quality of representative images of western blot is not always high: the bands are fuse and, consequently, the quantification is not reliable. For instance Fig S4 B, S4 G; in Fig 2 the representative image does not really represent the reported quantification.
• Please specify in the figure legends the meaning of the acronym in the axis title (for instance RFU, MFI or DCF) and in the axis title the unit of measure (for instance Count (?)).
• The authors always wrote "filed" in place of "field"
• In the figures 7 and 8 the letters for densitometry panel are missed.
• In the figure legend 8 the panel letters do not match the panels in the figure
• Figure 3I: replace "nucleis" with "nuclei"

Text editing:

• Line 64: Satellite cells (SCs) would be more appropriate than myoblasts
• Line 64: Please define more carefully the location of SCs
• Line 67: MyoD+/Myog+ would be more appropriate than Pax7+/Myog+
• Line 67: (Pax7+)/Myog+ "myocytes" in place of myotubes ... and fuse with each other to generate myotubes
• Line 69: Please add Myf6/MRF4 to MRFs list
• Line 112: replace "its" with "their"
• Lines 330-331: "promoted IL-6" "enhanced TNF", please insert secretion/ production
• Line 352-353: this sentence is not necessary

Significance

The study reports robust and interesting data applicable to both basic research and translational research, such as tissue engineering applications.

Keywords for field of expertise of this reviewer:

Skeletal muscle regeneration

Duchenne Muscular Dystrophy

Inflammation

Macrophages

Oxidative stress

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Reply to the reviewers

Firstly, we would like to thank the reviewers for their helpful and insightful comments.

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

In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus epithelial fractions postnatally. They hypothesize on this basis that the remodeling at postnatal stage is likely only dependent on the mesenchymal compartment and it involves interaction of laminins with integrity a6.

It seems that some of the presented data have already been described and could not be considered as « novel ».

For some of the statements, like this one « the basement-membrane produced by the epithelium is not sufficient to increase stem cell numbers and induce a morphological crypt formation », the conclusion is not sustained by provided experiments. To draw definitive conclusion on this particular point, authors could reproduce the experiment presented in Fig. 4d but using Cre recombinases specific for mesenchymal and epithelial compartments rather than the ubiquitous Cre line. It would be interesting to investigate if organoids grown from lamc1-/- mice can generate protruded organoids or not.

In addition, how interpret the fact that fetal organoids up is associated with « laminin interactions » in fig. 1c?

The statement that the epithelium-produced basement membrane is not sufficient to increase stem cell numbers is based on our in vitro observations. Analysis of the RNAseq data shows that the expression of several laminins is increased on collagen (see heatmap of laminin interactions below, which will be added to the manuscript). This is also the reason why ‘laminin interactions’ is highly significant in the gene set enrichment analysis (Fig. 1C). Despite this upregulation, we never observed morphological changes (or expression changes) as when laminin is added to the collagen-hydrogel. In addition, we showed that the vast majority of ECM components is produced by the mesenchyme in vivo, in line with previous literature as cited in the manuscript. The mentioned Cre lines to address the question in vivo are unfortunately not available to our collaborators with the Lamc1 k.o. mice and it would therefore take too long to perform these experiments.

However, to address this point in vitro we will grow organoids from Lamc1 fl/fl mice and induce loss of laminin in the pure epithelial cell culture. Organoids will then be analysed for morphological changes, as well as proliferation and gene expression changes.

One major point to address regards statistics. In material and methods, the paragraph describing statistical analyses is missing. Moreover, in the figures presenting qPRC data ( figs 1g 3b 3D 3g 4c and f), no statistic analysis is provided; and the number of samples for some conditions is extremely limited (n=2). In general, the term « independent experiment « should be clarified : does it correspond to one organoid line for which the experiment was repeated or one single experiment using different organoid lines?

In fig 4c , all collagen conditions are set to 1.

The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistics. In addition, for most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.

The term independent experiment was used mainly for repeated experiments with the same organoid lines (exception RNAseq data, different organoids derived from individual mice). While conducting these experiments, we realised that the variability of these experiments comes from time in culture, density of cells and even Matrigel variation. The experiment in Fig. 4c (n=4, each time with the all controls) was performed with longer intervals in between, and showed variation in the absolute levels of expression. However, relative to each control we believe the effect is clear.

As we will perform additional experiments for the revision of this paper, we will then perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.

Regarding the experiment presented in fig 4c, authors should include additional control conditions : anti-a6 integrity antibody in matrigel and use of an isotype antibody.

We will conduct additional experiments regarding the Itga6. In addition to including the mentioned controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.

Another point regards RNAscope data presented in Fig 4b, it is surprising to observe such difference in terms of expression between E19 and P0. Does this mean that birth dramatically unregulates Itga6 expression in few hours? Authors should comment this point if verified.

We do believe that birth is a timepoint where a dramatic change in the ECM and their receptors can be observed. The epithelial RNAseq data would already indicate that at 18.5 there is an increase in expression compared to E16. This upregulation of the receptor is in line with the dramatic remodelling of the ECM at birth, as is shown by the expression of the basement membrane components in Fig. 3d.

Authors should avoid the word « signaling » for laminin-integrin interactions as they do not study this aspect at all in their experiments.

The word signaling was used for the protein:receptor interaction and to distinguish it from changes to the physical characteristics of the hydrogel. But we agree with the reviewer, that we did not study laminin signaling per se and therefore will change the wording accordingly.

Regarding Col1a1, authors cannot claim that it's expression only slightly changed (fig 3d) as it is clearly upregulated between E17 and P0.

The reviewer is right, and we apologise for the misleading sentence. The contrast was meant to the basement membrane components that are very lowly expressed at E17 and then suddenly show the burst of expression at birth, whereas collagen seems to be continuously expressed with a peak at P7. We will rephrase the sentence.

Reviewer #1 (Significance (Required)):

Overall, the methodology used for the asked questions is accurate.

One potential problem for publication comes from the fact that some of the findings are already reported and hat the present data do not provide further advances.

for example, collagen and fetal-like expression profile, Ly6a sorting and replating in culture-Yui et al, 2018, Jabaji et al, 2013.

We obviously do not agree with the reviewer on this point. We build upon the work of Jabaji et al. 2013, and Wang 2017 to characterise the specific effect of collagen on the intestinal epithelium compared to a pure Matrigel culture. The emergence of Ly6a cells was nicely shown by Yui et al., however it was unclear if collagen changes the fate of all intestinal cells or only a few. We strongly feel that our data extends these findings as it associates the changes we observe in vitro to the development of the crypt morphology and intestinal stem cells.

The phenotype of Lamc1-/- mice and the observed reduced stem cell marker expression are also reported by Fields et al, 2019.

Indeed, as we cited this paper. However we predicted based on our in vitro model, that deletion of laminin would result in this specific fetal-like gene expression and hence were happy to include these findings in our manuscript.

Infine, authors do not interpret their ex vivo data in the context of fetal progenitors which grow as spheres in matrigel (containing laminin)?

Our ex-vivo (in vitro) data would suggest that adult epithelial cells express some genes that are characteristic for fetal organoids, however we do not think these cells completely revert back to a fetal stage. Regarding the comment of spheres, it is noteworthy that fetal cells from E14-16 stay as spheres in Matrigel, whereas fetal cultures from E19 initially grow as spheres and then develop into organoids within 30 days in vitro (M. Navis et al., “Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning,” EMBO Rep., vol. 20, no. 2, pp. 1–12, 2019.).

In figure 5, should we interpret that there is no laminin at all in the fetal mesenchyme?

We now see how the image is a bit misleading for that stage. The levels of laminin are lower in the fetal stage as can be seen by the IF image in Fig.3f and the image will be updated. We also apologise for the lack of labeling in Figure 3f, which should be E19, P7 and adult.

Also, authors do not cite a paper reporting on the role of the epithelium ( stem cells) in regulating its own extracellular matrix composition, this process modulating the stem cell number and fate (Fernandez-Vallone et al. 2020). As this is contradictory with the claim that only mesenchyme impacts on crypt morphogenesis, authors could discuss on this point.

In the paper by Fernandez-Vallone, deletion of Lgr5 in E16.5 embryos resulted in a decrease expression of several ECM genes. Further, the authors could show that the fetal epithelium does express for example Col1a1 at this point, which decreases with maturation. However even for the example of Col1a1 it is evident in their paper that the mesenchyme expresses Col1a1 at much higher levels. Our proposed experiments with Lamc1 k.o. In organoids will show if the produced laminins of the epithelium are essential.

This manuscript could be interesting for an audience in the stem cell and developmental fields ( my field of expertise).

**Referee Cross-commenting**

Considering the pertinent and sometimes overlapping comments of the two other reviewers, the estimated time is revised to 3-6 months.

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

**Summary:**

In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal epithelial cells, correlating with a transient increase in expression levels of collagen and laminin genes in vivo spanning time points of crypt formation. The authors reported the functional contribution of laminin signaling (Lamc1 KO) via integrin alpha 6 (antibody-blocking) to intestinal stem cell acquisition in vitro and in vivo.

There are a handful of comments/concerns that would need to be addressed before publication.

Major points:

1. The author claimed: "the effect of ECM components on gene expression is not due to difference in morphology (2D collogen vs. 3D Matrigel)". The conclusion of the 2D vs. 3D experiment should be toned down to that organoid morphology (2D vs. 3D) does not directly impact on the expression of fetal-like genes. Otherwise more analysis of RNAseq data with different group of genes (e.g., in different mechanosensing pathways) should be provided with Fig. S1D. Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.

We apologise for the too strong claim of structure of growth vs. signalling of the ECM and its effect on the transcriptome. Indeed, the main message is that a changed morphology from 3D to 2D is not responsible for the expression of fetal-like genes. The paragraph will be rephrased.

Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.

To address this point, we want to refer to the excellent idea of growing established organoids in collagen (3D) vs. Matrigel (3D) as suggested by this reviewer (and reviewer #3) in Minor points #5. This circumvents the need for Wnt3a addition, which affects stem cell and Paneth cell gene expression.

1. For Fig. 1f, the authors should include overlapping stainings of Lyz (or Olfm4, CD44 etc.) and Adolase B signal, or they could perform Aldolase B staining in Lgr-5-DTR-GFP and/or Lyz-RFP organoid line. From the current data provided one cannot draw clear conclusions on the crypt morphology as claimed by the authors. Additionally, when talking about crypt morphology and apical accumulation of Actin specifically in the Lyz+ cells, the authors should show a higher zoom in of the picture and either add an orthogonal slice to see apical and basal side and the specific accumulation in one of the cell types, or also co-label with apical polarity markers.

We will perform additional co-stainings to further highlight the differences in the spatial distribution of differentiated cells and undifferentiated-crypt-like cells. Further we will provide higher magnification images highlighting the apical accumulation of Actin in the crypt-like structures, which can also be seen in mature organoids.

1. Authors referred the organoid transient change to fetal-like state. To exam the similarity of ECM-induced reprogramming with the regenerative-type of reprogramming, it would be essential to compare the expression of the selected fetal-like genes (Anxa3, Ly6a/Sca1, Msln, Col4a2 et al.), as well as bulk and single-cell (if applicable) RNA-seq data.

Here, we would like to refer to the excellent study by Yui et al. ([S. Yui et al., “YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration,” Cell Stem Cell, vol. 22, no. 1, pp. 35-49.e7, 2018.). In this study the authors detected the same gene signature in the repairing epithelium. We can provide a GSEA for the Ly6a+ signature that was derived from this paper, if necessary.

1. For in vivo data, authors were looking at the normal development of intestine. Following the point of organoid culture recapitulates regeneration, it would be relevant to check the in vivo ECM change by staining in the process of intestinal regeneration or discuss would the fetal-like genes be involved in regeneration.

We will address this point in the discussion as it also involves the study by Yui et al.

1. For Fig2.d and e, it would be important to measure compactness vs. the emergence/probability of Ly6+ cells to see if there is correlation.

If we understand the reviewer correctly, this would address the important relationship between cell shape and cell fate/type. However, this is a topic that needs more attention than a simple correlation and would exceed the scope of this manuscript as we are not able to modulate cell shape to make any further points about its effect on the fetal gene expression program.

1. In Fig.2d, Ly6a expression is very obscure, and it would be important to show control staining for cell boundaries (eg. Phalloidin, PM) to visualize which nuclei show Ki67 staining and are high or low in Ly6a (plus quantification).

We will improve the image in Fig.2d and include the mentioned Actin staining. In addition we will perform an analysis via Flow Cytometry to quantify the level of Ly6a staining and EdU positivity.

1. In Fig. 2f-g, FACS-ed Ly6a+ and Ly6- cells embedded in Matrigel can grow into organoids with crypts. Here the imaging of Paneth cell staining is not clear, and a quantification on number of Paneth cells per crypt would be very helpful to confirm the phenotype. Also, authors should either provide data on the initial size of seeded cell clusters and report organoid growth and cell type composition in more detail when plating from Ly6a+ and Ly6- cells or report the variation in the respective populations.

This comment suggests that we may not have described the experimental settings properly. The sorted cells were embedded as single cells, not as clusters, in drops of matrigel (10k cells/25ul Matrigel). The emergence of Paneth cells together with a normal organoid architecture grown from Ly6a+ cells shows their stem cell capacity, as has been shown by Yui et al. before from the regenerating colon. In addition, organoids from both cell populations (Ly6a+ and Ly6a-) could be passaged, indicating presence of intestinal stem cells.

1. The authors could also test whether Ly6+ cells have any advantages over Ly6- cells when grown on collagen I instead of Matrigel.

We will sort Ly6a+ and Ly6- negative cells and plate them on collagen I. It will be interesting to see if the Ly6a+ cells can give rise to the other cell types when plated on collagen or if they stay Ly6+ cells. This will also answer whether Ly6a+ need the presence of Ly6a- cells in the cultures. In addition, the experiment proposed in #6 will also highlight any proliferative advantage of Ly6a cells compared to Ly6-negative cells on collagen.

1. In Fig.3f, a control of membrane protein staining should be added for the experiment. The increased Laminin signal can be caused by the global increase of protein when there are more cells, or tissues are more compact. When authors make conclusion of "Dramatic remodelling of ECM during crypt formation ", the experiment should also count cell numbers vs. Laminin (intensity). The phenotype can come from increased area of interface between epithelium and mesenchyme instead of active remodelling.

We agree with the reviewer that by itself the IF images are not enough to make such a claim. However, we would point to the qPCR data and RNA in situ, that can be more easily normalised and shows the dramatic increase in expression of all laminins at birth. To show that laminin protein is increasing is more difficult than we initially anticipated. However, in the study by De Arcangelis (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.) the authors use an EDTA assay to show that the epithelium detaches easily when Itga6 is deleted. Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth.

1. The authors claim that intestinal stem cells in vivo are controlled by Laminin signalling that goes via Integrin alpha 6. However, there is no evidence provided that supports the contribution of ITGA6 in the in vivo setting. So, the authors should either tone down on that point or show a convincing in vivo experiment (e.g., inhibit ITGA6 in vivo by inhibitor injections or by extracting the ECM of a wild-type mouse and seeding intestinal epithelial cells without vs. with ITGA6 blocking antibody which should recapitulate the phenotype in Fig. 4 c.

We apologise for this confusion. We are well aware about the limitations of our Itga6 blocking experiment in vitro and its relevance in vivo. We tried to get material of the inducible VilCreER Itga6 mouse as referenced in the discussion of the manuscript, without any luck so far. Therefore we will highlight further that any claims about the laminin:Itga6 interaction can only be made in vitro.

1. Fig. 4: For the data of ITGA6 expression and all sorts of analysis on protein expression with staining, normalization with cell numbers should be performed.

The RNAseq data that shows the upregulation of Itga6 in the epithelium at E18 is normalized within. Our RNAscope only further validates these expression changes and highlights the specific enriched expression at the bottom of the nascent crypts. We can add quantification of the RNAscope if required.

1. Two questions on mechanisms:

2. What is the mechanism from ITGA signaling to Ly6a+ cell fate?

3. And would/how Laminin induce ITGA expression? Depends on how much the authors would like to go deep with the project, could be addressed further with functional studies, or touch on the topics with discussion.

These are important questions, however we do agree that this will go to deep for the scope of this manuscript. We will address these open questions in the discussion and leave the experimental part for a follow-up study.

**Minor points:**

1. Text in Fig.S1d regarding 'in' or 'on' collagen, could be clearer by changing the terms to 2D and 3D correspondingly.

We agree and the text will be changed accordingly.

1. Fig. S1a, it is great the authors showed that similar stiffness in Matrigel and collagen I. It would be important to check the concentration of collagen I vs. stiffness (also for increasing concentrations of Laminin in Fig. 3b), since this is also the type of ECM change that might lead to the change of cell status in cancer progression or collective cell migration.

We will perform further stiffness measurements of the hydrogels and update the Fig. S1a.

1. When plating intestinal epithelial cells on collagen I, is the Ly6+ phenotype altered upon Wnt addition? This is not so clear from the RNAseq data Fig S1d., so authors should provide antibody stainings (stem cells/Paneth cells). This could give insight whether Ly6+ cells are still able to convert into stem cells/ Paneth cells by changing morphogen concentration vs. ECM composition.

We will reanalyse the RNaseq dataset further, specifically analysing the ratio of stem cell and Paneth cell gene expression. However, as mentioned before, Wnt3a specifically does reduce the expression of Paneth cell markers.

Similar to this point, also enteroendocrine cell fate is absent in collagen I condition (Fig2.ab), the authors could address this point by medium induced EE cell fate.

Due to the reduced number of secretory cells the clustering in Fig2 a/b does not separate all the different cell lineages. However, EE cells are present in the collagen cultures as characterised by expression of Chga, just reduced in their number (see Supl Fig. 2B).

1. It would be more informative to indicate the thickness of ECM layer in culture of 2D collagen I, as well as the image of the whole well, demonstrating the morphological variation in the middle and peripheral of the ECM layer.

The thickness of the collagen layer is about 1mm in a 6well plate and we do not observe any morphological differences in the cells between the periphery and center of the well.

1. After the formation of PC/SC clusters, would ECM contribute to maintenance? Putting mature organoids from Matrigel to Collagen I 3D would help to clarify.

This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.

1. Check secretome and individual culture of Mesenchyme, see if the increase of Laminin is epithelium independent.

We agree that the mesenchyme is key for laminin production, therefore these are important questions. Our prediction would be that epithelium from birth (P0) versus adult might result in different responses on the mesenchyme. However, we feel these experiments are better suited for a follow-up study.

1. In general, the authors should look at cell polarity markers to check the ECM contribution to cell polarity in different cell types.

We thank the reviewer for the suggestions and as mentioned above, we will perform additional stainings.

Reviewer #2 (Significance (Required)):

**Significance:**

The work highlights the role of ECM on stem cell niche and is of great interest to the organoid and stem cell community.

Our field of expertise is image- and seq-technology-based quantitative biology, regeneration and mechanics in organoid.

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

**Summary:**

Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the abstract could be much better supported: "This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells." The in vivo work was largely published previously and has caveats noted below, while the in vitro association of ITGA6 signaling with crypt formation is over-interpreted based upon an antibody blocking experiment and a lack of statistical rigor. Despite these concerns, this reviewer finds the work of considerable interest in an important area of the field (epithelial/stromal interactions of the intestine).

**Major Concerns:**

The use of the Ubc-Cre Lamc1-flox mouse model is an interesting way to test the impact of loss Lamc1 on intestinal development. However, with the Ubc-Cre, does the mouse model have other deleterious effects on the mouse beyond the intestine?

Can the authors use a more localized Cre to observe specifically the impacts of Lamc1 loss in the intestine? What is the fate of these mice? Can the authors show swiss roll, low mag sections to let the reader know the extent of this phenotype? OLFM4 and Ki67 IHC should be conducted over a timecourse to show how the changes occur over time after loss of Lamc1. How long does Lamc1's protein product perdure after tamoxifen treatment? More details of this exciting, in vivo validation of the authors' in vitro studies are key to elevating the impact of this work. However, it appears that much of this mouse model was previously published, but the previous findings are not well summarized in the current manuscript.

We will describe the model in more detail and refer readers to the excellent study of our collaborators which answers most of the raised questions. It is interesting to note that although a ubiquitous Cre was used to delete Lamc1 in adult mice, a phenotype was only observed in the intestine indicating a specific role for continuous laminin production here.

Can the authors show that ITGA6 loss has functional consequences in vivo with an epithelial knockout or via an organoid knockdown? A more rigorous genetic test of this proposed function would be important for substantiating the claims made in the abstract.

As referenced in the discussion of the manuscript, there is a VilCreER Itga6 mouse described in the literature (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.), which mainly focus on the colon. However, the authors use an EDTA assay in the small intestine to show that the epithelium detaches easily when Itga6 is deleted (Fig. 1J). Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth which is dependent on Itga6.

We tried to get material of the inducible VilCreER Itga6 mouse however without any luck so far.

We will conduct additional experiments regarding the Itga6 in vitro. In addition to including additional controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.

The authors state that the changes in gene expression are not due to differences in morphology, but rather are specific to the components of the environment. While the authors show that organoids treated with Wnt3a "in Matrigel" and "CollagenI" appear to have similar morphologies and yet still result in a different gene expression profiles, it would be of great interest to see whether that difference persists without Wnt3a when organoids are "in Matrigel" and "in CollagenI". While the reviewer understands the difficulties of culturing organoids in 3D Collagen without Wnt3a, organoids can be indeed be cultured in 3D using "floating collagenI rings" (Sachs et al 2017).

This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.

Similarly, while the authors indicate that increasing Matrigel concentrations altered the gene expression patterns in a dose-dependent manner, it is unknown whether this can fully be attributed to the Matrigel composition, or whether the layer of Matrigel is providing the capability to transition from 2D to 3D culture.

We are not entirely sure, we understood this point. The Matrigel and the collagen I are mixed before they solidify, therefore enabling a homogenous hydrogel. The different hydrogels are not layered (if that is what the reviewer is referring to).

The authors cultured organoids in different concentrations of Laminin/CollagenIV when mixed with CollagenI. Can organoids be sustained only on a matrix of CollagenIV and/or Laminin? Would this show more direct differences between CollagenI vs. Laminin cultured organoids?

Organoids cannot be grown in pure Collagen IV, but pure laminin should be feasible. We did initial experiments with 3-5mg/ml laminin in PBS and that was sufficient to allow organoid growth. We will perform additional experiments with pure laminin and show the impact on organoid growth.

With the reduction in stem-cell and Paneth cells in the Lamc1-KO mice, it would also be of interest to determine what cell types are now prominent within the heavily elongated intestinal "crypt" structures seen in the Lamc1-KO mice and whether populations are more TA-cells or enterocytes to consider differentiation status of the cells. Additionally, it would also of interest to see if the Itga6 expression is significantly altered in the absence of Lamc1.

We will test expression changes for Itga6 in the Lamc1-KO mice, in the epithelium via qPCR. Additionally we can stain tissue from these mice for Sox9, Ki67 and differentiated markers eg. CD44, AldolaseB, Villin etc .to determine whether the elongated, hyperproliferative crypts contain progenitor cells or secretory enterocytes.

**Minor concerns:**

Matrigel is a complex matrix derived from mouse tumors. In many instances in the manuscript, the authors portray it as a more simpler mix of laminin/Collagen4 (fig 3a). It should be made clearer to the reader that Matrigel is not a mix of recombinant proteins, but a more clear depiction of how Matrigel is derived will be critical for this study, given the focus on specific ECM components and how they affect intestinal epithelial growth.

We agree and will change the oversimplified view of Matrigel.

Some labels of specific conditions would be appreciated in the figures as opposed to only the figure legends (ie. Fig. 1b and 1d should be labeled with comparisons; Fig. 3f labels of fluorescence, Fig. 4b label of itga6 staining).

We apologise for this and the Figure labels will be updated.

With the light staining of Lamc1 in-situ, it is hard to appreciate the expression of laminin within the stroma of the intestine when compared to Col4. This reviewer is also curious of the biological relevance of the concentrations of Laminin/CollagenIV when culturing organoids in Fig. 4a.

Indeed, the Lamc1 due to its lower expression than Col4a1 is more difficult to see. Maybe the reviewer overlooked Suppl.Fig.4A, where the blue channel of these in situ images show more contrast. If required, we can try to optimise the hybridisation times to increase the signal a bit further.

When culturing the organoids with the mixture of collagen and laminin or collagen IV, the concentrations of the two single components were selected similar to their concentrations in Matrigel. Regarding the absolute concentrations of laminin/collagen IV in vitro versus their “concentration” in vivo is much harder to answer. In addition to the unknown concentrations in vivo, there are many more Laminin types present with specific localisation and even specific receptor interactions. For our in vitro studies we relied on the Laminin present in EHS tumours, which is Laminin alpha 1 beta 1 gamma 1. We are currently investigating decellularization protocols to purify the ECM from mouse intestinal tissue, but again this would be more suited for a follow up study.

It would be appreciated if gene expression analyses presented in figures would include p-values to provide context for differences in gene expression.

The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistical testing. For most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.

As we will perform additional experiments for the revision of this paper, we can perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.

In figure 3f, the authors report "immunofluorescence of laminin". How is this measured? Can more details be given about the antibody in the text and figure legend? Laminins are a family of genes, and it's not clear what's being demonstrated in this figure panel. Developmental stages of the samples are also not clear.

We apologise for the lack of labeling in Fig.3f. The details of the antibody were hidden in the Materials and Methods of the manuscript (Slides were incubated with Laminin Polyclonal Antibody (1/200, Thermo Fisher #PA5-22901) overnight at 4C ). This pan-laminin antibody reacts with most Laminin isoforms alpha1, alpha2, beta1, gamma1. We will declare it as a pan-laminin antibody in the Figure legend to help future readers.

Reviewer #3 (Significance (Required)):

This work is in an exciting "hot" area of research to understand the role of non-epithelial cells in intestinal epithelial development and function. The audience would be those in the GI field and those studying tissue-tissue interactions.

There's some concern that the in vivo portion of the manuscript (4th figure) uses a model that was previously characterized and published by this group, and that isn't clearly disclosed. The manuscript would benefit from more disclosure and detail about the in vivo phenotype. Such changes would substantially increase the impact and novelty of the study.

We would like to point out that we cited the paper of the original study that uses the model throughout the manuscript. We will disclose in more detail that this group did the study and that the reduction in stem cell genes was already mentioned in the original publication.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the abstract could be much better supported: "This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells." The in vivo work was largely published previously and has caveats noted below, while the in vitro association of ITGA6 signaling with crypt formation is over-interpreted based upon an antibody blocking experiment and a lack of statistical rigor. Despite these concerns, this reviewer finds the work of considerable interest in an important area of the field (epithelial/stromal interactions of the intestine).

Major Concerns:

The use of the Ubc-Cre Lamc1-flox mouse model is an interesting way to test the impact of loss Lamc1 on intestinal development. However, with the Ubc-Cre, does the mouse model have other deleterious effects on the mouse beyond the intestine? Can the authors use a more localized Cre to observe specifically the impacts of Lamc1 loss in the intestine? What is the fate of these mice? Can the authors show swiss roll, low mag sections to let the reader know the extent of this phenotype? OLFM4 and Ki67 IHC should be conducted over a timecourse to show how the changes occur over time after loss of Lamc1. How long does Lamc1's protein product perdure after tamoxifen treatment? More details of this exciting, in vivo validation of the authors' in vitro studies are key to elevating the impact of this work. However, it appears that much of this mouse model was previously published, but the previous findings are not well summarized in the current manuscript.

Can the authors show that ITGA6 loss has functional consequences in vivo with an epithelial knockout or via an organoid knockdown? A more rigorous genetic test of this proposed function would be important for substantiating the claims made in the abstract. The authors state that the changes in gene expression are not due to differences in morphology, but rather are specific to the components of the environment. While the authors show that organoids treated with Wnt3a "in Matrigel" and "CollagenI" appear to have similar morphologies and yet still result in a different gene expression profiles, it would be of great interest to see whether that difference persists without Wnt3a when organoids are "in Matrigel" and "in CollagenI". While the reviewer understands the difficulties of culturing organoids in 3D Collagen without Wnt3a, organoids can be indeed be cultured in 3D using "floating collagenI rings" (Sachs et al 2017). Similarly, while the authors indicate that increasing Matrigel concentrations altered the gene expression patterns in a dose-dependent manner, it is unknown whether this can fully be attributed to the Matrigel composition, or whether the layer of Matrigel is providing the capability to transition from 2D to 3D culture.

The authors cultured organoids in different concentrations of Laminin/CollagenIV when mixed with CollagenI. Can organoids be sustained only on a matrix of CollagenIV and/or Laminin? Would this show more direct differences between CollagenI vs. Laminin cultured organoids? With the reduction in stem-cell and Paneth cells in the Lamc1-KO mice, it would also be of interest to determine what cell types are now prominent within the heavily elongated intestinal "crypt" structures seen in the Lamc1-KO mice and whether populations are more TA-cells or enterocytes to consider differentiation status of the cells. Additionally, it would also of interest to see if Itga6 expression is significantly altered in the absence of Lamc1.

Minor concerns:

Matrigel is a complex matrix derived from mouse tumors. In many instances in the manuscript, the authors portray it as a more simpler mix of laminin/Collagen4 (fig 3a). It should be made clearer to the reader that Matrigel is not a mix of recombinant proteins, but a more clear depiction of how Matrigel is derived will be critical for this study, given the focus on specific ECM components and how they affect intestinal epithelial growth.

Some labels of specific conditions would be appreciated in the figures as opposed to only the figure legends (ie. Fig. 1b and 1d should be labeled with comparisons; Fig. 3f labels of fluorescence, Fig. 4b label of itga6 staining).

With the light staining of Lamc1 in-situ, it is hard to appreciate the expression of laminin within the stroma of the intestine when compared to Col4. This reviewer is also curious of the biological relevance of the concentrations of Laminin/CollagenIV when culturing organoids in Fig. 4a. It would be appreciated if gene expression analyses presented in figures would include p-values to provide context for differences in gene expression.

In figure 3f, the authors report "immunofluorescence of laminin". How is this measured? Can more details be given about the antibody in the text and figure legend? Laminins are a family of genes, and it's not clear what's being demonstrated in this figure panel. Developmental stages of the samples are also not clear.

Significance

This work is in an exciting "hot" area of research to understand the role of non-epithelial cells in intestinal epithelial development and function. The audience would be those in the GI field and those studying tissue-tissue interactions.

There's some concern that the in vivo portion of the manuscript (4th figure) uses a model that was previously characterized and published by this group, and that isn't clearly disclosed. The manuscript would benefit from more disclosure and detail about the in vivo phenotype. Such changes would substantially increase the impact and novelty of the study.

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Referee #2

Evidence, reproducibility and clarity

Summary:

In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal epithelial cells, correlating with a transient increase in expression levels of collagen and laminin genes in vivo spanning time points of crypt formation. The authors reported the functional contribution of laminin signaling (Lamc1 KO) via integrin alpha 6 (antibody-blocking) to intestinal stem cell acquisition in vitro and in vivo.

There are a handful of comments/concerns that would need to be addressed before publication.

Major points:

1. The author claimed: "the effect of ECM components on gene expression is not due to difference in morphology (2D collogen vs. 3D Matrigel)". The conclusion of the 2D vs. 3D experiment should be toned down to that organoid morphology (2D vs. 3D) does not directly impact on the expression of fetal-like genes. Otherwise more analysis of RNAseq data with different group of genes (e.g., in different mechanosensing pathways) should be provided with Fig. S1D. Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.
2. For Fig. 1f, the authors should include overlapping stainings of Lyz (or Olfm4, CD44 etc.) and Adolase B signal, or they could perform Aldolase B staining in Lgr-5-DTR-GFP and/or Lyz-RFP organoid line. From the current data provided one cannot draw clear conclusions on the crypt morphology as claimed by the authors. Additionally, when talking about crypt morphology and apical accumulation of Actin specifically in the Lyz+ cells, the authors should show a higher zoom in of the picture and either add an orthogonal slice to see apical and basal side and the specific accumulation in one of the cell types, or also co-label with apical polarity markers.
3. Authors referred the organoid transient change to fetal-like state. To exam the similarity of ECM-induced reprogramming with the regenerative-type of reprogramming, it would be essential to compare the expression of the selected fetal-like genes (Anxa3, Ly6a/Sca1, Msln, Col4a2 et al.), as well as bulk and single-cell (if applicable) RNA-seq data.
4. For in vivo data, authors were looking at the normal development of intestine. Following the point of organoid culture recapitulates regeneration, it would be relevant to check the in vivo ECM change by staining in the process of intestinal regeneration or discuss would the fetal-like genes be involved in regeneration.
5. For Fig2.d and e, it would be important to measure compactness vs. the emergence/probability of Ly6+ cells to see if there is correlation.
6. In Fig.2d, Ly6a expression is very obscure, and it would be important to show control staining for cell boundaries (eg. Phalloidin, PM) to visualize which nuclei show Ki67 staining and are high or low in Ly6a (plus quantification).
7. In Fig. 2f-g, FACS-ed Ly6a+ and Ly6- cells embedded in Matrigel can grow into organoids with crypts. Here the imaging of Paneth cell staining is not clear, and a quantification on number of Paneth cells per crypt would be very helpful to confirm the phenotype. Also, authors should either provide data on the initial size of seeded cell clusters and report organoid growth and cell type composition in more detail when plating from Ly6a+ and Ly6- cells or report the variation in the respective populations.
8. The authors could also test whether Ly6+ cells have any advantages over Ly6- cells when grown on collagen I instead of Matrigel.
9. In Fig.3f, a control of membrane protein staining should be added for the experiment. The increased Laminin signal can be caused by the global increase of protein when there are more cells, or tissues are more compact. When authors make conclusion of "Dramatic remodelling of ECM during crypt formation ", the experiment should also count cell numbers vs. Laminin (intensity). The phenotype can come from increased area of interface between epithelium and mesenchyme instead of active remodelling.
10. The authors claim that intestinal stem cells in vivo are controlled by Laminin signalling that goes via Integrin alpha 6. However, there is no evidence provided that supports the contribution of ITGA6 in the in vivo setting. So, the authors should either tone down on that point or show a convincing in vivo experiment (e.g., inhibit ITGA6 in vivo by inhibitor injections or by extracting the ECM of a wild-type mouse and seeding intestinal epithelial cells without vs. with ITGA6 blocking antibody which should recapitulate the phenotype in Fig. 4 c.
11. Fig. 4: For the data of ITGA6 expression and all sorts of analysis on protein expression with staining, normalization with cell numbers should be performed.
12. Two questions on mechanisms: a) What is the mechanism from ITGA signaling to Ly6a+ cell fate? b) And would/how Laminin induce ITGA expression? Depends on how much the authors would like to go deep with the project, could be addressed further with functional studies, or touch on the topics with discussion.

Minor points:

1. Text in Fig.S1d regarding 'in' or 'on' collagen, could be clearer by changing the terms to 2D and 3D correspondingly.
2. Fig. S1a, it is great the authors showed that similar stiffness in Matrigel and collagen I. It would be important to check the concentration of collagen I vs. stiffness (also for increasing concentrations of Laminin in Fig. 3b), since this is also the type of ECM change that might lead to the change of cell status in cancer progression or collective cell migration.
3. When plating intestinal epithelial cells on collagen I, is the Ly6+ phenotype altered upon Wnt addition? This is not so clear from the RNAseq data Fig S1d., so authors should provide antibody stainings (stem cells/Paneth cells). This could give insight whether Ly6+ cells are still able to convert into stem cells/ Paneth cells by changing morphogen concentration vs. ECM composition. Similar to this point, also enteroendocrine cell fate is absent in collagen I condition (Fig2.ab), the authors could address this point by medium induced EE cell fate.
4. It would be more informative to indicate the thickness of ECM layer in culture of 2D collagen I, as well as the image of the whole well, demonstrating the morphological variation in the middle and peripheral of the ECM layer.
5. After the formation of PC/SC clusters, would ECM contribute to maintenance? Putting mature organoids from Matrigel to Collagen I 3D would help to clarify.
6. Check secretome and individual culture of Mesenchyme, see if the increase of Laminin is epithelium independent.
7. In general, the authors should look at cell polarity markers to check the ECM contribution to cell polarity in different cell types.

Significance

Significance:

The work highlights the role of ECM on stem cell niche and is of great interest to the organoid and stem cell community.

Our field of expertise is image- and seq-technology-based quantitative biology, regeneration and mechanics in organoid.

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Referee #1

Evidence, reproducibility and clarity

In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus epithelial fractions postnatally. They hypothesize on this basis that the remodeling at postnatal stage is likely only dependent on the mesenchymal compartment and it involves interaction of laminins with integrity a6. It seems that some of the presented data have already been described and could not be considered as « novel ». For some of the statements, like this one « the basement-membrane produced by the epithelium is not sufficient to increase stem cell numbers and induce a morphological crypt formation », the conclusion is not sustained by provided experiments. To draw definitive conclusion on this particular point, authors could reproduce the experiment presented in Fig. 4d but using Cre recombinases specific for mesenchymal and epithelial compartments rather than the ubiquitous Cre line. It would be interesting to investigate if organoids grown from lamc1-/- mice can generate protruded organoids or not. In addition, how interpret the fact that fetal organoids up is associated with « laminin interactions » in fig. 1c?

One major point to address regards statistics. In material and methods, the paragraph describing statistical analyses is missing. Moreover, in the figures presenting qPRC data ( figs 1g 3b 3D 3g 4c and f), no statistic analysis is provided; and the number of samples for some conditions is extremely limited (n=2). In general, the term « independent experiment «  should be clarified : does it correspond to one organoid line for which the experiment was repeated or one single experiment using different organoid lines? In fig 4c , all collagen conditions are set to 1.

Regarding the experiment presented in fig 4c, authors should include additional control conditions : anti-a6 integrity antibody in matrigel and use of an isotype antibody.

Another point regards RNAscope data presented in Fig 4b, it is surprising to observe such difference in terms of expression between E19 and P0. Does this mean that birth dramatically unregulates Itga6 expression in few hours? Authors should comment this point if verified. Authors should avoid the word « signaling » for laminin-integrin interactions as they do not study this aspect at all in their experiments.

Regarding Col1a1, authors cannot claim that it's expression only slightly changed (fig 3d) as it is clearly upregulated between E17 and P0.

Significance

Overall, the methodology used for the asked questions is accurate.

One potential problem for publication comes from the fact that some of the findings are already reported and hat the present data do not provide further advances.

for example, collagen and fetal-like expression profile, Ly6a sorting and replating in culture-Yui et al, 2018, Jabaji et al, 2013.

The phenotype of Lamc1-/- mice and the observed reduced stem cell marker expression are also reported by Fields et al, 2019.

Infine, authors do not interpret their ex vivo data in the context of fetal progenitors which grow as spheres in matrigel (containing laminin)? In figure 5, should we interpret that there is no laminin at all in the fetal mesenchyme?

Also, authors do not cite a paper reporting on the role of the epithelium ( stem cells) in regulating its own extracellular matrix composition, this process modulating the stem cell number and fate (Fernandez-Vallone et al. 2020). As this is contradictory with the claim that only mesenchyme impacts on crypt morphogenesis, authors could discuss on this point.

This manuscript could be interesting for an audience in the stem cell and developmental fields ( my field of expertise).

Referee Cross-commenting

Considering the pertinent and sometimes overlapping comments of the two other reviewers, the estimated time is revised to 3-6 months.

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Reply to the reviewers

Reviewer #1

This manuscript by Gulyrutlu and co-workers addresses the role of CUG expanded repeat RNA associated with DM1 in regulating the formation of higher order RNP assemblies such as stress granules and P-bodies in the cell. The authors used lens epithelial cells (hLECs) derived from a DM1 patient

We used cell lines from several patients and age-matched controls to avoid effects of individual cell-line variation. We will make sure that this is clear in the text.

or a HeLa cell inducible model of DM1 to investigate whether expression of the CUG repeat-associated protein MBNL1 and CUGBP1 affected the formation and dispersal of stress granules and P-bodies. The authors show that MBNL1 and CUGBP1 are components of SGs and PBs in hLECs and HeLa cells. In cells expressing the CUG repeat, there are minor alterations in the dispersal of stress granules as well as in the formation of P-bodies.

The alterations in the formation and dispersal of stress granules are not minor. For example, in the HeLa cell model, stress granules take more than twice as long to form in cell expressing the CUGexp repeats associated with DM1 and disperse in half the time. These data are already in the results section, but we will highlight them in a revision and have included an additional representation of the data to the figure, using graphs of ‘proportion of cells with stress granules’ against time. The changes we see are as large, or larger, then results published elsewhere (see appendix below)

MBNL1 could affect the formation and dispersal of SGs independent of the CUG repeat.

In fact, we present data in HeLa cells with MBNL1 almost completely removed by shRNA revealing that this has a much smaller effect on stress granules than does the expression of CUGexp RNA. This is an important point, as it is widely assumed that most of the cellular defects in DM1 are caused by the ‘sequestration’ of MBNL1 in the CUGexp foci. Since only . This is not what our data show. In the hexanediol experiments, both cell lines over-express MBNL1 in similar amounts. The difference between them is that one cell line expresses a DMPK1 mini-gene with a CUG expansion and the other expresses a mini-gene without the expansion. Again, our results show that the alteration to P-body responses to 1,6-hexanediol can be attributed to the presence of the CUGexp RNA, rather than altered levels of MBNL1. We will revise the results and discussion to further emphasise this point.

Finally, in HeLa cells, overexpression of MBNL1 can reduce the dispersal of P-bodies upon 1,6-hexanediol treatment.

This is not what our data show. In the hexanediol experiments, both cell lines over-express MBNL1 in similar amounts. The difference between them is that one cell line expresses a DMPK1 mini-gene with a CUG expansion and the other expresses a mini-gene without the expansion. Again, our results show that the alteration to P-body responses to 1,6-hexanediol can be attributed to the presence of the CUGexp RNA, rather than altered levels of MBNL1. We will revise the results and discussion to further emphasise this point.

Major comments:

One limitation of the work is that the perturbations seen with stress granules or P-bodies are all relatively small, and no evidence for a functional consequence on gene expression is demonstrated. Specifically, the authors observe only minor alterations in the formation or disaggregation of PBs and SGs in these DM1 models. Further, some of the effects observed are independent of the CUG repeat expression, suggesting that MBNL1 and CUGBP1 might have independent roles in modulating some properties of SG and PB formation or dispersal.

As above, the changes we see in SG formation and dispersal are not small. There are already numerous studies of the effects of DM1 on gene expression and mRNA splicing. This is not what we set out to study: we are interested in perturbations to the organisation of cellular structures associated with the expression of the CUGexp repeat RNA characteristic of DM1. We do show some data relating specifically to the proteins MBNL1 and CUGBP1 in the paper. shRNA resulting in almost complete loss of these proteins has much smaller effects that the expression of CUGexp RNA, suggesting that the major part of the effects caused by expression of the CUGexp RNA is not mediated through changes in MBNL1 or CUGBP1 levels. MBNL1 and CUGBP1 levels may well contribute to alterations in SG dynamics, but our data suggest that they are minor contributors. This is an advance in our current knowledge

1. The authors could investigate whether the CUG repeat RNA itself is localized to SGs or PBs in their models, and whether the presence of the repeat RNA is absolutely necessary for regulating the dynamics of SG or PB formation.

We have now done this. The CUG repeat RNA is not localised in stress granules or PBs to a detectable extent. This suggests that the effect we see on these structures by expression of the expanded RNA occurs despite the absence of the RNA from the structures. This is similar to the effects of the ALS-associated paraspeckle protein FUS, which can affect the integrity of nuclear LLPS structures (gems) despite not co-localising with them https://doi.org/10.1016/j.celrep.2012.08.025 We have added these data to the manuscript, as part of draft figure 8, and will add text emphasising this as an additional example of disease-causing macromolecules affecting the structure of LLPS domains in which they are not found.

1. The authors use 1,6-hexanediol to suggests that PBs and SGs in HeLa cells show behavior analogous to LLPS. However, the use of 1,6,-hexanediol to establish an assembly as a LLPS is a relatively limited analysis (despite its widespread use in the field), since this compound can affect the formation of multiple cellular substructures that are not always LLPS (for example, see Wheeler et al, 2016, eLife).

We are aware of and have cited this publication. Our comments about LLPS structures are measured, as there is still controversy about how to definitively identify them in cells. SGs and PBs have, however, previously been widely published to be formed by LLPS. The rapid exchange of SG and PB components during FRAP and the ability of SGs to both fuse and bud (seen in our supplementary movies) are also supportive of these structures behaving as LLPS structures in our models. Wheeler et showed that, in yeast, the nuclear pore complex and some cytoskeletal structures were affected by 1,6-hexanediol but membrane-bound structures such as the ER and mitochondria were not. The disruption of the nuclear pore complex is not unexpected, since phase separation is involved in cargo shuttling through the NPC (reviewed in https://doi.org/10.1016/j.devcel.2020.06.033). We will revise our discussion to make it more clear that we are not relying only on the use of 1,6-hexanediol to define SGs and PBs as LLPS structures but also on other aspects of their dynamic behaviour and on extensive prior literature.

Significance

This study would be of interest to the field if the impact of the DM! repeat RNAs on PB and SG were more substantial...

As above, the effects we see on SG formation and loss are substantial. Tissue types affected in DM1 are prone to stress, particularly the lens of the eye, so alterations to cellular response to stress associated with the presence of CUG repeats are of key importance to understanding the cellular pathology of DM1.

...and if some functional consequences were demonstrated.

In terms of function, we show altered responses to stress caused by the expression of CUGexp RNA and probably mediated through alterations in the propensity of LLPS cytoplasmic structures (SGs and PBs) to form and be resolved. Additionally, we can now show that SGs in HeLa cells expressing CUGexp RNA contain less total polyA RNA than is seen in controls, and that ‘docking’ events between SGs and PBs are compromised in cells with CUGexp RNA. These docking events are proposed to mediate transfer of RNA from SGs to PBs (reviewed in https://doi.org/10.1007/978-1-4614-5107-5_12). These new data demonstrate functional impairment of SGs and PBs associated with DM1. We have included this as an additional draft figure 8.

The lack of a strong effect on SG or PB formation in the DM1 models, along with the CUG repeat-independent effect of MBNL1 on the formation and dispersal of these complexes, argues that MBNL1/CUGBP1 may not significantly affect the formation or dispersal of SGs and PBs.

We are actually not arguing that MBNL1 and CUGBP1 are the main effectors in the changes we see to SGs and PBs, but that the CUGexp RNA is the key player, so are a little confused by this comment.

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Reviewer #2:

In the current study, the authors compared the dynamics of P-bodies (PBs) and stress granules (SGs) between control and several DM1 cell lines. They found that MBNL1 and CUGBP1, two CUG repeat RNA-binding proteins that are primarily nuclear, could also co-localize with PBs in the cytoplasm and re-localize to SGs under stress. Small differences were observed in SG assembly and disassembly dynamics between control and DM1 HLECs, between HeLa cells expressing either CTG12 or CTG960, and between HeLa cells with and without shRNAs targeting CUGBP1 or MBNL1.

As detailed above, the alterations in SG assembly and disassembly in cells expressing CUGexp RNA are not small, in contrast to those in cells will lowered expression of MBNL1 and CUGBP1, which are much smaller suggesting that the changes caused by CUGexp RNA largely do not result from loss of MBNL1 (or CUGBP1). We have inserted additional graphs of ‘proportion of cells with stress granules’ against time' and will modify the text to emphasise both of these points.

Overall, the experiments were clearly described and the results properly presented. However, critical controls, as detailed below, are missing in multiple analyses. The mechanisms underlying these apparent differences are also unknown.

We do not consider that any ‘critical controls’ are missing, but can supply all of the additional analysis of our data that the reviewer requests below. We can also now provide additional mechanistic insight and will add an additional figure showing lowered amount of polyA RNA in stress granules in cells expressing CUGexp RNA and compromised docking events between stress granules and P-bodies, suggesting impaired communication between them.

Major concerns:

1. Throughout the study, the authors compared MBNL1 and CUGBP1 association with PBs and SGs without considering the potential differences in their cytoplasmic abundance between control and DM1 cell lines, which seems to be case for MBNL1 abundance in CTG960-expressing HeLa cells (Fig. 3). Provided that PBs and SGs exchange components with the cytosol at an equilibrium, if the cytoplasmic abundance of, for example, MBNL1 is decreased in DM1, one would expect the equilibrium being shifted resulting in less MBNL1 associated with PB/SG. Therefore, before measuring the association or the assembly/disassembly kinetics of PB and SG, the authors should first test whether MBNL1 and CUGBP1 abundance may be different between control and DM cell lines.

There is, in fact, no difference in the relative cytoplasmic abundance of GFP-MBNL1 between CTG12 and CTG960- expressing HeLa cells. Each has approximately a 50/50 split between nucleus and cytoplasm, with <3% of nuclear GFP-MBNL1 found in nuclear CUGexp foci when they are present. We have added a graph demonstrating this to the supplementary data. The abundance of total endogenous MBNL1 is also not altered in DM1 patient-derived lens cell lines compared to controls, as shown by semi-quantitative western blot analysis, which we have also added to the supplementary data. However, if the expression of CUGexp RNA did cause a major loss of cytoplasmic MBNL1, this change would be reflective of the situation seen in DM1 and would not invalidate our results or conclusions.

The same caveat applies to MBNL1/CUGBP1 knockdown experiments, where knocking down one may change the abundance of the other.

To carry out FRAP experiments or live cell analysis of SG formation and loss, it is necessary to over-express a tagged version of the protein being studied. For the knockdown experiments shown in figure 6, therefore, when we knocked down MBNL1, CUGBP1 was present in excess as a GFP-tagged protein and when we knocked down CUGBP1, MBNL1 was present in excess as a GFP-tagged protein. Thus, any effects of the knockdowns on expression of the endogenous proteins being analysed would be highly unlikely to influence the results.

1. Similarly, the authors did not consider the possibility that changes in SG/PB dynamics may be due to changes in the abundance/availability of essential SG/PB components such as GE1 and G3BP1.

From our immunofluorescence experiments, there was certainly no obvious reduction in GE1 or TIA1 abundance (we did not assess G3BP1). We have quantitative proteomic analysis (unpublished) from a similar pair of cell lines, expressing CUGexp RNA alongside GFP rather than GFP-MBNL1. This shows no change in GE1 or G3BP1, so we would not expect to see any here either. We can easily carry out a quantitative western blot analysis to confirm and will add this to the supplementary data

1. Most of the observed differences between control and DM cell lines were modest, leaving one wonder whether it could be simply due to cell line-to-cell line variability. Whenever possible, the authors should present results for each individual lines. For example, in Fig.2, 3 DM1 lines and 2 control lines were used. Was the difference in SG disassembly (Fig. 2B) observed in each of the 3 lines?

Some of the alterations were modest and there is cell line-to-cell line variability in the lens cell lines. This is why we pooled the data: on average, DM1 cells disperse their SGs more quickly than control cell lines do on average. This is not an unusual way to present data from patient cell lines of diverse genetic background. We have added data for stress granule loss in the individual cell lines to the supplementary data. These data show a consistent trend towards quicker dispersal of stress granules in patient cell lines. The variability between the patient lens cell lines was also the primary reason for us to develop the inducible system in HeLa cells, on a fixed genetic background, as explained in the manuscript.

Minor points:

1. Western blot in Fig. 3 shows two protein products from both endogenous and overexpressed MBNL1. Please explain.

Many of the commercially available anti-MBNL1 antibodies show this double-band in some cell lines as evidenced in numerous publications and on manufacturers’ websites (for example https://abclonal.com/catalog-antibodies/MBNL1RabbitmAb/A5149, https://www.ptglab.com/products/MBNL1-Antibody-66837-1-Ig.htm). We haven’t analysed the two bands in detail, but assume this to be a result of a post translational modification of some sort. Since GFP-MBNL1 and endogenous MBNL1 show the same thing, we do not consider it to be a major concern. We do mention the double-band as ‘characteristic’ in the figure legend for figure 3 so are not seeking to conceal anything here.

1. No data were shown to substantiate the statement that "MBNL1 localises to CUGexp foci and CUGBP1 does not" (page 6).

This has been published many times and is shown in figure 1A. However, we will add in a citation for this and have added an additional supplementary figure showing the lack of co-localisation in the foci from figure 1A more clearly together with separate data confirming that MBNL1 and CUGexp RNA do not co-localise with CUGBP1 in the nuclei of line HeLa_CTG960_GFPMBNL1.

1. The y-axis of Fig. 4D should not go beyond 1.

We will trim the axis. There are no data points above 1.0, just the indicator of statistical significance

Significance:

The nature of the current study is highly descriptive with little mechanistic insights.

Our work is not descriptive, as we observed a change in stress granules in patient cells, which we could then replicate (and enhance) in a novel inducible model of DM1 designed to abrogate the unavoidable variation in patient-derived cell lines. We also now have additional mechanistic insights (see above) and have added an additional figure (draft figure 8) detailing these.

For the subtle differences observed between control and DM1 cell lines, it remains unclear whether it may be due to cell line-to-cell line variation (see above).

We cannot completely rule out an influence of cell line-to-cell line variation in the patient-derived lens cell lines (see above), though we think this unlikely as we saw the same effect repeated and amplified in the inducible HeLa-derived cell model, which was designed to minimise this concern. Furthermore, for stress granule loss, we see a larger effect in the HeLa cell model after 72hrs of induction than after 24hrs (figure 5C). This argues strongly that the effects seen are due to the expression of CUGexp RNA and we will emphasise this point more strongly.

Some difference appear to be specific to one model but not the others (e.g., SG formation is slower in HeLa-CTG960 cells but not in DM1 HLECs).

Even for the observations that seem consistent between models, the current results yielded little novel biological insights into whether and how these subtle differences in PB/SG dynamics may relate to DM1 pathogenesis. Collectively, these weaknesses render the current study incremental at best.

The key biological insight the results provide is that the presence of the CUGexp repeat RNA results in defects in LLPS structures that are largely separable from any sequestration of MBNL1 in nuclear foci. With many researchers attributing the cellular defects in DM1 simply to the loss of MBNL1 by sequestration into nuclear foci, both this separation of altered stress response from MBNL1 levels and the involvement of altered LLPS formation (evidenced by the changes in PB behaviour on 1,6-hexanediol treatment) are novel biological insights into the cellular pathology of DM1. Additionally, our results shift the emphasis from nuclear effects to those seen in the cytoplasm.

In terms of specific DM1 pathogenesis, the eye lens is subject to constant repeated stress and is subject to continued growth throughout the life span, relying on lens epithelial cells as a stem cell pool. Epithelial cells are also vital to the homeostatic regulation of ions, growth factor and nutrient flow from the aqueous humor to the underlying fibre cells. Any alterations in the response of lens epithelial cells, in particular, to stress is highly relevant to the pathology of cataract seen in DM1. We will revise our discussion to emphasise these key points more strongly.

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Reviewer #3

The manuscript entiled "Phase-separated stress granules and processing bodies are compromised in Myotonic Dystrophy Type 1" by Gulyurtlu et al., characterizes the composition and ydnamics of stress granules and P-bodies in two Myotonic Dystrophy Type 1 (DM1) cell models, human lens epithelial cells from DM1 patients and age-matched controls and HeLa_CTG12_GFPMBNL1 and HeLa_CTG960GFPMBNL1 cell lines. The manuscript is somewhat descriptive with lack of functional data and some discrepancies. For example, in the discussion section, the authors conclude that "MBNL1 appears to be absent from P-bodies in cells with CUGexp foci in their nuclei. This observation suggests that the role of MBNL1 in P-bodies may be disrupted by the presence of CUGexp RNA." Figure 4A shows that "P-bodies in the DM1 model line, HeLa_CTG960GFPMBNL1 do not contain detectable amounts of GFPMBNL1". However, Figure 4E shows similar levels of total cellular MBNL1 per PB between the control CTG12 and mutated CTG960 lines.

There is no discrepancy here. The reviewer has misinterpreted our data. PBs in the HeLa CTG960 cell line do not contain detectable amounts of GFP-MBNL1 under normal growth conditions, as shown in figure 4A. The data shown in figure 4E concern arsenite-treated cells, where some PBs in the CTG960 line do contain detectable levels of GFP-MBNL1, but significantly less than in the control CTG12 cells. We will reword these sections to make sure this is clear.

Most importantly, in Figure S3 the authors show that CUGexp foci are present in 1-2 % of the cells. The claim appears to be too strong for the data presented in the manuscript.

This is not what is shown in figure S3. The reviewer has misinterpreted our data. Figure S3 shows that in cells from line CTG960, only 1-2% of the total nuclear GFP-MBNL1 signal is found in the CUGexp foci, despite the intensity of the signal within them. Virtually all of the cells from the CTG960 cell line contain CUGexp foci (>95%). We will add a statement to this effect into the results section. We would not have continued working with a cell line in which only 1-2% of cells showed the DM1 phenotype of nuclear CUGexp RNA foci.

Although the findings are interesting and of potential impact for a better understanding of the implications of RNA-protein condensate dynamics in the pathogenesis of DM1, the work presented here is still descriptive and preliminary in my opinion. In summary, the conclusions are not so convincing and additional experiments are essential to support the authors claims.

This reviewer seems to have misinterpreted several of our data sets, including the specific points above, leading to the assertion that our conclusions are not convincing.

Several months of works will be required to consolidate data and reorganize and ameliorate the manuscript, including the way data are presented and quantified.

We already have data with which to address the majority of the queries posed, so should be able to make the adjustments relatively quickly.

Specific comments:

"On removal of stress, clearance of stress granules is mediated largely by a form of autophagy." This statement is not correct since the majority of stress granules disassemble and are not targeted to autophagy; in healthy cells only 5 % (or less) of the total SGs tend to persist in presence of autophagy or lysosome inhibitors, while the vast majority disassembles. Please rephrase carefully.

The degree and manner of dispersal of stress granules in healthy cells on removal of stress is not well understood, but is known to differ depending on the type and duration of the stress (DOI: 10.1126/science.abj2400). We do not yet know how this may be altered in DM1, however, compromised autophagy is implicated in cataract formation, which is of relevance to our study. We will re-phrase this section of the discussion carefully to reflect the complex situation.

Figure 1: RNA-protein complexes have heterogeneous composition. In HLECs, do all PBs colocalize with MBNL1 and CUGBP1 or only a fraction of them?

We do not routinely see PBs without MBNL1 or CUGBP1 in the HLECs, in contrast to the situation in the HeLa CTG960 line. We have data available in order to quantify this and will add the numbers to the text of the results.

Figure 2: Stress granules and P-Bodies are known to touch each-other, a process referred to as a "kissing event". The authors have studied the mobility of GFP-MBNL1 inside these two types of assemblies. It would be important also to quantify the "kissing" events. Is this altered in DM1 cells?

We couldn’t find reference in the literature to ‘kissing events’ between SGs and PBs, but found several references to ‘docking’ events. We have noticed such interactions between PBs and SGs in our models. We are currently quantifying this and our first experiments (one in the HeLa cell model and one comparing one of the patient-derived lens cell lines to a control) suggest that there is a change in the frequency and/or size of such interactions in the HeLa CTG960 cell line compared to the CTG12 control and in the DM1 lens cell line derived to control. If this holds true in our repeat experiments (currently in progress), this would also provide the mechanistic insight requested by reviewer 2. We have included this, together with our data showing a decrease in total polyA RNA in stress granules in HeLa cells expressing CUGexp RNA, as an additional draft figure 8.

Figure 3: In HeLa cells overexpressing CTG960_GFPMBNL1, beside the accumulation of one bright CUGexp puncta, several intranuclear GFPMBNL1 protein foci are visible. This subcellular distribution is different from the one observed in the control HeLaCTG12 GFPMBNL1. Can the author describe what these intranuclear GFPMBNL1 protein foci are?

In most cells expressing CUGexp RNA, several nuclear foci form (usually one large and several smaller) and all of them contain MBNL1 (or GFP-MBNL1 in the HeLa_CTG960_GFPMBNL1 cell line). Figure S3 shows object identification using MBNL1 in this cell line, with two clear foci detected as the reviewer points out. We have added an additional panel to supplementary figure 1 to confirm that the additional foci are also CUGexp RNA foci and will clarify in the text of the results that there is not a single focus of CUGexp RNA in each nucleus.

Is GFPMBNL1 accumulating at the level of splicing speckles? Or paraspeckles? Or other types on intranuclear condensates such as e.g. PML nuclear bodies? The different intranuclear distribution of GFPMBNL1 should be better characterized.

The sub-nuclear distribution of MBNL1 is, indeed, very complex. MBNL1 also sometimes co-localises to splicing speckles/interchromatin granule clusters as we have previously reported in lens epithelial cell lines (DOI: 10.1042/BJ20130870 ) . The details of differences in the nuclear distribution of MBNL1, beyond its accumulation in CUGexp RNA foci, in DM1 cells compared to controls is the subject of another manuscript we have in preparation but are beyond the scope of the current study.

Moreover, the % of cells expressing CTG960_GFPMBNL1 and forming intranuclear CUGexp foci is only mentioned in the discussion (Figure S3); for clarity it should be reported in the main text when describing Figure 3.

The number of cells forming nuclear CUGexp foci on expression of CTG960_GFP-MBNL1 is >95% and we will add this to the text of the results section.

"Figure S2: Quantitation of GFPMBNL1 in P-bodies in HeLa cell model of DM1." The authors report in the legend "Some, but not all, of these P-bodies contain detectable amounts of GFPMBNL1". However, the figure only shows a representative image of cells without quantification. Quantitation should be provided.

We have data available to provide this simple quantitation. Approximately 38% of PBs in arsenite-treated cells from line HeLa_CTG960_GFPMBNL1 contain detectable levels of GFPMBNL1 using a manually-assigned cut-off intensity. We will add this to the relevant figure legend (now figure S5). However, this method of analysis requires an intensity to be manually set above which GFP-MBNL1 signal is considered ‘detectable’. This is hugely subjective, and in our opinion, the automatically generated quantitative comparison of “% total cellular MBNL1 per P-body” as shown in figure 4E is a more experimentally robust way to demonstrate a small loss of MBNL1 from P-bodies in cells from line HeLa_CTG960_GFPMBNL1 treated with arsenite compared to the relevant control.

The authors report "a subtle change in stress granule architecture associated with the presence of CUGexp RNA". This statement is not supported by experimental data and should be omitted.

We will qualify this statement to make it clear that we are referring to a subtle alteration in the co-localisation between CUGBP1 and MBNL1 specifically in the SGs, as our experimental data shown in figure 4D clearly support that, showing a statistically significant increase in the Pearson’s co-efficient of cololcalisation between MBNL1 and CUGBP1 in cell containing CUGexp RNA compared to the relevant control (0.90+/-0.05 for CTG960; 0.87+/-0.07 for CTG12).

Figure 4. MBNL1 and CUGBP1 co-localise in P-bodies. What is the % of colocalization?

We’re not sure exactly what is being requested here or what biological question the reviewer is asking us to address. MBNL1 and CUGBP1 co-localise in virtually all PBs (except in the HeLa CTG960 line where MBNL1 is undetectable in PBs under normal growth conditions). Figure 4E shows that, in cells with PBs upregulated by sodium arsenite, the mean amount of total cellular MBNL1 per PB is 0.1%, so it will be similar in cells grown under normal conditions as the PB sizes are similar and they appear to be of similar brightness by immunofluorescence. Again, this would be straightforward to quantify with our existing data if this is, indeed what the reviewer is requesting, but we question the biological significance. We would be reluctant to derive a Pearson’s co-efficient for the degree of co-localisation between CUGBP1 and MBNL1 in P-bodies as the structures are too small in size for this to be meaningful within the limits of imaging capabilities. We could, however, provide this if this is a specific request.

Figure 5: "Treatment with sodium arsenite was then carried out under time-lapse microscopy, with Z-stacks of images taken every 4 minutes until stress granule formation was clearly seen (Fig.5A). This revealed a pronounced delay in formation of stress granules in cells containing CUGexp foci (HeLa CTG960 GFPMBNL1, 36 min +/- 12) compared to those without (HeLa CTG12 GFPMBNL1, 15 min +/- 2) (Fig.5B)." Data representation in Figure 5 is unclear and the pronounced delay in stress granule formation is not appreciated. Since the authors performed a live imaging taking pictures every 4 minutes, it would me more informative to plot the data and show the assembly and disassembly kinetics over time for both control and CTG960_ GFPMBNL1 cell lines (similar to what shown in e.g. Gwon et al., Science 2021, Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner, Figure 2G).

The bar graph in figure 5B shows that cells from the CTG960 line take more than twice as long to form SGs compared to controls and are lost in half the time, with the precise numbers given in the text. A simple bar graph seemed the clearest way to present this. However, we have plotted our existing data in a similar manner similar to that in the cited reference and added this to figure 5. These graphs clearly show that the differences we see are at least as great as in other published literature, including the reference given by the reviewer (see below).

Concerning Figure 1, the authors report no difference in the kinetic of stress granule formation in HLECs. However, they only report data after 45 and 60 min of arsenite treatment; at these time-points the assembly step is maximal. Thus, for consistency, the authors should include earlier time-points to the analysis of stress granule assembly also in HLECs, similar to what done in HeLa cells in Figure 5.

The assembly step is not ‘maximal’ in these cell lines after 45 minutes. Figure 2A clearly shows that only ~30% of cells have SGs after 45 minutes of treatment, compared with 100% of cells after 90 minutes shown in figure 2B. We have additional data at 10, 20 and 30 minutes all showing no significant differences. We had omitted them to keep the graph simple, but have now included them as a graph of ‘% of cells with stress granules against time’ in figure 2.

"Having established that MBNL1 and CUGBP1 co-localise closely in stress granules": the authors investigated the colocalization of each of these two proteins with stress granule markers but they did not verify whether MBNL1 and CUGBP1 co-localise.

In figure 1B we show that endogenous CUGBP1 and endogenous MBNL1 both co-localise with the stress granule marker TIA1 in stress granules in lens epithelial cells. It would, therefore, be highly unlikely that CUGBP1 and MBNL1 would not co-localise with each other in stress granules. We have also previously verified that GFPMBNL1 behaves identically to its endogenous counterpart (Coleman et al, 2014). Furthermore, in figure 4C and 4D, we show close co-localisation between endogenous CUGBP1 and GFPMBNL1 in stress granules in our HeLa cell model, using high-resolution AiryScan microscopy for which we provide detailed quantitation.

This aspect should be addressed experimentally since the authors also conclude that "a complex relationship between MBNL1 and CUGBP1 in stress granules" exists. Thus, the authors need to assess the colocalization of GFPMBNL1 with endogenous CUGBP1 in stress granules and the one of GFPCUGBP1 with endogenous MBNL1.

The complex relationship we propose is based on the effects of CUGBP1 or MBNL1 knockdown on the dynamic behaviours of each other by FRAP assay and not solely on their co-localisation, although we have already analysed their co-localisation in detail as above.

Figure 6: Please add antibody labeling to microscopy panels A and B.

Certainly, this was an accidental omission and has been added

Moreover, specify is the numbers refer to minutes in panel F. The data representation is also unclear - see comment above, Figure 5.

As stated in the figure legend and on the graph axes, these numbers have been normalised to the mean time taken for SG formation/loss in the control CTG12 cell line (set at 100%). The precise numbers in minutes for mean and SD are given in the text. We have added additional graphs of ‘% of cells with stress granules against time’ to this figure, with the values in minutes given to clarify the exact time-scale.

Figure 7: was 1,6-hexanediol added in presence of arsenite? Or was arsenite removed?

Arsenite was not removed (neither was Doxycycline) as we wanted to examine the effect of 1,6-hexanediol on SGs and PBs without the added complication of the effects of stress removal. We will clarify this point in the methods/results.

Aberrant persistent stress granules have been implicated in age-related (Mateju et al., 2017) and neurodegenerative diseases (Protter and Parker, 2016), such as ALS and FTD (Jain et al., 2016; Markmiller et al., 2018; Zhang et al., 2018). These are proposed to result from increased liquid-to-solid phase transitions within the stress granules (Mateju et al., 2017)." The authors should better define what are aberrant stress granules (e.g. see Ganassi et al., 2016; Turakhiya et al., 2018, PMID: 29804830).*

We will expand on this subject in the discussion

"Persistent stress granules have long been associated with degenerative conditions, notably ALS (Li et al., 2013)". I suggest updating the reference adding a more recent one.

We selected this 2013 review to emphasise that there is a long history of association of persistent stress granules with degenerative conditions. We will add in an additional, more recent review.

Significance

The work is descriptive; thus, in this form I do not consider that it is strongly advancing the field.

Having noted alterations to stress granule disassembly in lens epithelial cells from DM1 patients, we went on to develop a novel inducible model in which we replicated and enhanced these effects by expressing the large CUGexp RNA that causes DM1 as part of a DMPK mini-gene mimicking the genetic mutation seen in DM1 patients. This is not purely descriptive. Furthermore, we are now in a position to add an additional figure showing two pieces of evidence for functional defects in stress granules associated with CUGexp RNA expression 1) reduced accumulation of total PolyA RNA in stress granules indicating compromised function and 2) compromised ‘docking’ events between stress granules and P-bodies, a process proposed to be integral to the function of both structures.

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Referee #3

Evidence, reproducibility and clarity

The manuscript entiled " Phase-separated stress granules and processing bodies are compromised in Myotonic Dystrophy Type 1" by Gulyurtlu et al., characterizes the composition and ydnamics of stress granules and P-bodies in two Myotonic Dystrophy Type 1 (DM1) cell models, human lens epithelial cells from DM1 patients and age-matched controls and HeLa_CTG12_GFPMBNL1 and HeLa_CTG960_GFPMBNL1 cell lines. The manuscript is somewhat descriptive with lack of functional data and some discrepancies. For example, in the discussion section, the authors conclude that "MBNL1 appears to be absent from P-bodies in cells with CUGexp foci in their nuclei. This observation suggests that the role of MBNL1 in P-bodies may be disrupted by the presence of CUGexp RNA." Figure 4A shows that "P-bodies in the DM1 model line, HeLa_CTG960_GFPMBNL1 do not contain detectable amounts of GFPMBNL1". However, Figure 4E shows similar levels of total cellular MBNL1 per PB between the control CTG12 and mutated CTG960 lines. Most importantly, in Figure S3 the authors show that CUGexp foci are present in 1-2 % of the cells. The claim appears to be too strong for the data presented in the manuscript.

Although the findings are interesting and of potential impact for a better understanding of the implications of RNA-protein condensate dynamics in the pathogenesis of DM1, the work presented here is still descriptive and preliminary in my opinion. In summary, the conclusions are not so convincing and additional experiments are essential to support the authors claims. Several months of works will be required to consolidate data and reorganize and ameliorate the manuscript, including the way data are presented and quantified.

Specific comments:

"On removal of stress, clearance of stress granules is mediated largely by a form of autophagy." This statement is not correct since the majority of stress granules disassemble and are not targeted to autophagy; in healthy cells only 5 % (or less) of the total SGs tend to persist in presence of autophagy or lysosome inhibitors, while the vast majority disassembles. Please rephrase carefully.

Figure 1: RNA-protein complexes have heterogeneous composition. In HLECs, do all PBs colocalize with MBNL1 and CUGBP1 or only a fraction of them?

Figure 2: Stress granules and P-Bodies are known to touch each-other, a process referred to as a "kissing event". The authors have studied the mobility of GFP-MBNL1 inside these two types of assemblies. It would be important also to quantify the "kissing" events. Is this altered in DM1 cells?

Figure 3: In HeLa cells overexpressing CTG960_GFPMBNL1, beside the accumulation of one bright CUGexp puncta, several intranuclear GFPMBNL1 protein foci are visible. This subcellular distribution is different from the one observed in the control HeLaCTG12 GFPMBNL1. Can the author describe what these intranuclear GFPMBNL1 protein foci are? Is GFPMBNL1 accumulating at the level of splicing speckles? Or paraspeckles? Or other types on intranuclear condensates such as e.g. PML nuclear bodies? The different intranuclear distribution of GFPMBNL1 should be better characterized. Moreover, the % of cells expressing CTG960_GFPMBNL1 and forming intranuclear CUGexp foci is only mentioned in the discussion (Figure S3); for clarity it should be reported in the main text when describing Figure 3.

"Figure S2: Quantitation of GFPMBNL1 in P-bodies in HeLa cell model of DM1." The authors report in the legend "Some, but not all, of these P-bodies contain detectable amounts of GFPMBNL1". However, the figure only shows a representative image of cells without quantification. Quantitation should be provided.

The authors report "a subtle change in stress granule architecture associated with the presence of CUGexp RNA". This statement is not supported by experimental data and should be omitted.

Figure 4. MBNL1 and CUGBP1 co-localise in P-bodies. What is the % of colocalization?

Figure 5: "Treatment with sodium arsenite was then carried out under time-lapse microscopy, with Z-stacks of images taken every 4 minutes until stress granule formation was clearly seen (Fig.5A). This revealed a pronounced delay in formation of stress granules in cells containing CUGexp foci (HeLaCTG960 GFPMBNL1, 36 min +/- 12) compared to those without (HeLaCTG12 GFPMBNL1, 15 min +/- 2) (Fig.5B)." Data representation in Figure 5 is unclear and the pronounced delay in stress granule formation is not appreciated. Since the authors performed a live imaging taking pictures every 4 minutes, it would me more informative to plot the data and show the assembly and disassembly kinetics over time for both control and CTG960_ GFPMBNL1 cell lines (similar to what shown in e.g. Gwon et al., Science 2021, Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner, Figure 2G). Concerning Figure 1, the authors report no difference in the kinetic of stress granule formation in HLECs. However, they only report data after 45 and 60 min of arsenite treatment; at these time-points the assembly step is maximal. Thus, for consistency, the authors should include earlier time-points to the analysis of stress granule assembly also in HLECs, similar to what done in HeLa cells in Figure 5.

"Having established that MBNL1 and CUGBP1 co-localise closely in stress granules": the authors investigated the colocalization of each of these two proteins with stress granule markers but they did not verify whether MBNL1 and CUGBP1 co-localise. This aspect should be addressed experimentally since the authors also conclude that "a complex relationship between MBNL1 and CUGBP1 in stress granules" exists. Thus, the authors need to assess the colocalization of GFPMBNL1 with endogenous CUGBP1 in stress granules and the one of GFPCUGBP1 with endogenous MBNL1.

Figure 6: Please add antibody labeling to microscopy panels A and B. Moreover, specify is the numbers refer to minutes in panel F. The data representation is also unclear - see comment above, Figure 5.

Figure 7: was 1,6-hexanediol added in presence of arsenite? Or was arsenite removed?

Minor comments:

Aberrant persistent stress granules have been implicated in age-related (Mateju et al., 2017) and neurodegenerative diseases (Protter and Parker, 2016), such as ALS and FTD (Jain et al., 2016; Markmiller et al., 2018; Zhang et al., 2018). These are proposed to result from increased liquid-to-solid phase transitions within the stress granules (Mateju et al., 2017)." The authors should better define what are aberrant stress granules (e.g. see Ganassi et al., 2016; Turakhiya et al., 2018, PMID: 29804830).

"Persistent stress granules have long been associated with degenerative conditions, notably ALS (Li et al., 2013)". I suggest updating the reference adding a more recent one.

Significance

The work is descriptive; thus, in this form I do not consider that it is strongly advancing the field.

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 the current study, the authors compared the dynamics of P-bodies (PBs) and stress granules (SGs) between control and several DM1 cell lines. They found that MBNL1 and CUGBP1, two CUG repeat RNA-binding proteins that are primarily nuclear, could also co-localize with PBs in the cytoplasm and re-localize to SGs under stress. Small differences were observed in SG assembly and disassembly dynamics between control and DM1 HLECs, between HeLa cells expressing either CTG12 or CTG960, and between HeLa cells with and without shRNAs targeting CUGBP1 or MBNL1. Overall, the experiments were clearly described and the results properly presented. However, critical controls, as detailed below, are missing in multiple analyses. The mechanisms underlying these apparent differences are also unknown.

Major concerns:

1. Throughout the study, the authors compared MBNL1 and CUGBP1 association with PBs and SGs without considering the potential differences in their cytoplasmic abundance between control and DM1 cell lines, which seems to be case for MBNL1 abundance in CTG960-expressing HeLa cells (Fig. 3). Provided that PBs and SGs exchange components with the cytosol at an equilibrium, if the cytoplasmic abundance of, for example, MBNL1 is decreased in DM1, one would expect the equilibrium being shifted resulting in less MBNL1 associated with PB/SG. Therefore, before measuring the association or the assembly/disassembly kinetics of PB and SG, the authors should first test whether MBNL1 and CUGBP1 abundance may be different between control and DM cell lines. The same caveat applies to MBNL1/CUGBP1 knockdown experiments, where knocking down one may change the abundance of the other.
2. Similarly, the authors did not consider the possibility that changes in SG/PB dynamics may be due to changes in the abundance/availability of essential SG/PB components such as GE1 and G3BP1.
3. Most of the observed differences between control and DM cell lines were modest, leaving one wonder whether it could be simply due to cell line-to-cell line variability. Whenever possible, the authors should present results for each individual lines. For example, in Fig.2, 3 DM1 lines and 2 control lines were used. Was the difference in SG disassembly (Fig. 2B) observed in each of the 3 lines?

Minor points:

1. Western blot in Fig. 3 shows two protein products from both endogenous and overexpressed MBNL1. Please explain.
2. No data were shown to substantiate the statement that "MBNL1 localises to CUGexp foci and CUGBP1 does not" (page 6).
3. The y-axis of Fig. 4D should not go beyond 1.

Significance

The nature of the current study is highly descriptive with little mechanistic insights. For the subtle differences observed between control and DM1 cell lines, it remains unclear whether it may be due to cell line-to-cell line variation (see above). Some difference appear to be specific to one model but not the others (e.g., SG formation is slower in HeLa-CTG960 cells but not in DM1 HLECs). Even for the observations that seem consistent between models, the current results yielded little novel biological insights into whether and how these subtle differences in PB/SG dynamics may relate to DM1 pathogenesis. Collectively, these weaknesses render the current study incremental at best.

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Referee #1

Evidence, reproducibility and clarity

This manuscript by Gulyrutlu and co-workers addresses the role of CUG expanded repeat RNA associated with DM1 in regulating the formation of higher order RNP assemblies such as stress granules and P-bodies in the cell. The authors used lens epithelial cells (hLECs) derived from a DM1 patient or a HeLa cell inducible model of DM1 to investigate whether expression of the CUG repeat-associated protein MBNL1 and CUGBP1 affected the formation and dispersal of stress granules and P-bodies. The authors show that MBNL1 and CUGBP1 are components of SGs and PBs in hLECs and HeLa cells. In cells expressing the CUG repeat, there are minor alterations in the dispersal of stress granules as well as in the formation of P-bodies. MBNL1 could affect the formation and dispersal of SGs independent of the CUG repeat. Finally, in HeLa cells, overexpression of MBNL1 can reduce the dispersal of P-bodies upon 1,6-hexanediol treatment.

Major comments:

One limitation of the work is that the perturbations seen with stress granules or P-bodies are all relatively small, and no evidence for a functional consequence on gene expression is demonstrated. Specifically, the authors observe only minor alterations in the formation or disaggregation of PBs and SGs in these DM1 models. Further, some of the effects observed are independent of the CUG repeat expression, suggesting that MBNL1 and CUGBP1 might have independent roles in modulating some properties of SG and PB formation or dispersal.

1. The authors could investigate whether the CUG repeat RNA itself is localized to SGs or PBs in their models, and whether the presence of the repeat RNA is absolutely necessary for regulating the dynamics of SG or PB formation.
2. The authors use 1,6-hexanediol to suggests that PBs and SGs in HeLa cells show behavior analogous to LLPS. However, the use of 1,6,-hexanediol to establish an assembly as a LLPS is a relatively limited analysis (despite its widespread use in the field), since this compound can affect the formation of multiple cellular substructures that are not always LLPS (for example, see Wheeler et al, 2016, eLife).

Significance

This study would be of interest to the field if the impact of the DM! repeat RNAs on PB and SG were more substantial, and if some functional consequences were demonstrated. The lack of a strong effect on SG or PB formation in the DM1 models, along with the CUG repeat-independent effect of MBNL1 on the formation and dispersal of these complexes, argues that MBNL1/CUGBP1 may not significantly affect the formation or dispersal of SGs and PBs.

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Reply to the reviewers

Reviewer #1:

This paper puts together a nice set of data showing that a specific gene called Resf1 when deleted effects the ability of ESCs to self-renew and proceed to germline fates. I believe the data are sound and that they provide the evidence needed for the authors to make their conclusions.While I think the data are presented well and the manuscript is well-written, the "modest" functional results suggest this work would be more suited for a specialized journal.

We thank Reviewer 1 for their supportive comments.

• *

Reviewer #2:

1. In the presence of LIF, there is no difference between Resf1 knockout mESCs and WT mESCs except the expression of Esrrb, Nanog and Pou5f1. What about other genes? RNA-seq is needed to distinguish the two cell lines.

Fukuda et al. have shown that deletion of Resf1 leads to misregulation of ~1000 genes (adj. p-value 2) in presence of LIF. This highlights large differences between transcriptomes of Resf1 KO and WT cells that occur despite only a marginal difference in self-renewal efficiency between Resf1 KO cells and WT in the presence of LIF. It is therefore questionable whether the time and resources required to perform the requested RNA-seq would produce data that could unambiguously identify the potential causative effector difference downstream of Resf1.

As an alternative approach, we have reanalysed the Fukuda et al RNA-seq data. We find that Esrrb is significantly downregulated (in agreement with our Q-RT-PCRs), as are Klf4 and LifR (FDR 1.5). However, our meta-analysis of the Fukuda et al data did not show Pou5f1 and Nanog to be differentially expressed (FDR 1.5). This is in line with the lower level of downregulation of Pou5f1 and Nanog, compared to Esrrb in our Q-RT-PCR data. Notably, our gene expression analyses were performed in 5 biological replicates, whereas Fukuda et al. performed RNA-seq in two biological replicates. We can include the meta-analysis of the Fukuda et al data in our submission. As the change in ESC self-renewal that we see at low LIF concentrations could result from a decrease in Lifr expression, we will verify the change in expression of Lifr by Q-RT-PCR. Importantly, we will do this in a way that discriminates between expression of the transmembrane Lifr and soluble LifR, since the latter acts antagonistically (PMID: 9396734, Chambers, BJ, 1997).

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1. The authors showed Resf1 is not required for Nanog function, so how does Resf1 regulate the expression of pluripotency genes? Through epigenetic modifications or signaling pathways? The authors should design experiments to explain the detailed mechanisms.

The strength of the immunoblot signal for RESF1 is low, even when Resf1 is expressed episomally. Therefore, although we could try to co-immunoprecipitate with the Resf1-v5 cell line and endogenous Nanog, the expression level of RESF1 may mean this effort is unsuccessful. Given the fact that the result will not affect the conclusions of our study, we do not think this effort is justifiable.

1. The authors showed that Resf1 interacts with Nanog, but they used forced expressed proteins. Does the endogenous Resf1 interacts with endogenous Nanog? Do they bind to some same DNA sequences?

This are important questions to answer. However, many more experiments would be required to reach firm conclusions. The reviewer is right to say that the mechanisms by which Resf1 affects pluripotency are unknown and remain to be answered in future. We therefore propose to improve the text discussing similarities in pluripotency phenotype between deletions of Trim28, SETDB1, YTHDC1 and RESF1. As deletion of RESF1 partner SETDB1 or other proteins involved in repression of retrotransposons lead to downregulation of pluripotency genes and in some cases collapse of ESCs (e.g. PMID: 19884255, Bilodeau et al. 2009; PMID: 19884257, Yuan et al. 2009), we hypothesise that the RESF1 phenotype may be explained by affecting SETDB1 chromatin binding and therefore repression of SETDB1 targets. The mild phenotype of RESF1 KO indicates that RESF1 would not be an essential component of this repressor complex but rather “a modulatory protein”.

It is also worth noting that the meta-analysis of the RNA-seq data from Fukuda et al. suggests that Resf1-null ESCs may express reduced levels of LifR mRNA, and this is something we plan to investigate.

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1. In figure 5C, some Resf1 positive cells showed Nanog negative. Are these Nanog negative cells pluripotent?

Nanog-null ESCs are pluripotent (PMID: 18097409, Chambers et al., 2007). In addition, NANOG-negative cells in FCS/LIF cultures can retain pluripotency. Our purpose in this figure was therefore not to say whether NANOG-negative:RESF1-positive cells are pluripotent but to draw attention to the broader expression of RESF1 in FCS/LIF compared to NANOG. Such broader expression has also been noted for other heterogeneously expressed factors (PMID: 31582397, Pantier et al. 2019).

1. In figure 6A, the naïve mESCs are induced to EpiLCs. Is the transition efficiency of Resf1 knockout cells the same with WT mESCs? The finally obtained PGCLCs should be identified.

We show that the key TFs of EpiLC state are expressed similarly in WT and Resf1 KO cells (Supplementary figure 4) and we have data showing that WT and Resf1KO EpiLCs have a similar morphology. Together this suggests an efficient transition to an EpiLC state. Our analysis has identified expression of Blimp1/Ap2g/Prdm14 in Resf1-null cultures. Compared to wild-type cells these levels are reduced up to 3-fold. As this is from an unsorted population and the number of SSEA1/CD61-positive cells is decreased around 2x, this suggests that the PGCLC population formed by Resf1-null cells is reduced in proportion but is otherwise normal.

We will add photographs of EpiLC colonies formed by Resf1 KO and WT cells.

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1. in figure 5c, the scale bar is missing.

We will add missing scale bars in the figure 5C.

Reviewer #3:

1. What was less clear was an explanation of why colonies 4 and 24 were chosen. Were there other colonies with the desired expression? Was this amount of expression repeated in replicative experiments with approximately 2 colonies only available to be selected?

Approximately 30 colonies were selected for analysis. Of these, only 2 had deletion of both Resf1 alleles. We will make this point clearer in the text.

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1. Figure 1C, 5C and S2B with microscopic images should include a scale bar.

Missing scalebars in the Figure 1C will be added. Unfortunately the microscopy setup used to collect the images in Figures 5C and S2B did not allow scalebars to be added at the time of imaging and these cannot be added retrospectively. However, we do not think that inclusion of scalebars, even were it possible would affect the conclusions of our manuscript.

1. Figure 1E needs a better explanation of the significance, "less clear cut" is not adequate. Reporting statistics, or lack of significance, on the graph would help.

We will update the manuscript and the Figure 1E to include results of a statistical analysis (Wilcoxon-rank sum test) comparing formation of AP+ colonies between Resf1 KO and WT cells at different LIF concentrations. These results show that both Resf1 KO cell lines have lower median number of AP+ colonies than WT cells at LIF concentrations 0 and 1 (p.adj. *

1. It's translatability to medicine, although perhaps that is not the intention, is somewhat lacking. Is there a naturally occurring situation where LIF is absent that would require this pathway to be used? These were mouse ESC's, perhaps this study could incorporate information about relevant translation to a human condition to aid in the significance. This manuscript suggests a mechanistic evaluation by which self-renewal can occur other than the canonical pathway, which is interesting and can inform the field.

Our results suggest that RESF1 directly or indirectly supports self-renewal of ESCs. Interestingly, Human cell atlas identified RESF1 expression as a negative predictor of survival of renal cancer and was found to be expressed in testis cancer cells and other cancer tissues. Therefore, RESF1 could promote self-renewal of cancer cells similarly to ESCs. However, this is speculative and needs further studies. As this is both outside of the scope of this manuscript and our expertise, we do not think it prudent for us to pursue this line of inquiry. However, we agree that further studies could evaluate RESF1 function in human tissues, especially pluripotent cells and germ cells. As we show that RESF1 deletion leads to reduced induction of PGCLCs and previous studies showed infertility of Resf1 KO mice, investigating link between human fertility and RESF1 could have implications in reproductive medicine.

We will improve our discussion to highlight the possible significance of RESF1 function in human fertility.

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Referee #3

Evidence, reproducibility and clarity

The authors aimed to study RESF1 in ESC's to understand it's role in germ cell specification and PGCLC differentiation under in vitro experimental challenges.

The experiments performed and reported were thorough and convincing. Data and methods were clearly explained.

What was less clear was an explanation of why colonies 4 and 24 were chosen. Were there other colonies with the desired expression? Was this amount of expression repeated in replicative experiments with approximately 2 colonies only available to be selected?

Figure 1C, 5C ad S2B with microscopic images should include a scale bar.

Figure 1E needs a better explanation of the significance, "less clear cut" is not adequate. Reporting statistics, or lack of significance, on the graph would help.

Significance

Understanding the specific interactions and suggested role of RESF1 in self-renewal is informative on a molecular biology and developmental biology level.

It's translatability to medicine, although perhaps that is not the intention, is somewhat lacking. Is there a naturally occurring situation where LIF is absent that would require this pathway to be used? These were mouse ESC's, perhaps this study could incorporate information about relevant translation to a human condition to aid in the significance. This manuscript suggests a mechanistic evaluation by which self-renewal can occur other than the canonical pathway, which is interesting and can inform the field.

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Referee #2

Evidence, reproducibility and clarity

The authors uncovered the new roles of Resf1 in mESC self-renewal and germline entry. They showed that Resf1 deletion reduced mESC self-renewal, and it's not required for Nanog function. In addition, the efficiency of PGCLC specification of Resf1 knockout mESC is less than WT mESC. However, these conclusions are too preliminary and the underlying mechanism is missing.

Major comments:

1. In the presence of LIF, there is no difference between Resf1 knockout mESCs and WT mESCs except the expression of Esrrb, Nanog and Pou5f1. What about other genes? RNA-seq is needed to distinguish the two cell lines.
2. The authors showed Resf1 is not required for Nanog function, so how does Resf1 regulate the expression of pluripotency genes? Through epigenetic modifications or signaling pathways? The authors should design experiments to explain the detailed mechanisms.
3. The authors showed that Resf1 interacts with Nanog, but they used forced expressed proteins. Does the endogenous Resf1 interacts with endogenous Nanog? Do they bind to some same DNA sequences?
4. In figure 5C, some Resf1 positive cells showed Nanog negative. Are these Nanog negative cells pluripotent?
5. In figure 6A, the naïve mESCs are induced to EpiLCs. Is the transition efficiency of Resf1 knockout cells the same with WT mESCs? The finally obtained PGCLCs should be identified.

Minor comments:

1. in figure 5c, the scale bar is missing.

Significance

The authors uncovered the new roles of Resf1 in mESC self-renewal and germline entry.

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Referee #1

Evidence, reproducibility and clarity

This paper puts together a nice set of data showing that a specific gene called Resf1 when deleted effects the ability of ESCs to self-renew and proceed to germline fates. I believe the data are sound and that they provide the evidence needed for the authors to make their conclusions.

Significance

While I think the data are presented well and the manuscript is well-written, the "modest" functional results suggest this work would be more suited for a specialized journal.

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Reply to the reviewers

First of all, we would like to thank the each of the expert reviewers for their effort in evaluating our study. We are confident that we can positively address each of the issues and queries raised by the reviewers.

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** This study investigates the functions of the tricalbin proteins in S. cerevisiae, which are homologs extended synaptotagmins in mammals. It suggests the tricalbins modulate plasma membrane phospholipid composition and are particularly important for surviving shift to elevated temperature. The tricalbins are proposed to directly or indirectly promote phosphatidylserine transport from the ER to the plasma membrane during heat stress. They also promote the localization of the kinase Pkh1 to the plasma membrane during heat stress.

**Major comments:**

General Response: We thank the reviewer for the comments and opinions. While they are starkly different from the other two reviewers’, they have caused us to consider how we can add additional support for our conclusions and to consider alternative possibilities.

1. To determine whether the tricalbins and other tethering proteins play a role in phospholipid homeostasis, lipid distribution is measured using biosensors (FLARES) and phospholipid levels are determined by mass spec. The experiments are well done but say little about what role the tricalbins or other tethering proteins play in homeostasis. There are no measurements of lipid transport or rates of phospholipid production, degradation or modification. It is reasonable to propose the tricalbins transport lipids, since other proteins with SMP domains do, but this study does not present any evidence that they do.

Response: We thank the reviewer for stating that the quantitative microscopy and lipidomics experiments in our study are well done. However, we strongly disagree that the results do not provide new information on the roles of the tricalbins or other tethering proteins in membrane lipid homeostasis. In fact, the lipidomics results definitively show that Ist2 and Scs2/22 control phosphatidylserine (PS) levels. In contrast, loss of the tricalbins does not significantly affect PS levels. We will provide a new figure to make this even more clear to expert and non-expert readers.

While the tricalbins do not regulate PS levels, the data clearly show that the tricalbins control PS acyl chain saturation and cellular distribution. A PS reporter is reduced at the plasma membrane (PM) upon loss of the tricalbins and we have now confirmed increased localization at the endoplasmic reticulum (ER). These new results will be included in a revised manuscript. As the lipid desaturase Ole1 is localized in the ER, the lipidomics are consistent with increased ER residency of PS species. Thus, the data indicate that while the tricalbins do not regulate PS levels, they control either delivery of PS from the ER to the PM, and/or they may control the organization and stability of PS at the PM which would be a novel finding on its own.

The reviewer must be aware that there are currently no in vivo cell assays that directly (only) measure lipid transport. These experiments are subject to several factors including lipid metabolism, anterograde transport rates, bilayer organization, lipid accessibility, and retrograde transport rates. While our findings clearly show that the Tcb proteins do not control PS levels, we agree that there are alternative possibilities to explain the changes in PS distribution. In our revised manuscript, we will perform additional experiments to distinguish between these possibilities. New experiments will include mutant forms of Tcb3 bearing substitutions in the SMP domain. We will also examine whether the Tcb proteins control PS organization, availability/accessibility, and stability at the PM (also see Reviewer #3, comment 2). This latter possibility may reveal a novel concept regarding Tcb/E-Syt protein function that goes beyond their proposed conventional role as lipid transfer proteins. Based on the outcome of these experiments, we shall adjust the final cartoon model and conclusions in the discussion accordingly.

The study convincingly demonstrates that there are fewer Pkh1 puncta formed after temperature shift in cells lacking tricalbins than in wild-type cells (Fig. 6 C,D). However, there is no demonstration that this change in localization alters Pkh1 function or signaling.

Response: Regulation of Pkh1 by lipids is outside the scope of our current study that is focused on providing new understanding of Tcb protein function. However, the decrease in heat-induced Pkh1 puncta may provide insight into the PM integrity defects in cells lacking Tcb1/2/3 (as Pkh1 is required for PM integrity). To test whether Pkh1 function is compromised in the tcb1/2/3 mutant cells, we can test whether constitutively active Ypk1 (which acts downstream of Pkh1) rescues the PM integrity defects in tcb1/2/3 mutant cells.

There is no demonstration that association of tricalbins and Skh1 (Fig. 4) has any functional significance or affects phosphoinositide metabolism.

Response: We thank the reviewer for raising this issue. If the association of Tcb3 and Sfk1 has functional significance, then one would expect that loss of the proteins should phenocopy one another. Deletion of the Sfk1 cytoplasmic domain necessary for co-localization with Tcb3 should also phenocopy loss of Tcb3. This is exactly what we find. Localization of the PS probe is decreased at 42oC upon loss of Sfk1 or truncation of the Sfk1 cytoplasmic tail, similar to cells lacking Tcb3. Furthermore, we find that Tcb3 regulates sterol homeostasis at the PM (using the D4H probe), as has been recently reported for Sfk1 (Kishimoto et al, 2021). Thus, Tcb3 and Sfk1 not only co-localize, but they also share common functions in PM lipid organization. These new results will be presented in our revised manuscript.

The reviewer also inquired about potential roles for Tcb3 and Sfk1 in phosphoinositide lipid homeostasis, as Sfk1 has reportedly been implicated in heat-induced PI(4,5)P2 synthesis. However, while we find clear roles for Sfk1 and the tricalbins in PS and sterol homeostasis, we did not find a requirement for Sfk1 or the tricalbins in PI(4,5)P2 homeostasis upon heat stress conditions. These findings will be included in our revised manuscript. Importantly, our results indicate that the tricalbins and Sfk1 primarily control PS and sterol homeostasis at the PM, and may regulate phosphoinositides indirectly, and thus provide new insight into the key role of these proteins.

The study proposes the tricalbins directly or indirectly promote phosphatidylserine transport after temperature shift, but transport has not measured and other possibilities are not ruled out.

Response: While the Tcb3 SMP domain has been shown to transfer phospholipids in vitro (Qian et al., 2021), we agree that a role in PS transfer in vivo should be examined in more detail and that other possible roles in PS homeostasis should also be considered (also see responses to Reviewer #3, comment 2).

Upon heat shock, we not only observe a decrease in relative levels of the PS reporter at the PM in the tcb1/2/3 mutant cells (as shown in our original manuscript), but also a corresponding increase in relative levels of the PS probe at the ER and vacuole membrane (also see response to Reviewer #3, comment 5). This could reflect impaired delivery of PS from the ER to the PM and reduced stability of PS at the PM (i.e. increased internalization of PS into the cell).

In our original manuscript, we showed that deletion of the SMP domain (the lipid transfer domain), phenocopies deletion of the full-length Tcb3 protein in terms of PS distribution and PM integrity following heat shock. To more rigorously test whether lipid transport activity of the SMP domain is responsible for these phenotypes, we will generate amino acid substitutions within the SMP domain of Tcb3 that maintains its overall structure but impairs its ability to transport lipids (by targeting conserved key residues identified in Saheki et al., 2016). We will then assess whether SMP-mediated lipid transfer is necessary for PS homeostasis and PM integrity under heat stress.

We also agree that other possibilities should be examined. First, to rule out a defect in PS production upon heat stress, we are performing new mass spectrometry lipidomics experiments to measure levels of individual phospholipid species in the tcb1/2/3 mutant and wild type cells after heat stress.

Second, we have considered whether the Tcb proteins control phospholipid bilayer distribution (e.g. flip and flop). However, cells lacking the Tcbs are not hypersensitive to duramycin (Omnus et al. 2016) and thus phosphatidylethanolamine exposure on the extracellular leaflet is not increased. Moreover, cells lacking the Tcbs (and Scs2/22 and Ist2) are not impaired in the uptake of exogenous NBD-labelled phospholipids (and thus flip across the PM bilayer is not impaired). Possibly, there may be increased lipid ‘flop’ in the mutant cells at high temperature. We can test whether there is increased phospholipid exposure in the extracellular leaflet at high temperature, but our results thus far indicate accumulation of PS on internal membrane compartments (the ER and vacuole membrane).

Another potentially exciting possibility is that the tricalbin proteins bind and stabilize PS within the cytosolic leaflet of the PM and prevent its internalization by endocytosis or non-vesicular transfer. This mechanism would be completely independent of lipid transport to the PM and would constitute new mechanistic insight into Tcb function. We will test whether PS (and sterol) becomes more accessible (less stable or reduced sequestered pools at the PM) and internalized into the cell, upon removal of the tricalbin proteins. For example, we will monitor PS distribution in cells where endocytosis is blocked with latrunculin A.

As mentioned, there currently no cellular lipid transport assay that directly (only) measure anterograde transport. However, if the Tcb3 SMP domain mutants are impaired in PS homeostasis and PM integrity, then we can consider monitoring PS transfer in vivo. By performing the experiments outlined here, we will have thoroughly characterised the roles of the tricalbin proteins in PS homeostasis at the PM. Moreover, the new findings may even reveal novel roles that are independent of transport.

Reviewer #1 (Significance (Required)): While this study is likely to be of interest to those studying the tricalbins or phospholipid homeostasis, it is incremental and provides little conceptual advance on what is already known about the tricalbins and extended synaptotagmins. They have already been implicated in lipid homeostasis in the plasma membrane and this study provides no new mechanistic insight into how this occurs. Similarly, it has already been shown that the tricalbins play a role in maintaining cell integrity during heat stress and there is little new insight into what role the tricalbins play. Perhaps the most notable part of the study is the idea that tricalbins are necessary for phosphatidylserine transport during stress, but considerable additional work is necessary to make a strong case for this claim.

Response: We strongly disagree with the reviewer’s opinions. Indeed, Reviewer #2 found our study “novel and detailed” and Reviewer #3 found the results in our study to be “highly valuable” and “interesting”.

In contrast to the reviewer’s claims, there are certainly novel findings in our study. Foremost, this is the first study that demonstrates a role of the tricalbins in PS homeostasis. Previous studies have implicated E-Syt family members in diacylglycerol and phosphoinositide regulation. Our results indicate that the tricalbins and Sfk1 primarily control PS and sterol homeostasis at the PM, not phosphoinositides, and thus provide new insight into the key role of these proteins. Second, while a previous study by Collado et al reported a role of the tricalbins in PM integrity upon heat stress, this work did not provide mechanistic insight into this process. We performed the PM integrity assays for the Collado et al study (as co-authors). Our current study now shows that Tcb function is needed for PS homeostasis and Pkh1 recruitment at the PM upon heat stress; both factors are needed for PM integrity under these conditions. As such, our current study does provide new insight into roles of the Tcbs in PM integrity. Finally, we are exploring roles of the Tcb proteins in PS homeostasis that go beyond their proposed functions as lipid transfer proteins. We are convinced that our study will provide novel and deep mechanistic understanding of the Tcb/E-Syt protein family.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): The study examines the roles of tricalbins in signalling in yeast. By using several mutations they are able to evaluate whether the interactions are caused by tethering the PM and ER or by other mechanisms. Particualrly powerful is their use of cryo-EM tomography and shot-gun mass spectrometery. They look at all the major lipids of the ER and PM and consider their interconversions. They identify large changes in lipid species with one double bond and with two, particularly for PS.

Reviewer #2 (Significance (Required)): There is little information about membrane physical properties and how it changes as a result of changes in lipid molecular species. Nevertheless, the information provided is novel and detailed. The topic of ER-PM contact sites is new and evolving and this paper advances our understanding of the yeast system considerably. It also looks at protein-protein interactions by fluorescence methods and studies the consequences of heat shock.

Response: We are pleased that the reviewer concluded that our study on the Tcb proteins “advances our understanding of the yeast system considerably” and found our use of lipidomics to be “powerful”.

This reviewer only had only one critique; there “is little information about membrane physical properties and how it changes as a result of changes in lipid molecular species”. In our revised manuscript, we will provide new data showing changes in levels of sterol (ergosterol) accessibility/availability at the PM in cells lacking the tricalbin proteins. Sterol lipids exist in distinct pools in the PM bilayer (extracellular vs. cytoplasmic leaflet, accessible vs. sequestered) that control the biophysical and mechanical properties of the PM (packing order, permeability, etc.). Moreover, PS and sterol lipids are proposed to undergo mutual associations whereby PS controls sterol accessibility (the ‘umbrella’ model) and sterol in turn stabilizes PS in the cytoplasmic leaflet of the PM. Our findings demonstrate that the primary function of the Tcb proteins is PS and sterol organization in the PM, providing new mechanistic insight into regulatory mechanisms for membrane homeostasis. We will attempt to further characterize changes in PM mechano-chemical and biophysical properties to further understand how changes in membrane lipid composition affect membrane integrity.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): The Tcb/ESyt proteins play important role in contact site formation and non-vesicular lipid transport. However, their exact functions remain highly controversial. This study by Stefan and colleagues revealed a new role for the yeast Tcb proteins, especially Tcb3, in regulating plasma membrane phosphatidylserine, as well as PIP2. These results are highly valuable to people working on membrane contact sites and lipid trafficking. Overall, the results are fairly convincing and interesting. There are however some concerns and suggestions:

Response: We are pleased that the reviewer stated that our study has “revealed a new role for the yeast Tcb proteins” and that the findings are “fairly convincing and interesting”. We also thank the reviewer for providing constructive criticisms and helpful suggestions.

1. Fig. 4, for Tcb3 and Sfk1 interaction, what about Tcb1/2, which would be good controls for the specificity of the interaction.

Response: We agree that it would be useful to determine if the Tcb3-Sfk1 interaction is specific. We will perform additional BiFC experiments to address whether Tcb1 and Tcb2 associate with Sfk1. Our previous work suggested that Tcb3 forms heterodimers with Tcb1 and Tcb2 necessary for PM integrity (Collado et al., 2019). However, the functional association between Sfk1 and Tcb3 may be specific to Tcb3, and we will test this possibility. It would be interesting to identify a function specific to an individual Tcb protein.

A central question is whether Tcb3 transfers PS by itself through the SMP domain or requires other lipid transfer proteins. One possibility is that the transfer is mediated by Osh6 and Osh7. Did the expression level of Osh6/7 change between the delta tether and the ist2scs2/22 null strain? Under normal and stressful conditions?

Response: We agree that it is important to address whether Tcb3 transfers PS via its SMP domain (an issue also raised by Reviewer #1) or whether this activity is carried out by another lipid transfer protein, such as Osh6 and Osh7. We have considered several alternative possibilities, as well as designed and performed new experiments, as described below (two key experiments are in italics).

To rigorously test whether SMP domain-mediated lipid transport activity is required for PS homeostasis at the PM under heat stress, we will generate amino acid substitutions within the Tcb3 SMP domain that maintain its overall structure but impair its ability to transport lipids (targeting conserved key residues described in Saheki et al., 2016 & Bian et al., 2018). We will then assess whether SMP-mediated lipid transfer is necessary for PS homeostasis and PM integrity under heat stress.

As suggested by the reviewer (and discussed in our original manuscript), the tricalbins might serve as scaffolds for PS transfer proteins, including the Osh6 and Osh7 proteins, under stress conditions. Osh6 and Osh7 are recruited to ER-PM contacts through interactions with the Ist2 tether protein where they mediate PS delivery to the PM under non-stress conditions (D’Ambrosio et al., 2020). However, strong lines of evidence suggest that Ist2, Osh6, and Osh7 are not required for PS homeostasis at the PM under stress conditions. First, loss of Ist2 has no impact on the PS probe under heat stress conditions (this result will be included in the revised manuscript). Therefore, the Ist2-Osh6/7 interaction is not required for PS homeostasis upon heat stress. Ist2 is not required for PM integrity upon heat stress either (Omnus et al., 2016).

These results do not rule out the possibility that the tricalbins serve as scaffolds for other PS transfer proteins, such as Osh6 and Osh7, under stress conditions. In this scenario, a switch between Osh6/7 tethering proteins may occur: from Ist2 under normal growth conditions to the Tcb proteins during stress conditions. However, our findings also suggest that Osh6 and Osh7 function is impaired upon stress conditions, including heat and nutrient starvation. Notably, Osh6 and Osh7 become mis-localized from the PM under upon heat stress (we can provide this data in the revised manuscript). The mechanisms for Osh6/7 attenuation upon stress conditions is outside the scope of our current study, but our preliminary results suggest that changes in cytoplasmic pH and ion homeostasis are involved; this will be a focus of a future study. This is also in line with results from a previous study (Omnus et al., 2020) that showed Osh3 forms intracellular aggregates in response to heat stress. The activity of the Osh proteins and other lipid transfer proteins, in general, may be impaired upon stress conditions (see below).

To directly determine whether Osh6 and Osh7 are required for PS homeostasis at the PM under heat stress conditions, we will monitor the distribution of the PS probe in cells lacking the Osh6 and Osh7 (osh6 osh7 double mutant cells) upon heat stress. This is a key experiment that will directly address the reviewer’s concerns.

As suggested by the reviewer, we can also examine the expression and localization of GFP-tagged Osh6 and/or Osh7 in tcb1/2/3, scs2/22 ist2, and ‘delta tether’ mutant cells. However, we have not observed any changes in other Osh proteins, including Osh2 and Osh3, in the ‘delta tether’ mutant strain.

Finally, we have considered yet another possibility. PS transfer to the PM may be generally attenuated under heat stress conditions and a key role of the Tcb proteins may be to bind and stabilize PS at the PM. In other words, the Tcb proteins may act as a ‘buttress’ to stabilize and maintain pre-existing pools of PS at the PM under stress conditions. Consistent with this idea, the Tcb3 C2 domains are required for PS homeostasis and PM integrity upon heat stress. If the Tcb3 SMP domain mutants are not impaired in PS homeostasis or PM integrity, then this alternative mechanism may be very relevant to PM quality control and organization in response to membrane stress. To address this possibility, we will address whether PS is internalized or removed (extracted) from the PM upon heat stress in cells lacking the Tcb proteins. This may uncover a novel role of the Tcb proteins that is independent of SMP domain-mediated lipid transfer.

Figure 5A. It is not obvious that the intensity of C2 decreases in tcb null cells at 42 degree. Perhaps there is more internal staining.

Response: We thank the reviewer for pointing out this issue. We think Figures 5A and 5B together convincingly show increased cytoplasmic localization of the PS reporter in the tcb1/2/3 mutant cells upon heat stress. More importantly, we thank the reviewer for pointing out the increased localization at internal membrane compartments. We realized it would be important to identify the PS-containing membrane compartments in the tcb1/2/3 mutant cells upon heat stress. We have now confirmed that the PS probe localizes at the ER and vacuole membrane (to be included in the revised manuscript). The example in Figure 5A also shows accumulation of the PS probe at both the nuclear ER and vacuole membrane. Thus, whilst wild type cells show little PS reporter localization at the ER or vacuole membrane, loss of the tricalbin proteins leads to an increase in ER and vacuole membrane PS probe localization after heat shock. Accumulation at the ER may reflect impaired PS delivery from the ER to the PM, and possibly rerouting to the vacuole membrane. Alternatively, as discussed above, vacuole membrane localization may be due increased PS removal from the PM and delivery to the vacuole membrane in tcb1/2/3 cells upon heat stress.

It is important to determine the primary function of Tcb3 since both defects in both PIP2 and PS were observed. If the change in PIP2 is due to a lack of PS, can overexpressing Osh6/7 rescue the PIP2 defect in the tetherless mutant?

Response: We agree that is important to determine whether the primary function of the Tcb proteins is regulation of PS or PI(4,5)P2 homeostasis. Our new findings definitively indicate that their primary function is PS regulation, not PI(4,5)P2 regulation. A clear effect in the distribution of the PS reporter was observed following heat shock in the tcb1/2/3 mutant cells. In contrast, there is no difference in the localization of the PI(4,5)P2 reporter in tcb1/2/3 cells compared to wild type after heat shock (also see response to reviewer #1, comment 3). In addition, cells lacking the Tcb proteins were not impaired in heat-induced PI(4,5)P2 synthesis, as assessed by metabolic labelling and HPLC analysis. These findings will be included in our revised manuscript, as they indicate that the Tcb proteins primarily control PS at the PM, not phosphoinositides, and thus provide new insight into the main role of these proteins.

Both PS and sterol are required for the proper recruitment of type I PIP5K to the PM (Nishimura et al., 2019). Therefore, defects in PS distribution could be responsible for the PI(4,5)P2 effects observed in Figure 2. However, overexpression of Osh7 in the ‘delta tether’ mutant did not significantly rescue localization of the PI(4,5)P2 reporter (included in the revision plans). Sterol organization is also perturbed in the ‘tether’ mutant cells (Quon et al., 2018), and this may explain why Osh7 expression did not rescue. Accordingly, Osh2/3/4 (sterol transfer proteins) rescue the PI(4,5)P2 effects.

The detection of PS by LactC2 has been well-established. However, an alternative approach would be to use the 2XPH in permeabilized cells. See PMID: 33929485 for some detailed discussions on the techniques. It is not a requirement for the authors to adopt the 2XPH.

Response: We thank the reviewer for suggesting another technique to confirm the results of the LactC2 domain as a PS probe. In this study, we have primarily used a genetically encoded LactC2 probe to observe PS distribution within live, intact cells. Whilst this approach was sufficient to identify accumulation of PS on cytosolic membrane leaflets of the ER and vacuole (see above), the addition of an exogenous probe to permeabilized cells may allow the detection of PS on luminal and extracellular membrane leaflets. Therefore, we plan to repeat our heat shock experiments using permeabilized cells and a purified tagged form of the LactC2 protein. This may allow for improved imaging of intracellular PS localisation and bilayer distribution. However, these experiments are technically challenging, and fixation and permeabilization conditions have not yet been optimized for yeast cell experiments. It is not yet known whether we will be able to optimize these protocols in a reasonable amount of time while completing revisions to the manuscript.

**Minor:**

1. the discussion seems to be a bit long

Response: We will shorten the discussion and modify our final conclusions based on the results from the new experiments.

Reviewer #3 (Significance (Required)): These proteins are highly important in cell biology/contact sites. The redundancy made it difficult to pinpoint their function. Previous studies have had a number of models. The current study proposed a new function of these proteins, i.e. PS transfer, and this is very interesting and valuable. There will be a good audience for this work. I specialize in lipid storage and trafficking, lipid droplets, cholesterol and phosphatidylserine.

Response: We are pleased that this expert reviewer found our study to be “very interesting and valuable”.

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Referee #3

Evidence, reproducibility and clarity

The Tcb/ESyt proteins play important role in contact site formation and non-vesicular lipid transport. However, their exact functions remain highly controversial. This study by Stefan and colleagues revealed a new role for the yeast Tcb proteins, especially Tcb3, in regulating plasma membrane phosphatidylserine, as well as PIP2. These results are highly valuable to people working on membrane contact sites and lipid trafficking. Overall, the results are fairly convincing and interesting. There are however some concerns and suggestions:

Major:

1.Fig. 4, for Tcb3 and Sfk1 interaction, what about Tcb1/2, which would be good controls for the specificity of the interaction.

1. A central question is whether Tcb3 transfers PS by itself through the SMP domain or requires other lipid transfer proteins. One possibility is that the transfer is mediated by Osh6 and Osh7. Did the expression level of Osh6/7 change between the delta tether and the ist2scs2/22 null strain? Under normal and stressful conditions?
2. Figure 5A. It is not obvious that the intensity of C2 decreases in tcb null cells at 42 degree. Perhaps there is more internal staining.
3. It is important to determine the primary function of Tcb3 since both defects in both PIP2 and PS were observed. If the change in PIP2 is due to a lack of PS, can overexpressing Osh6/7 rescue the PIP2 defect in the tetherless mutant?
4. The detection of PS by LactC2 has been well-established. However, an alternative approach would be to use the 2XPH in permeabilized cells. See PMID: 33929485 for some detailed discussions on the techniques. It is not a requirement for the authors to adopt the 2XPH.

Minor:

1. the discussion seems to be a bit long

Significance

These proteins are highly important in cell biology/contact sites. The redundancy made it difficult to pinpoint their function. Previous studies have had a number of models. The current study proposed a new function of these proteins, i.e. PS transfer, and this is very interesting and valuable. There will be a good audience for this work. I specialize in lipid storage and trafficking, lipid droplets, cholesterol and phosphatidylserine.

Referee Cross-commenting

I was asked to include my expertise in the Significance part. Other reviewers did not include it. Maybe my last sentence should be removed?

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Referee #2

Evidence, reproducibility and clarity

The study examines the roles of tricalbins in signalling in yeast. By using several mutations they are able to evaluate whether the interactions are caused by tethering the PM and ER or by other mechanisms. Particualrly powerful is their use of cryo-EM tomography and shot-gun mass spectrometery. They look at all the major lipids of the ER and PM and consider their interconversions. They identify large changes in lipid species with one double bond and with two, particularly for PS.

Significance

There is little information about membrane physical properties and how it changes as a result of changes in lipid molecular species. Nevertheless, the information provided is novel and detailed. The topic of ER-PM contact sites is new and evolving and this paper advances our understanding of the yeast system considerably. It also looks at protein-protein interactions by fluorescence methods and studies the consequences of heat shock.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This study investigates the functions of the tricalbin proteins in S. cerevisiae, which are homologs extended synaptotagmins in mammals. It suggests the tricalbins modulate plasma membrane phospholipid composition and are particularly important for surviving shift to elevated temperature. The tricalbins are proposed to directly or indirectly promote phosphatidylserine transport from the ER to the plasma membrane during heat stress. They also promote the localization of the kinase Pkh1 to the plasma membrane during heat stress.

Major comments:

1. To determine whether the tricalbins and other tethering proteins play a role in phospholipid homeostasis, lipid distribution is measured using biosensors (FLARES) and phospholipid levels are determined by mass spec. The experiments are well done but say little about what role the tricalbins or other tethering proteins play in homeostasis. There are no measurements of lipid transport or rates of phospholipid production, degradation or modification. It is reasonable to propose the tricalbins transport lipids, since other proteins with SMP domains do, but this study does not present any evidence that they do.
2. The study convincingly demonstrates that there are fewer Pkh1 puncta formed after temperature shift in cells lacking tricalbins than in wild-type cells (Fig. 6 C,D). However, there is no demonstration that this change in localization alters Pkh1 function or signaling.
3. There is no demonstration that association of tricalbins and Skh1 (Fig. 4) has any functional significance or affects phosphoinositide metabolism.
4. The study proposes the tricalbins directly or indirectly promote phosphatidylserine transport after temperature shift, but transport has not measured and other possibilities are not ruled out.

Significance

While this study is likely to be of interest to those studying the tricalbins or phospholipid homeostasis, it is incremental and provides little conceptual advance on what is already known about the tricalbins and extended synaptotagmins. They have already been implicated in lipid homeostasis in the plasma membrane and this study provides no new mechanistic insight into how this occurs. Similarly, it has already been shown that the tricalbins play a role in maintaining cell integrity during heat stress and there is little new insight into what role the tricalbins play. Perhaps the most notable part of the study is the idea that tricalbins are necessary for phosphatidylserine transport during stress, but considerable additional work is necessary to make a strong case for this claim.

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Reply to the reviewers

1. General Statements We are grateful to all reviewers for the critical comments and valuable suggestions that have helped improve our paper. We provide point-to-point answers to the comments and added detailed explanations in the preliminary revised manuscript. We put the comments made by the reviewer in italics with our responses below.

Description of the planned revisions

Reviewer #1

\*Major comments:***

- The authors thought almost all iRFP forms a holo-complex with BV when HO1 is expressed in fission yeast. This should be proved by calculating the percentage of BV-iRFP in yeast. It is meaningful to compare the percentage of BV-iRFP and PCB-iRFP in vitro and in yeast.

We would like to thank the reviewer for this valuable comment. We plan to quantify the percentage of iRFP holo-complex in vitro and in yeast by using fluorescence correlation spectroscopy (FCS). In FCS, the fluctuation of fluorescence emitted from a very tiny space (i.e., confocal volume, ~ 1 fL) in solution is measured and statistically analyzed by autocorrelation, providing the average number of fluorescent molecules in the confocal volume (Krichevsky and Bonnet, 2002, Rep. Prog. Phys.). The fusion proteins of iRFP with mNeonGreen (mNG) are purified or expressed in yeast, and thus the stoichiometry of iRFP to mNG must be 1:1. The numbers of iRFP and mNG are measured by FCS in vitro or in yeast. If iRFP forms 100% holo complex with BV or PCB, the number of fluorescent iRFP is equal to that of mNG. We have used FCS to measure the concentration of fluorescent protein in mammalian cells (Sadaie et al., 2014, MCB; Komatsubara et al., 2020, JBC), and therefore have no technical difficulties. One possible limitation is the maturation efficiency of mNG in vitro and in yeast, although it would not disturb at least the qualitative comparison of holo-complex formation between iRFP-BV and iRFP-PCB. In this preliminary version of the revised manuscript, we added the data of FCS measurement in yeast, but not yet in vitro, in Supplementary Figure S5A. The numbers of fluorescent iRFP molecules relative to the numbers of mNG were comparable between iRFP-BV (HO1 expressing cell) and iRFP-PCB (SynPCB2.1 expressing cell). Of note, the number of fluorescent iRFP molecules in cells treated with external BV was significantly lower than that in cells expressing HO1, suggesting the low permeability of BV.

In addition to the FCS measurement, we prepare the following backup experiments; recombinant iRFP proteins are mixed with sufficient amounts of BV or PCB, and used them as a reference. Then, the iRFP proteins are expressed and purified from yeast, and subjected to Zinc blot with the reference iRFP proteins to compare the percentage of iRFP holo-complex in vitro and in yeast. This method is feasible, but we are not able to quantitatively determine the percentage of iRFP holo-complex with BV or PCB.

-The brightness of fluorescent proteins in organisms often depends on the molecular brightness (fluorescence quantum yield and extinction coefficient) and the amounts of fluorescent proteins. The authors indicated that iRFP-PCB is brighter than iRFP-BV at the molecular level. To calculate the amounts of iRFP-PCB and iRFP-BV when different proteins are expressed in yeast, it is better to explain the results that phycocyanobilin was better than biliverdin for the imaging in fission yeast.

We agree with the reviewer’s comment. We will quantify the amounts of iRFP in the fission yeast cells expressing iRFP, iRFP/HO1, and iRFP/SynPCB2.1 by western blot analysis and fluorescent imaging. With regard to western blot analysis, the iRFP proteins are normalized by Tubulin in western blot analysis, which has been used as the reference protein.

During this preliminary revision period, we assessed the expression levels by fluorescence microscopy. The fusion proteins of iRFP fused with mNG were expressed in yeast and the expression levels were quantified by the fluorescence signals of mNG. In this preliminary version of the revised manuscript, we only added fluorescence imaging data in Supplementary Figure S4. The fluorescence intensities of mNG among cells expressing iRFP, iRFP/HO1, and iRFP/SynPCB2.1 were comparable as well as cells treated with BV or PCB. We will further confirm this result by western blotting analysis.

To test the application of PCB as chromophore in mammalian cells, a HO1 gene knock out mammalian cells should be used.

We would like to thank the reviewer for this excellent suggestion. It is indeed interesting to examine the effect of an HO1 gene knock-out on the iRFP fluorescence and the application of PCB as a chromophore in mammalian cells. We plan to knock out the HO1 gene in HeLa and HeLa/BVRA KO cells by conventional CRISPR/Cas9 genome editing techniques. After the establishment of HO1-KO HeLa cell lines, iRFP fluorescence is assessed in the same way as we did in Figure S9. In addition, we will carefully investigate the effect of BV in serum on iRFP fluorescence.

The authors may use the all-in-one plasmids carrying SynPCB2.1 and iRFP fusion protein genes to image the target proteins in mammalian cells.

According to the reviewer’s suggestion, we will construct an all-in-one plasmid carrying SynPCB2.1 and an iRFP fusion protein gene for iRFP imaging in mammalian cells.

To this end, the IRES and iRFP genes are inserted downstream of the SynPCB2.1 gene cassette, and iRFP fluorescence is confirmed in mammalian cells.

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

Reviewer #1

\*Minor comments:***

- In line 35. iRFP is derived from bacteriophytochromes.

We have replaced “phytochrome” with “bacteriophytochrome”.

- In line 62. The reference of Rodriguez et al. deals with allophycocyanin instead of bacteriophytochromes.

We have included the following note in the revised manuscript.

(Page 3, line 56)

“Near-infrared fluorescent proteins have been developed through the engineering of phytochromes, which are photosensory proteins of plants, bacteria, and fungi (Chernov et al., 2017), or allophycocyanin, which is a light-harvesting phycobiliprotein of cyanobacteria (Rodriguez et al., 2016). “

- In lines 65-66. Bacteriophytochromes bind BV, phycocyanin, allophycocyanin and cyanobacterial phytochromes bind PCB, and plantal phytochromes bind PΦB.

Thank you for the notification. We have added the explanation in the revised manuscript as follows.

(Page 3, line 65)

“Unlike the canonical fluorescent proteins derived from jellyfish or coral, phytochromes and allophycocyanin require a linear tetrapyrrole as a chromophore such as biliverdin IXα (BV), phycocyanobilin (PCB), or phytochromobilin (PΦB); bacteriophytochromes bind to BV, allophycocyanin and cyanobacterial phytochromes bind to PCB, and plantal phytochromes bind to PΦB.”

- In lines 67-71. The authors described the biosynthesis of BV, PCB and PΦB. But it missed many references.

We agree with this reviewer’s comment, and have included the references in the revised manuscript as follows.

(Page 3, line 70)

“These linear tetrapyrroles are produced from heme (Terry and Lagarias 1991; Beale 1993). Heme-oxygenase (HO) catalyzes oxidative cleavage of heme to generate BV with the help of ferredoxin (Fd), an electron donor, and ferredoxin-NADP+ reductase (Fnr) (Cornejo, Willows, and Beale 1998). In cyanobacteria, PCB is produced from BV through PcyA, Fd, and Fnr, while in plants PΦB is synthesized from BV using HY2, Fd, and Fnr (Muramoto et al. 1999; Frankenberg et al. 2001; Kohchi et al. 2001).“

- In line 270. One full stop should be deleted in "cells.. Fission".

We have corrected this mistake.

- In line 272. How did the authors add the high concentration of PCB (625 microM) into the culture? PCB is insoluble.

As the reviewer pointed out, 625 microM PCB seem to be insoluble in PBS solution and fission yeast culture medium, because insoluble PCB debris was observed in the medium. We have included the following note in Materials and Methods of the revised manuscript.

(Page 20, line 505)

“Of note, 625 μM PCB or 625 μM BV is insoluble in PBS solution and fission yeast culture medium, and 25 μM PCB or 25 μM BV is insoluble in mammalian cell culture medium, because insoluble PCB or BV debris is observed.”

- In line 401. smURFP is derived from allophycocyanin instead of cyanobacteriochromes.

We have corrected this mistake.

- In line 474. As BV and PCB are insoluble, how do the authors add the pigments into DMEM?

We directly added BV or PCB dissolved in DMSO into the culture medium of HeLa cells, i.e., DMEM, 10% FBS, and maintained cells for 3 hours. We have included the following note in the figure legend of the revised Supplementary Information.

Fig. S9 legend

“Of note, 25 μM BV or PCB remained unsolved in the cultured medium for mammalian cells, and therefore chromophores in the medium could be saturated under these conditions.”

- In line 509. In which solvent are BV and PCB dissolved?

BV or PCB dissolved in DMSO was added into His-iRFP PBS solution. We have added the explanations as follows.

(Page 22, line 555)

“Purified His-iRFP in PBS solution was mixed with an excess amount (1:5 molar ratio) of BV or PCB dissolved in DMSO, followed by size exclusion chromatography with NAP-5 Columns (Cytiva, 17085301) to remove free BV or PCB.”

- In lines 546, 547. What is ROI?

We apologize for the reviewer’s confusion. ROI is the abbreviation of “region of interest.” We have included the full expression in the revised manuscript.

- The authors should indicate whether codon optimization was necessary when HO1, PcyA, Fd and Fnr were expressed in yeast or in mammalian cells.

We would like to thank the reviewer for pointing out this issue. The codon optimization for expressing SynPCB genes was needed in mammalian cells (Uda et al., PNAS, 2017). In fission yeast, we used the same SynPCB gene optimized for human codon usage, but not for fission yeast codon usage. As far as we used, there have been no problems with PCB synthesis in fission yeast. We have included this important note in the revised manuscript as follows.

(Page 20, line 483)

“The nucleotide sequence of these genes and SynPCB were optimized for human codon usage (see Benchling link; Table S1). ”

- Figure S1 should show the software and algorithm for the construction of phylogenetic.

Thank you for pointing out the ambiguity in our statement. We have not constructed the species tree on our own; instead, we have manually drawn the tree by Adobe Illustrator, based on the latest genome-scale phylogeny of fungi (Yuanning Li et al. 2021), where results of multiple algorithms were compared to evaluate the reliability of phylogenies. To make it explicit, we revised the manuscript as follows.

(Page 24, line 627)

“We have manually drawn the evolutionary relationship among representative species (Nguyen et al. 2017) based on a recent genome-scale phylogeny (Yuanning Li et al. 2021), which is consistent with the current consensus view of the fungal tree of life (James et al. 2020).”

Here, to validate that the tree in Figure S1 is consistent with the current consensus phylogeny, we have added a reference to a recent review on the fungal tree of life (Timothy Y. James et al., Ann Rev Microbiol, 2020). Additionally, we modified the tree in Figure S1 to represent the remaining ambiguity among subdivisions in Basidiomycota (Yuanning Li et al. 2021, Figure 4G).

- In Figure 1C and Figure 2C. The authors should explain the reasons why the fluorescence of iRFP was lower when yeast was treated with excessive BV and PCB.

The decrease in iRFP intensity under 625 μM PCB or 625 μM BV could be due to cell death and/or toxicity by the excess DMSO. We have included the explanation in the revised manuscript.

Figure 1 legend

“The decrease in iRFP intensity under 625 μM BV could be due to cell death and/or toxicity by the excess DMSO”

Figure 2 legend

“The decrease in iRFP intensity under 625 μM PCB or BV could be due to cell death and/or toxicity by the excess DMSO”

- In Supplementary Information, line 51. What is "teh"?

We have corrected this mistake.

Reviewer #2

\*Minor comments:***

- In the paragraph headed "iRFP brightens iRFP more efficiently..." there is mention of "NLS-iRFP-NLS". Please introduce the abbreviation (nuclear localisation sequence?) and, if possible, why the sequence is present N- and C-terminally.

We would like to thank the reviewer for this comment. The abbreviation of NLS (nuclear localization signal) has been already included in the previous version of manuscript (page 5, line 113). Two NLSs are fused with iRFP because the addition of a single NLS does not sufficiently localize the protein at the nucleus. We have included this note in the revised manuscript (page 5, line 114).

- The fluorescence intensity increase upon PCB compared to BV treatment of S. pombe cultures expressing iRFP (Fig. 2C,D) appears about 7-fold, which is far more than the factor of 2 measured on the level of fluorescence quantum yield, and the protein levels were determined to be comparable. Are there any factors conceivable that further enhances the signal of PCB-bound iRFP in S. pombe?

As we have discussed in the previous version of the manuscript, we presume the three factors enhancing fluorescence of iRFP-PCB in fission yeast; (1) the increase in quantum yield (~ 1.61-fold, Fig. 3F), (2) blue-shifted excitation and emission spectrum of iRFP-PCB, which are beneficial for our microscopic setup, and (3) the efficiency of chromophore formation (~ 1.75-fold) (Rumyantsev et al. 2015). During this revision, we calculated to what extent the second factor potentially enhances iRFP-PCB fluorescence compared to iRFP-BV. Based on the emission spectrum (Fig. S3C, left), iRFP-PCB is approximately 1.3-fold more effectively excited by 640 nm of excitation laser than iRFP-BV. Similarly, the detection of iRFP-PCB fluorescence is about 2.0-fold more efficient than that of iRFP-BV with our emission filter (665-705 nm emission filter). Based on these data, the rough estimation yields 1.61*1.3*2*1.75 = 7.3-fold increase, which is comparable with the experimental results showing 5~10-fold increase in iRFP-PCB (Figs. 2C, 2D, 3G). We have included this additional discussion in the revised manuscript as follows:

(page 17, line 429)

“Based on the emission and excitation spectrum (Fig. S3C), iRFP-PCB is approximately 1.3-fold more effectively excited by 640 nm of the excitation laser, and detected about 2.0-fold more efficiently with our emission filter (665-705 nm emission filter) in comparison to iRFP-BV.”

(page 17, line 433)

“Based on these data, the rough estimation yields 1.61*1.3*2*1.75 = 7.3-fold increase, which is comparable with the experimental results showing the 5~10-fold increase in iRFP-PCB fluorescence compared to iRFP-BV (Figs. 2C, 2D, 3G).”

- In the spectral data of Fig. 3D,E, there appears to be a spectrometer artifact at around 450 nm in all spectra, which is not commented on. It is certainly not part of the cofactor / holoprotein spectra.

The peaks around 450 nm in spectra are the artifact of our spectrometer for unknown reasons. We have added an explanation of this artifact in the revised manuscript.

(page 10, line 287,290)

“Of note, there is a spectrometer artifact at around 450 nm in all spectra.”

- Lines 292/296: "Fig. 3H" should read "Fig. 2G".

We have corrected these mistakes.

- The reader may wonder whether it is a common or rather rare phenomenon that the exchange of BV by PCB or some other bilin as (usually covalently bound) cofactor in iRFPs (or, more general, also in the parental phytochromes from different branches of the tree of Life) can be performed. Are there comparable studies available in the literature in addition to the work of Rumyantsev et al. (2015)?

According to the reviewer’s suggestion, we examined the fluorescence of miRFP670 and miRFP703 in fission yeast cells under the conditions of DMSO, BV, or PCB treatment, or SynPCB2.1 expression. We found that the fluorescence intensities of miRFP670 and miRFP703 with PCB treatment or SynPCB2.1 expression showed much higher values than those with BV treatment as observed in iRFP fluorescence in this study (Figure S7). These data indicate that the replacement of BV with PCB is beneficial for enhancing the fluorescence intensity of other iRFPs. We have included the data in the revised manuscript as follows:

(page 12, line 340)

“To explore the generality of the application of PCB and SynPCB2.1 system to other near-infrared fluorescent proteins, we measured fluorescence intensities of miRFP670 and miRFP703, which are derived from a different branch of bacteriophytochrome RpBphP1 (Shcherbakova et al. 2016), in fission yeast treated with BV or PCB or expressing SynPCB2.1 (Fig. S7). The fluorescence intensities of both miRFP670 and miRFP703 were enhanced by the addition of PCB and the expression of SynPCB2.1 compared to the addition of BV in a similar manner iRFP (Fig. S7). From these data, we concluded that PCB biosynthesis by SynPCB2.1 is suitable for imaging with near-infrared fluorescent proteins in fission yeast.”

Reviewer #3

(1) In the Fig. 1D, authors tried to measure the BV incorporation into fission yeast cells. How about the time after 180 min, is it still a increase for the fluorescence?

According to the reviewer’s suggestion, we did the same experiments in Figure 1D for up to 24 hours. As the reviewer expected, iRFP fluorescence still increased gradually for up to 24 hours. We replaced Figure 1D with the new data. We do not have any good idea why iRFP fluorescence gradually increases in the presence of BV.

(2) The authors used the Fig. 3H in the manuscript, but I did not find the H in the Fig. 3.

We apologize for the reviewer’s confusion. We have corrected these mistakes.

(3) The information for the BVRA KO HeLa cells need to be provided.

We have included the information of the BVRA KO HeLa cells in the materials and methods.

(page 21, line 519)

“BVRA KO HeLa cells have been established previously (Uda et al. 2017)”

Description of analyses that authors prefer not to carry out

Reviewer #1

- What is the affinity (KD) of BV and PCB to iRFP? Whether is this related to the fluorescence intensity of iRFP in yeast?

The attachment of BV or PCB to iRFP is an irreversible reaction, i.e., BV or PCB is covalently attached to iRFP. For this reason, it is technically impossible to measure equilibrium dissociation constant (Kd) values to evaluate the affinity of BV or PCB to iRFP.

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Referee #3

Evidence, reproducibility and clarity

In this manuscript, entitled "Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin", Sakai and co-workers report that phycocyanobilin (PCB) could function as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast. They first found that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence due to the lack of BV and HO gene. Upon the addition of external BV, the iRFP fluorescence signal could be recovered. In addition, expression of HO1 in fission yeast cells also resulting in the iRFP fluorescence. Next, they found that PCB brightens iRFP more efficiently than BV in fission yeast and in vitro, while the fluorescence excitation and emission spectra were 10 nm blue-shifted in iRFP-PCB compared to iRFP-BV. Finally, they introduced a system named SynPCB2.1 for efficient PCB biosynthesis in fission yeast and concluded that PCB biosynthesis by SynPCB2.1 is ideal for iRFP imaging in fission yeast based on the experiments data. They also developed all-in-one plasmids carrying SynPCB2.1 and iRFP fusion protein genes to image the target proteins in fission yeast. In the final part of this manuscript, PCB was tested as an iRFP chromophore in HeLa cells, however it does not offer significant advantage over BV. Overall, this work provides a system for efficient iRFP imaging in fission yeast. Yet, several issues have been identified and need to be addressed in order to strengthen or even validate some of the conclusions made by the authors.

(1) In the Fig. 1D, authors tried to measure the BV incorporation into fission yeast cells. How about the time after 180 min, is it still a increase for the fluorescence?

(2) The authors used the Fig. 3H in the manuscript, but I did not find the H in the Fig. 3.

(3) The information for the BVRA KO HeLa cells need to be provided. To test the application of PCB as chromophore in mammalian cells, a HO1 gene knock out mammalian cells should be used. The authors may use the all-in-one plasmids carrying SynPCB2.1 and iRFP fusion protein genes to image the target proteins in mammalian cells.

Significance

This work provides new information regarding the chromophore of iRFP. It shows that PCB brightens iRFP more efficiently than BV in fission yeast and in vitro. The all-in-one plasmids system developed in this work supplies a tool for iRFP imaging in the organisms without BV production. Although the iRFP-PCB produces a brighter fluorescence compared with iRFP-BV, the fluorescence excitation and emission spectra were ~15 nm blue-shifted compared to that of iRFP-BV, which might restrict its applications in tissues imaging. As the abundant of BV exist in the mammalian cells, iRFP-PCB does not offer significant advantage over iRFP-BV.

Referee Cross-commenting

All the reviewers gave reasonable suggestions.

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Referee #2

Evidence, reproducibility and clarity

Summary:

iRFPs have been engineered from the chromophore-binding domains of bacterial phytochromes to expand the wavelength range of genetically-encoded markers for fluorescence imaging and sensing applications into the far-red or near-infrared. In contrast to fluorescent proteins from jellyfish or corals, in which the chromophore is formed autocatalytically, iRFPs require a bilin chromophore for fluorescence such as biliverdin IX alpha (BV), phycocyanobilin (PCB) or phytochromobilin (P[phi]B). While available in some, these molecules may be not available in sufficient amounts in the particular cell type under study, and, therefore, frequently requires the introduction of at least one further gene such as heme oxygenase (HO, to generate BV from heme) or in addition PcyA (which produces PCB from BV). In this study, the authors show that one of the previously described iRFPs (denoted as iRFP713 in the original studies), which is non-fluorescent upon expression in the fission yeast Schizosaccharomyces pombe due to the lack of an endogenous HO gene, is able to complement with PCB in S. pombe when added externally (to the culture medium) or if produced intracellularly with the help of a plasmid developed for PCB synthesis (termed SynPCB2.1). It is shown that iRFP with PCB bound as cofactor exhibits brighter fluorescence than the BV-bound original iRFP713, which is due to a nearly two-fold increased fluorescence quantum yield as shown by in vitro reconstituted and in vivo generated iRFP with bound PCB. S. pombe cells harbouring the SynPCB2.1 system for PCB synthesis even release ("leak") PCB into the surrounding medium to be taken up by cells not producing PCB. Furthermore, the authors report the generation of a plasmid for C-terminal tagging of a protein of interest by iRFP, novel genome integration vectors and all-in-one plasmids harbouring the genes for PCB synthesis and iRFP-fused marker proteins. The genome integration vectors ensure that stable one-copy integration into the genome occurs at different uncritical loci on each of the chromosomes (different from the common Z-locus), which are in gene-free regions and distant enough for crossing strains, and at sites that do not affect the auxotrophy characteristics of cells. The plasmid system, termed pSKI, harbours elements for propagation in E. coli, constitutive or inducible promoters, a multiple cloning site, a selection marker cassette and homology arms separated by a unique restriction enzyme cutting site for plasmid linearization. The viability of the C-terminal fluorescence tagging system relying on PCB-bound iRFP in S. pombe is shown with 10 different proteins of variant and highly specific intracellular location, which all show the expected cellular distribution pattern by fluorescence imaging. Multi-spectral fluorescence labelling schemes (manifold of five) were successfully tested in S. pombe with four specifically localized proteins harbouring different conventional FP tags, and, in addition, one labeled with iRFP(PCB). Finally, it was tested whether PCB can be exploited to yield a brighter iRFP fluorophore also in a mammalian model cell line, HeLa cells. Here, treatment of cells with externally added BV or PCB produced a similar increase in iRFP fluorescence and also knockout cells devoid of the BV breakdown enzyme BVRA did not show different fluorescence levels upon BV or PCB treatment. Therefore, it is concluded that PCB is applicable to iRFP imaging in mammalian cells though offering no advantage, which to some extent limits the advantages to cells which naturally do not produce BV from heme.

Major comments:

The claim that the developed plasmid system for multi-spectral fluorescence imaging applications in fission yeast S. pombe, which relies on PCB synthesis and incorporation of PCB into iRFP, is fully justified by the data shown in the manuscript. Interpretation and the derived conclusions are put forward in a decent and balanced way. No additional experiments are required to justify the claims.

Minor comments:

• In the paragraph headed "iRFP brightens iRFP more efficiently..." there is mention of "NLS-iRFP-NLS". Please introduce the abbreviation (nuclear localisation sequence?) and, if possible, why the sequence is present N- and C-terminally.
• The fluorescence intensity increase upon PCB compared to BV treatment of S. pombe cultures expressing iRFP (Fig. 2C,D) appears about 7-fold, which is far more than the factor of 2 measured on the level of fluorescence quantum yield, and the protein levels were determined to be comparable. Are there any factors conceivable that further enhances the signal of PCB-bound iRFP in S. pombe?
• In the spectral data of Fig. 3D,E, there appears to be a spectrometer artifact at around 450 nm in all spectra, which is not commented on. It is certainly not part of the cofactor / holoprotein spectra.
• Lines 292/296: "Fig. 3H" should read "Fig. 2G".
• The reader may wonder whether it is a common or rather rare phenomenon that the exchange of BV by PCB or some other bilin as (usually covalently bound) cofactor in iRFPs (or, more general, also in the parental phytochromes from different branches of the tree of Life) can be performed. Are there comparable studies available in the literature in addition to the work of Rumyantsev et al. (2015)?

Significance

The observation of an increased fluorescence quantum yield upon PCB insertion into iRFP is a technical advance with value as such, which will stimulate more detailed studies by spectroscopists (fluorescence, IR, Raman in particular) to clarify the principles underlying the increased quantum yield. While the findings and the developments of this study present a clear advance for imaging applications in fission yeast (available for cell biologists), at least the data on HeLa cells show that the method may not present an advance in terms of delivering a better (i.e. brighter) near-infrared chromophore for mammalian cells. It seems that the ability of a cell line to produce BV (from heme with the help of a HO) limits the potential of PCB addition to iRFP-expressing cells, since BV is readily taken up by the protein shortly after protein biosynthesis. Thus, more elaborate interventions in heme metabolism may be required to fully exploit the potential of the findings in mammalian cell systems and to fulfil the claim that also the optogenetics toolbox may benefit from the findings in the future.

My own expertise relates to spectroscopic analyses (fluorescence, IR, Raman) of iRFP and mutant variants thereof in search of the determinants for increased fluorescence quantum yield, fluorescence lifetime analyses and photoreceptor research.

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Referee #1

Evidence, reproducibility and clarity

• This paper provided a method for near-infrared imaging using iRFP and proved that phycocyanobilin was better than biliverdin for the imaging in fission yeast.

Major comments:

• What is the affinity (KD) of BV and PCB to iRFP? Whether is this related to the fluorescence intensity of iRFP in yeast?
• The authors thought almost all iRFP forms a holo-complex with BV when HO1 is expressed in fission yeast. This should be proved by calculating the percentage of BV-iRFP in yeast. It is meaningful to compare the percentage of BV-iRFP and PCB-iRFP in vitro and in yeast. -The brightness of fluorescent proteins in organisms often depends on the molecular brightness (fluorescence quantum yield and extinction coefficient) and the amounts of fluorescent proteins. The authors indicated that iRFP-PCB is brighter than iRFP-BV at the molecular level. To calculate the amounts of iRFP-PCB and iRFP-BV when different proteins are expressed in yeast, it is better to explain the results that phycocyanobilin was better than biliverdin for the imaging in fission yeast.

Minor comments:

• In line 35. iRFP is derived from bacteriophytochromes.
• In line 62. The reference of Rodriguez et al. deals with allophycocyanin instead of bacteriophytochromes.
• In lines 65-66. Bacteriophytochromes bind BV, phycocyanin, allophycocyanin and cyanobacterial phytochromes bind PCB, and plantal phytochromes bind PΦB.
• In lines 67-71. The authors described the biosynthesis of BV, PCB and PΦB. But it missed many references.
• In line 270. One full stop should be deleted in "cells.. Fission".
• In line 272. How did the authors add the high concentration of PCB (625 microM) into the culture? PCB is insoluble.
• In line 401. smURFP is derived from allophycocyanin instead of cyanobacteriochromes.
• In line 474. As BV and PCB are insoluble, how do the authors add the pigments into DMEM?
• In line 509. In which solvent are BV and PCB dissolved?
• In lines 546, 547. What is ROI?
• The authors should indicate whether codon optimization was necessary when HO1, PcyA, Fd and Fnr were expressed in yeast or in mammalian cells.
• Figure S1 should show the software and algorithm for the construction of phylogenetic.
• In Figure 1C and Figure 2C. The authors should explain the reasons why the fluorescence of iRFP was lower when yeast was treated with excessive BV and PCB.
• In Supplementary Information, line 51. What is "teh"?

Significance

• near-infrared fluorescent protein broadens the spectrum of fluorescent protein and facilitates deep tissue imaging. Based on iRFP, this work successfully constructed the near-infrared fluorescent protein with PCB as chromophore in fission yeast by using the method of synthesizing PCB, which has been realized in mammalian cells by this group. The method of synthesizing PCB in yeast not only facilitates fluorescence imaging, but also meaningful for optogenetics experiments in yeast. This work is useful for the study of fluorescence imaging, optogenetics and biosynthesis when using yeast as a model organism.

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Reply to the reviewers

Manuscript number: RC-2021-00831

Corresponding author(s): Lu, Gan

Reviewer comments are in regular font. Our rebuttal is in bolded font. Experiments that we plan for the full revision are preceded with “FULL:”. In the revision files, the changes are highlighted in yellow.

General Statements

We thank the reviewers for their detailed feedback. There are two major concerns. First, the manuscript lacks functional analysis of the meiotic triple helices (MTHs). Second, the manuscript makes claims about the properties of synaptonemal complexes (SCs) and MTHs that are inadequately supported. In order to address the first concern, we would need extensive experiments to first identify and then perturb the genes associated with the MTH. Such experiments are beyond the scope of this manuscript and are the focus of future studies. We will address the second concern with mostly text revisions. We will also improve some of the imaging analysis with new experiments that can be done in a few months’ time.

2. Description of the planned revisions

We will acquire new cryo-ET data of pachytene-arrested cell cryolamellae using our new K3-GIF camera. These new data have higher signal-to-noise ratios and allow us to generate a higher-resolution subtomogram average of the MTHs. The achievable resolution will depend on the conformational homogeneity of the MTH segments and on the number of cryotomograms we can capture. If we are able to achieve a subnanometer-resolution reconstruction, we will narrow down the possible identities of the subunits. Even if we cannot achieve subnanometer resolutions, the new data will allow us to test if ladder-like densities were missed in our lower-resolution older data, thereby improving our understanding of the SC’s structure. We will also perform subtomogram analysis of purified ribosomes as a control to strengthen our handedness determination.

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

The Reviewers’ original comments are reproduced in regular font. Line numbers refer to the preliminary revision.

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

Ma and coworkers report studies of budding yeast undergoing meiosis by cryo-ET. They fail to detect structures interpretable as synaptonemal complex, and instead detect feather-like bundles of what appears to be a triple helix. These structures do not appear to be related to the synaptonemal complex, as spo11 mutants that do not initiate recombination, red1 mutants that lack axial elements, and zip1 mutants that lack the central element of the SC still make these bundles. These structures are absent from cells treated with latrunculin A, which depolymerizes actin filaments, but expected structures are not visible in light microscopy of cells treated with two different F-actin-staining reagents. However, it should be noted that another study (Takagi et al, 2021, bioRxiv) did detect actin associated with these structures by immunogold labeling. The structures are also reversibly dissolved in 7% hexanediol.

This part of the paper's findings is well supported by data and is certainly of interest, although interest is somewhat limited by the unknown nature of these structures-what they contain, let alone their function, remains to be determined-in fact, it is not even determined whether or not they are made of protein. However, as an initial report of a previously unknown phenomenon, the paper is of some value.

__Thank you for raising the issue of whether MTHs are composed of protein or not. Aside from the proteins, the only other materials capable of forming large bundles of linear polymers are polysaccharides and DNA. Yeast polysaccharides are found in the cell wall, so they are unlikely to be a candidate for the MTHs. In the nucleus, DNA is abundant. While we favor that MTHs are composed of protein, we cannot rule out that the MTHs are non-chromatin DNA-protein complexes. Depending on the resolution of future subtomogram averages, we may get a better idea of the MTH’s composition.__

There is, unfortunately, a second aspect of the paper that cannot be supported. Although it is clear that synaptonemal complex is present in the cells examined (by standard cytological methods) the authors cannot detect structures consistent with SC in their cryo-ET images. Unfortunately, authors then extrapolate from their inability to detect SC to conclusions about SC, such as that it is not crystalline, and even go so far as to suggest that their failure to detect SC invalidates two models for crossover interference, and that the ladder-like structure reported for SC in many organisms using many difference approaches may be a fixation artifact. Authors show remember that the absence of evidence is not evidence for absence; the speculation described above should be removed from both the abstract and discussion.

We have removed the speculation about crossover interference and limited the scope of our discussion on SCs to budding yeast only.

The differences between traditional EM and cryo-EM are not trivial. In the introduction, we added more details to explain the differences in both the sample preparation and contrast generation:

Lines 79-87: “Meiotic nuclei have been studied for decades by traditional EM (Fawcett, 1956; Moses, 1956), but not by cryo-ET. Cryo-ET can reveal 3-D nanoscale structural details of cellular structures in a life-like state because the samples are kept unfixed, unstained, and frozen-hydrated during all stages of sample preparation and imaging (Ng and Gan, 2020). The densities seen in cryo-ET data come from electron scattering of the biological macromolecules. In comparison, the densities seen in traditional EM from electron scattering of heavy metals such as uranium, tungsten, and osmium, which have adhered to a subset of biological macromolecules that were not extracted in earlier steps.”

We have also removed the term ‘artifact’ from lines 201-202:

Original: “The ladder model is based on images of traditional EM samples, which are vulnerable to fixation and staining artifacts.”

Revised: “The ladder model is based on images of traditional EM samples.”

Reviewer #1 (Significance (Required)):

This paper reports a previously unreported structure in the nuclei of yeast cells undergoing meiosis. The composition and function of this structure remain to be determined. This considerably limits the significance of the paper.

**Referee Cross-commenting**

I agree with the concerns of the other reviewers. I also agree with reviewer 3 that to raise the significance of the paper would require much work. But I think that the raw observation is of value, albeit in a journal of record. So I would stick with my recommendation of text changes, keeping in mind that there may not be a suitable journal in LSA's portfolio.

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

This work describes helical filamentous structures observed in budding yeast nuclei that were cryosectioned and imaged using cryo-electron tomography (cryoET).

The goal of this work seems to have been to conduct an ultrastructural analysis of the synaptonemal complex (SC), a meiosis-specific protein structure that holds chromosomes together during meiosis and is thought to regulate meiotic recombination. In conventional TEM images of fixed, embedded, and stained sections obtained from pachytene nuclei, SCs usually appear as long, thin, transversely striated structures. At pachytene, SCs extend along the full length of a thin (100-nm) gap between paired chromosomes (typically 1-6 µm long in yeast cells). Surprisingly, the authors did not observe SCs, perhaps because these structures do not produce much contrast in cryo-EM images. Instead, they observe abundant triple helical structures in the nucleoplasm, which they designate as "meiotic triple helices" (MTHs). The authors report that these triple helices assemble at the same time as but independently of the SC. They publised a preprint in which they indicated that these structures were somehow related to SCs, but this the revised version reports that they appear independently of SCs. They further report that treatment of cells with the F-actin depolymerizing drug Latrunculin-A (LatA) resulted in a lack of detectable triple helical structures in the nucleus, suggesting that these structures may be a form of actin or, alternatively, that they may require F-actin for their assembly.

While the work is technically mostly sound, its significance is unclear because the reported structures have no known function. Many, if not most proteins will form helical structures if their concentration rises above a threshold defined by their binding affinity for themselves (see https://doi.org/10.1186/1741-7007-11-119 and references therein), so this may simply be an example of an abundant nuclear protein that polymerizes to form helical filaments under the conditions that trigger yeast sporulation.

Thank you for raising the interesting possibility that MTHs form helices because their subunits have exceeded a critical concentration threshold. In the revised text, we discuss the possibility that the MTH is simply a protein that is highly expressed in meiotic cells and polymerize either due to exceeding a critical concentration, or having undergone a biochemical change like a post-translational modification:

Lines 408-415: “Note it is possible that the MTHs may not be directly involved in meiosis, but are instead a protein that is at a sufficient concentration or has the right biochemical modifications to form helices in pachytene because it is known that many proteins can form a helix under the right conditions {Crane, 1950; Pauling, 1953; Theriot, 2013}. These MTHs also have lateral interactions that allow them to pack with crystalline density. Their sensitivity to 1,6-hexanediol suggests that the polymerization both within and between MTHs are based on hydrophobic interactions. Further work will be needed to determine the identity of the MTH’s subunits and their potential function.”

The authors perform cryosectioning and cryoEM on yeast cells undergoing meiosis to show that the assembly and disassembly of MTHs follow a similar time course as that of the SC. The observation of these triple-helical filaments in meiotic nuclei has also been reported in another study (https://www.biorxiv.org/content/10.1101/778100v2.full.pdf+html), which proposed, based on immuno-EM labeling, that they may be actin cables. This study reports that the structures are not detected using phalloidin or Lifeact-mCherry. However, treatment with LatA did eliminate detection of MTH structures, suggesting that they may be comprised of actin.

In my view, there are a number of issues that should be addressed before publication. Many of these relate to the presentation of the findings. Detailed comments below:

1. The presentation of the work is very confusing. The authors clearly expected to observe SC structures, but did not. They conclude that MTHs are not SCs, since they do not depend on molecular components required for SC assembly. They should describe their findings in a more straightforward way rather than veering from introducing the SC to describing the MTHs.

We have restructured the manuscript to tone down the discussion about the SC. However, we have to start with the SC because it is the most iconic feature of pachytene cells and a major organizer of chromatin in meiosis. Furthermore, its presence, as indicated by Zip1-GFP signals, was key to establishing that our cells were indeed arrested in pachytene. It would have been confusing to then overlook the absence of structural features as conspicuous and prevalent as what was expected of SCs. The other sections did have room for improvement. In the sections below, we describe the changes point by point.

Similarly, the discussion section on "recombination and chromosome segregation" seems inappropriate and irrelevant, since no data are presented in this study regarding the functions of the MTHs, and there is no reason to think that they contribute to crossover interference, chromosome segregation, or other aspects of meiosis. Additionally, most of the ideas presented in this section seem very muddled. I recommend deleting this section.

We have deleted the section entitled "Recombination and chromosome segregation".

Throughout the text, we have also changed the term “meiosis-specific” to “meiosis-related” when describing MTHs. Doing so allows for the possibility that MTHs might just form as a consequence of being expressed to a high enough concentration as discussed above.

Along the same lines as comment #1: The title should be changed - absence of evidence for "ladders" is not evidence of absence. Prior work using TEM and superresolution fluorescence microscopy has clearly shown that ladder-like SC structures exist in pachytene nuclei of budding yeast and many other organisms, although they apparently cannot be visualized using the methods described here.

We have changed the title to be less forceful, yet report what we see and don’t see by cryo-ET:

“Cryo-ET detects bundled triple helices but not ladders in meiotic budding yeast”

The authors should clarify whether cryo-sectioning was performed through the full volume of pachytene nuclei.

This comment refers to our attempt at serial cryosectioning, as shown in Fig. S8. We have revised the text in lines 332-334 to reflect the estimated volume covered:

“We successfully reconstructed six sequential sections from one ndt80Δ cell (Figure S8), which represents approximately one third of a nucleus (assuming a spherical shape).”

We also changed Fig. S8’s title so that it doesn’t sound like we reconstructed an entire nucleus:

“MTH bundles are extensive throughout the cell nucleus.” → “MTH bundles are extensive.”

It is not clear from the manuscript which camera/microscope configuration was used to acquire the cryoET data that were used for sub-tomogram averaging. The authors state in the methods that Falcon II and K3-GIF was used for projection images, but it's not clear if this applies to all images. These technical details should be clarified.

We have added a new column to Table S4 that reports the camera used for all the projection imaging and tomography experiments. In the original MS, all of the subtomogram averaging was done using Falcon II data.

FULL: In the full revision, we plan to incorporate new subtomogram averages of MTHs in situ, using K3-GIF data of cryolamellae.

The analysis of the handedness of the helices seems to be questionable as the resolution of the reconstructions for 80S are also quite low. I am uncertain whether this can be used to state with confidence that the MTH are right-handed.

FULL: In the full revision, we will use purified 70S ribosomes, imaged on the same K3-GIF camera and using the same software workflow as for the new subtomogram averaging of MTHs in situ. We expect higher resolution for both ribosomes and MTHs, which will make the handedness assignment unambiguous.

The authors claim that treatment with Latrunculin-A (LatA) leads to disappearance of MTHs. However, they support this with projection images of cells treated with LatA. The projection images are of poor quality and the vitrification in these cells (as well as the DMSO treated cells) do not look appropriate. They should present data for LatA-treated cells and DMSO-treated controls obtained using the same approach and ideally imaged in parallel with untreated cells. They should also quantify the number of sections and cells imaged for all conditions.

Once we realized that the MTH bundles were visible in projections, we chose to report detections of MTH bundles by projection imaging instead of the costly tilt series. The apparent poor quality and questionable vitrification comes from the fact that the projection images show the cryosection’s crevasses and knife marks, which reside on opposite cryosection surfaces. These sectioning artifacts are computationally excluded from tomographic slices. The following line was added to the figure caption to explain this:

“These image features are not devitrification artifacts; they are absent from the tomographic slices in other figures because they can be computationally excluded.”

The quantification of the MTHs in Lat-A vs control cells are in Table 2. We have now added these numbers to both the text and the figure caption.

The similarities between the MTHs and SCs - that both are present in meiotic nuclei and sensitive to hexanediol - seem unlikely to be functioanlly relevant. Again, I think the presentation suffers from being focused on the SC which was not seen, rather than on the MTHs.

We have toned down the discussion on SCs throughout the manuscript. We have retained the motivation for using 1,6-hexanediol to probe the MTHs physico-chemical properties and the fact the previous work on SCs provided motivation for this perturbation experiment. However, we removed the comparison of their relative sensitivities to 1,6-hexanediol (see reply to point #10 below).

The absence of 100-nm-wide zones containing nucleosomes is again not evidence for lack of SCs. SCs are ribbon-like structures - they are about 100 nm in one dimension but the thickness has not been characterized reliably; even if SCs do exclude nucleosomes (which is not certain) the excluded volume might be much smaller than the authors imagine.

We did not argue for “lack of SCs”; these structures clearly exist in our cells given the fluorescent linear structures seen in Zip1-GFP expressing cells. We only say that the textbook portrayal of SCs needs revision, though we should have restricted our statement to yeast. In the literature and textbooks: whenever the SC’s central element is drawn, it is depicted without internal nucleosomes and being densely packed with SC proteins.

Does Lifeact-mCherry enter the nucleus? This information is important in interpreting the failure to detect MTHs using this probe.

While Lifeact-mCherry is small enough to passively diffuse through the nuclear pore, our data cannot rule out that this molecule is excluded from the nucleus. We added the following sentence as a caveat:

Lines 244-245 “Note that we cannot rule out that Lifeact-mCherry is excluded from the nucleus.”

The sensitivity of the MTH structures to 1,6-hexanediol treatment is potentially interesting, but it does not reveal anything about their structure or function, only that their assembly likely depends in part on hydrophobic interactions. Caution should be used in interpreting these findings.

We have toned down the discussion about the meaning of the MTH bundles’ 1,6-hexanediol sensitivity by removing this line from the original Results:

“MTH bundles are therefore sensitive to a slightly higher concentration of 1,6-hexanediol than SCs are and reform when 1,6-hexanediol is removed.”

We have also added the following line to more clearly say what sensitivity to 1,6-hexanediol means:

Lines 412-414: “Their sensitivity to 1,6-hexanediol suggests that the polymerization both within and between MTHs are based on hydrophobic interactions”

The figure legends and/or Methods sections should clarify what is represented in each figure, and how the data were acquired. In particular, cryotomographic slices of varying dimensions (6nm, 10 nm or 12 nm or 70 nm) are mentioned in the captions of several figures (2-6, and S1, S2, S3). However, is often unclear whether these represent physical or computational sections.

“Tomographic slice” refers to a rendering of a computationally extracted slice from a reconstructed tomogram. To make it clearer, we have added the term “computational” to describe the tomographic slices in each figure caption.

Page 23 has a supplemental figure but no captions. Is this the same as Fig. S8?

Yes, this is a copy of Fig. S8 that appeared due to MS Word’s jumping-figure bugs. We will manually edit the PDF in the future revision.

I do not find the model figure (Figure 7) to be helpful. Additionally, the failure to detect SCs and the presence of MTHs do not warrant a "revised model of the meiotic yeast nucleus."

We now call panel A and B the “Traditional EM” and the “Cryo-EM” models, respectively. The figure therefore reports the large nuclear bodies seen by the two methods and no longer implies correctness.

We also changed the related sentence in the Introduction:

Original: “Our work strongly suggests that current models of pachytene nuclear cytology need revision.”

Revised: “Our analysis shows that MTHs coexist with SCs, which have an unknown cryo-ET structure.”

The absence of MTHs in haploid cells induced to undergo meiosis should perhaps be studied in more detail. Even SCs are present in haploid meiotic cells, so the absence of these structures may be informative as to their function. Haploid cells should also be stained for SCs and imaged by immunofluorescence to verify that they are in meiosis.

The haploid strain that was treated with sporulation media cannot enter meiosis. Haploid cells that are capable of entering meiosis need to be disomic for chromosome III, with each copy having a different mating type at the Mat locus. We believe that the construction and studies of such strains would be more meaningful after we identify the MTH’s subunits and determine its function in diploid cells.

The yeast strain is SK1, not SK-1.

Thank you. This mistake is corrected.

What are "self-pressurized-frozen samples" (p.2)?

Self-pressurized-frozen samples are generated by an alternative to the conventional machine-based high-pressure-freezing method. We have added more details in the new lines 135-141:

“Self-pressurized freezing is a simpler and lower-cost alternative to conventional high-pressure freezing, which requires a dedicated machine that consumes large amounts of cryogen. In the self-pressurized freezing method, the sample is sealed in a metal tube and rapidly cooled in liquid ethane. The material in direct contact with the metal cools first and expands by forming crystalline ice, which exerts pressure on the material in the center of the tube (Leunissen and Yi, 2009; Yakovlev and Downing, 2011; Han et al., 2012).”

Reviewer #2 (Significance (Required)):

The observation of MTHs is novel but (as stated above) of unclear significance, given that their molecular identity and function are unknown.

This work may be of interest to the meiosis field, with the caveats described above that the functional relevance is currently unclear.

This review was co-written by referees with expertise in meiosis, chromosome organization, SC structure and function, and cryoEM.

**Referee Cross-commenting**

The reviews are strikingly concordant so I don't think much needs to be added.

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

The authors report the observation of filamentous structures (further termed meiotic triple helices MTH) in the nucleoplasm during meiosis in yeast cells. Those structures are visualized using Cryo-ET. While the authors initially seem to assume an association of those structures with synaptonemal complexes, they discover that those structures rely on filamentous actin and are not affiliated with synaptonemal complexes after all.

While I think that the observation of those MTH by Cryo-ET is interesting, the overall structure of the paper and presentation of the data are not very well done. As the authors find throughout their experiments that the MTHs are not associated with synaptonemal complexes the strong focus in the first figures on synaptonemal complexes as well as the title of the paper are very mis-leading. The authors try to initially make the point that other labs have observed ladder like structures by transmission electron microscopy and want to make the claim that those observed structures might be an artifact of sample preparation, hence the title: Meiotic budding yeast assemble bundled triple helices but not ladders. However, at the end those structures seem unrelated to synaptonemal complexes.

In addition, several labs have reported the presence of nuclear actin in meiosis and mitosis and have even succeeded to show those structures by transmission electron microscopy, questioning the "artifact" argument.

Presumably, the Reviewer means intranuclear actin, as opposed to perinuclear (cytoplasmic) actin. If so, then we have only seen one paper, the one from the Takagi et at 2021 (bioRxiv) that has reported seeing structures associated with nuclear actin. Note however, that the revised Takagi bioRxiv paper is very careful in saying that the filament bundles contain actin, which is not the same as saying that the filaments are polymers of actin.

Our “artifact” argument – now removed – referred to SCs, not to nuclear actin.

In the revised manuscript, we use the term “intranuclear” to make it clear that we are referring to structures inside the nucleus. We also use the term F-actin, where appropriate, to refer to the best-studied actin polymer, which resembles a double helix. Doing so eliminates confusion about other forms of actin: G-actin, which cryo-ET cannot yet directly visualize due to its small size; and non-canonical actin polymers, for which there are no previous experimental X-ray or cryo-EM structures for comparisons.

The story line of the paper is weak and I think the authors would have been better of reporting their cryo-ET structures and making a better link to actin or determining what else they think might be a component of those structures. Immuno-EM (as actually shown in Reference 41) of actin would have been much more convincing. The authors could also use the power of Cryo-ET and the achievable higher resolution to describe those filaments in much more detail. In my opinion this would have been a much better and more exciting paper.

We disagree that “making a better link to actin” is the right approach because doing so presupposes that the structures are composed of actin, for which the present evidence is inadequate. We do agree that determining what the MTHs are (or are not) would be valuable.

FULL: Now that we have better access to a K3-GIF camera and a cryo-FIB-SEM, we will attempt higher-resolution subtomogram averaging analysis. If we are able to achieve subnanometer resolution, we will attempt to narrow down the fold of the MTH subunit. Note that this goal will require that the MTHs are conformationally homogenous and that we can image sufficient copies of the MTHs.

In summary: While I think the Cryo-ET images of those structures could be very exciting the paper unfortunately does not do a very good way in presenting this data and is at times misleading trying to proof or rather dis-proof a connection to synaptonemal complexes. Based on this I think that the paper can not be published in the current form and needs major revisions that would require a significant amount of time.

**Minor comments:**

Figure 1: The choice of timepoints is confusing and makes it hard to compare. While wild type is shown at 0,1,3,4,5,8h, the mutant is shown at 0,2,3,4,5,6,7h. It would be appropriate to select the same timpepoints for both conditions.

FULL: We will recollect fluorescence images of the mutant cells at the same time points as the wild type.

Figure 3 and 4 need a quantification of the number of observed MTHs, in particular as only selected regions of the nuclei are shown.

These images were taken from single cryosections instead of serial cryosections, which would have been too difficult to do for multiple conditions and multiple cells. Therefore, quantification would be obfuscated by the fact each cryosection samples a small fraction of the nuclear volume. We believe that reporting the number of cell cryosections that are MTH-positive (Table 2) is at present the best way to characterize their abundance and ability to polymerize. Once we are able to identify the MTH gene products, we will be able to perform GFP tagging experiments and thereby get a much better estimate of the polymer mass as a function of biochemical perturbations.

Fig 7. The data certainly does not support a "REVISED" model of the yeast nuclear organization.

We have changed the Figure title to “A cryo-ET model of the meiotic yeast nucleus.” We now also refer to panels A and B as “Traditional EM model” and “Cryo-ET model”, respectively.

Reviewer #3 (Significance (Required)):

Several publications have already shown the presence of actin in meiotic and mitotic nuclei and have even succeeded in observing those structures by transmission electron microscopy. Based on this it is not clear why the authors have not tried to put their work in context to all these observations and used their technology to obtain novel information on the structure, which might be helpful to identify which proteins compose the MTHs. Based on how the data is presented I do not think that this paper contributes anything new to the field.

Presumably, the reviewer means that F-actin has been imaged in yeast cells, because for actin to exist in nuclei in mitosis/meiosis, the organism would have to undergo closed mitosis/meiosis. Furthermore, for actin to be observable by transmission electron microscopy, it would have to be in the filamentous (F-actin) form. We could not find any publications that report transmission electron microscopy of F-actin in yeast cells. We therefore cannot relate our results to F-actin in the meiotic nucleus.

My field of expertise is meiosis and mitosis as well as imaging (light and electron microscopy).

**Referee Cross-commenting**

All reviewers seem to agree that the general observation of these structures is interesting but that there is a reduced significance as the function and identity of these structures remains unknown.

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

The main unanswered question is: what is the function of the MTH bundles? To address this question, we would first need to identify the gene products that are needed for MTH assembly. Next, we would need to do genetic perturbation experiments to actually determine the MTHs’ function. These experiments would constitute a complete study, which is better suited for a separate, future manuscript.

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Referee #3

Evidence, reproducibility and clarity

The authors report the observation of filamentous structures (further termed meiotic triple helices MTH) in the nucleoplasm during meiosis in yeast cells. Those structures are visualized using Cryo-ET. While the authors initially seem to assume an association of those structures with synaptonemal complexes, they discover that those structures rely on filamentous actin and are not affiliated with synaptonemal complexes after all. While I think that the observation of those MTH by Cryo-ET is interesting, the overall structure of the paper and presentation of the data are not very well done. As the authors find throughout their experiments that the MTHs are not associated with synaptonemal complexes the strong focus in the first figures on synaptonemal complexes as well as the title of the paper are very mis-leading. The authors try to initially make the point that other labs have observed ladder like structures by transmission electron microscopy and want to make the claim that those observed structures might be an artifact of sample preparation, hence the title: Meiotic budding yeast assemble bundled triple helices but not ladders. However, at the end those structures seem unrelated to synaptonemal complexes. In addition, several labs have reported the presence of nuclear actin in meiosis and mitosis and have even succeeded to show those structures by transmission electron microscopy, questioning the "artifact" argument. The story line of the paper is weak and I think the authors would have been better of reporting their cryo-ET structures and making a better link to actin or determining what else they think might be a component of those structures. Immuno-EM (as actually shown in Reference 41) of actin would have been much more convincing. The authors could also use the power of Cryo-ET and the achievable higher resolution to describe those filaments in much more detail. In my opinion this would have been a much better and more exciting paper. In summary: While I think the Cryo-ET images of those structures could be very exciting the paper unfortunately does not do a very good way in presenting this data and is at times misleading trying to proof or rather dis-proof a connection to synaptonemal complexes. Based on this I think that the paper can not be published in the current form and needs major revisions that would require a significant amount of time.

Minor comments:

Figure 1: The choice of timepoints is confusing and makes it hard to compare. While wild type is shown at 0,1,3,4,5,8h, the mutant is shown at 0,2,3,4,5,6,7h. It would be appropriate to select the same timpepoints for both conditions.

Figure 3 and 4 need a quantification of the number of observed MTHs, in particular as only selected regions of the nuclei are shown.

Fig 7. The data certainly does not support a "REVISED" model of the yeast nuclear organization.

Significance

Several publications have already shown the presence of actin in meiotic and mitotic nuclei and have even succeeded in observing those structures by transmission electron microscopy. Based on this it is not clear why the authors have not tried to put their work in context to all these observations and used their technology to obtain novel information on the structure, which might be helpful to identify which proteins compose the MTHs. Based on how the data is presented I do not think that this paper contributes anything new to the field. My field of expertise is meiosis and mitosis as well as imaging (light and electron microscopy).

Referee Cross-commenting

All reviewers seem to agree that the general observation of these structures is interesting but that there is a reduced significance as the function and identity of these structures remains unknown.

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Referee #2

Evidence, reproducibility and clarity

This work describes helical filamentous structures observed in budding yeast nuclei that were cryosectioned and imaged using cryo-electron tomography (cryoET).

The goal of this work seems to have been to conduct an ultrastructural analysis of the synaptonemal complex (SC), a meiosis-specific protein structure that holds chromosomes together during meiosis and is thought to regulate meiotic recombination. In conventional TEM images of fixed, embedded, and stained sections obtained from pachytene nuclei, SCs usually appear as long, thin, transversely striated structures. At pachytene, SCs extend along the full length of a thin (100-nm) gap between paired chromosomes (typically 1-6 µm long in yeast cells). Surprisingly, the authors did not observe SCs, perhaps because these structures do not produce much contrast in cryo-EM images. Instead, they observe abundant triple helical structures in the nucleoplasm, which they designate as "meiotic triple helices" (MTHs). The authors report that these triple helices assemble at the same time as but independently of the SC. They publised a preprint in which they indicated that these structures were somehow related to SCs, but this the revised version reports that they appear independently of SCs. They further report that treatment of cells with the F-actin depolymerizing drug Latrunculin-A (LatA) resulted in a lack of detectable triple helical structures in the nucleus, suggesting that these structures may be a form of actin or, alternatively, that they may require F-actin for their assembly.

While the work is technically mostly sound, its significance is unclear because the reported structures have no known function. Many, if not most proteins will form helical structures if their concentration rises above a threshold defined by their binding affinity for themselves (see https://doi.org/10.1186/1741-7007-11-119 and references therein), so this may simply be an example of an abundant nuclear protein that polymerizes to form helical filaments under the conditions that trigger yeast sporulation.

The authors perform cryosectioning and cryoEM on yeast cells undergoing meiosis to show that the assembly and disassembly of MTHs follow a similar time course as that of the SC. The observation of these triple-helical filaments in meiotic nuclei has also been reported in another study (https://www.biorxiv.org/content/10.1101/778100v2.full.pdf+html), which proposed, based on immuno-EM labeling, that they may be actin cables. This study reports that the structures are not detected using phalloidin or Lifeact-mCherry. However, treatment with LatA did eliminate detection of MTH structures, suggesting that they may be comprised of actin.

In my view, there are a number of issues that should be addressed before publication. Many of these relate to the presentation of the findings. Detailed comments below:

1. The presentation of the work is very confusing. The authors clearly expected to observe SC structures, but did not. They conclude that MTHs are not SCs, since they do not depend on molecular components required for SC assembly. They should describe their findings in a more straightforward way rather than veering from introducing the SC to describing the MTHs. Similarly, the discussion section on "recombination and chromosome segregation" seems inappropriate and irrelevant, since no data are presented in this study regarding the functions of the MTHs, and there is no reason to think that they contribute to crossover interference, chromosome segregation, or other aspects of meiosis. Additionally, most of the ideas presented in this section seem very muddled. I recommend deleting this section.
2. Along the same lines as comment #1: The title should be changed - absence of evidence for "ladders" is not evidence of absence. Prior work using TEM and superresolution fluorescence microscopy has clearly shown that ladder-like SC structures exist in pachytene nuclei of budding yeast and many other organisms, although they apparently cannot be visualized using the methods described here.
3. The authors should clarify whether cryo-sectioning was performed through the full volume of pachytene nuclei.
4. It is not clear from the manuscript which camera/microscope configuration was used to acquire the cryoET data that were used for sub-tomogram averaging. The authors state in the methods that Falcon II and K3-GIF was used for projection images, but it's not clear if this applies to all images. These technical details should be clarified.
5. The analysis of the handedness of the helices seems to be questionable as the resolution of the reconstructions for 80S are also quite low. I am uncertain whether this can be used to state with confidence that the MTH are right-handed.
6. The authors claim that treatment with Latrunculin-A (LatA) leads to disappearance of MTHs. However, they support this with projection images of cells treated with LatA. The projection images are of poor quality and the vitrification in these cells (as well as the DMSO treated cells) do not look appropriate. They should present data for LatA-treated cells and DMSO-treated controls obtained using the same approach and ideally imaged in parallel with untreated cells. They should also quantify the number of sections and cells imaged for all conditions.
7. The similarities between the MTHs and SCs - that both are present in meiotic nuclei and sensitive to hexanediol - seem unlikely to be functioanlly relevant. Again, I think the presentation suffers from being focused on the SC which was not seen, rather than on the MTHs.
8. The absence of 100-nm-wide zones containing nucleosomes is again not evidence for lack of SCs. SCs are ribbon-like structures - they are about 100 nm in one dimension but the thickness has not been characterized reliably; even if SCs do exclude nucleosomes (which is not certain) the excluded volume might be much smaller than the authors imagine.
9. Does Lifeact-mCherry enter the nucleus? This information is important in interpreting the failure to detect MTHs using this probe.
10. The sensitivity of the MTH structures to 1,6-hexanediol treatment is potentially interesting, but it does not reveal anything about their structure or function, only that their assembly likely depends in part on hydrophobic interactions. Caution should be used in interpreting these findings.
11. The figure legends and/or Methods sections should clarify what is represented in each figure, and how the data were acquired. In particular, cryotomographic slices of varying dimensions (6nm, 10 nm or 12 nm or 70 nm) are mentioned in the captions of several figures (2-6, and S1, S2, S3). However, is often unclear whether these represent physical or computational sections.
12. Page 23 has a supplemental figure but no captions. Is this the same as Fig. S8?
13. I do not find the model figure (Figure 7) to be helpful. Additionally, the failure to detect SCs and the presence of MTHs do not warrant a "revised model of the meiotic yeast nucleus."
14. The absence of MTHs in haploid cells induced to undergo meiosis should perhaps be studied in more detail. Even SCs are present in haploid meiotic cells, so the absence of these structures may be informative as to their function. Haploid cells should also be stained for SCs and imaged by immunofluorescence to verify that they are in meiosis.
15. The yeast strain is SK1, not SK-1.
16. What are "self-pressurized-frozen samples" (p.2)?

Significance

The observation of MTHs is novel but (as stated above) of unclear significance, given that their molecular identity and function are unknown.

This work may be of interest to the meiosis field, with the caveats described above that the functional relevance is currently unclear.

This review was co-written by referees with expertise in meiosis, chromosome organization, SC structure and function, and cryoEM.

Referee Cross-commenting

The reviews are strikingly concordant so I don't think much needs to be added.

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Referee #1

Evidence, reproducibility and clarity

Ma and coworkers report studies of budding yeast undergoing meiosis by cryo-ET. They fail to detect structures interpretable as synaptonemal complex, and instead detect feather-like bundles of what appears to be a triple helix. These structures do not appear to be related to the synaptonemal complex, as spo11 mutants that do not initiate recombination, red1 mutants that lack axial elements, and zip1 mutants that lack the central element of the SC still make these bundles. These structures are absent from cells treated with latrunculin A, which depolymerizes actin filaments, but expected structures are not visible in light microscopy of cells treated with two different F-actin-staining reagents. However, it should be noted that another study (Takagi et al, 2021, bioRxiv) did detect actin associated with these structures by immunogold labeling. The structures are also reversibly dissolved in 7% hexanediol.

This part of the paper's findings is well supported by data and is certainly of interest, although interest is somewhat limited by the unknown nature of these structures-what they contain, let alone their function, remains to be determined-in fact, it is not even determined whether or not they are made of protein. However, as an initial report of a previously unknown phenomenon, the paper is of some value.

There is, unfortunately, a second aspect of the paper that cannot be supported. Although it is clear that synaptonemal complex is present in the cells examined (by standard cytological methods) the authors cannot detect structures consistent with SC in their cryo-ET images. Unfortunately, authors then extrapolate from their inability to detect SC to conclusions about SC, such as that it is not crystalline, and even go so far as to suggest that their failure to detect SC invalidates two models for crossover interference, and that the ladder-like structure reported for SC in many organisms using many difference approaches may be a fixation artifact. Authors show remember that the absence of evidence is not evidence for absence; the speculation described above should be removed from both the abstract and discussion.

Significance

This paper reports a previously unreported structure in the nuclei of yeast cells undergoing meiosis. The composition and function of this structure remain to be determined. This considerably limits the significance of the paper.

Referee Cross-commenting

I agree with the concerns of the other reviewers. I also agree with reviewer 3 that to raise the significance of the paper would require much work. But I think that the raw observation is of value, albeit in a journal of record. So I would stick with my recommendation of text changes, keeping in mind that there may not be a suitable journal in LSA's portfolio.

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Reply to the reviewers

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

Saha and colleagues investigated the functions of the long non-coding RNA (lncRNA) DRAIC in malignant glioma. They find that DRAIC expression decreases cell migration/invasion and tumorsphere/colony formation in vitro, and tumor growth in vivo using established cell lines. Mechanistically, DRAIC is known to inhibit NF-kB signaling and the authors demonstrate that DRAIC activates AMPK leading to repression of mTOR, which decreases protein synthesis and increases autophagy. This is a solid study highlighting a potentially interesting pathway of tumor growth and invasion in brain tumors.

Answer: We appreciate Reviewer 1 for the positive feedback of our study

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Major Comments:

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1) It is unclear whether the presented values (mean +/- SD) in the histograms refer to repeat measurements (in which case n = 1) or independent experiments (n>1). The number of replicate experiments is not stated in the methods or figure legends. This must be included.

Answer: We want to thank Reviewer 1 for pointing out this omission. We have now included this information in the Materials and methods and in figure legends section.

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2) I don't think the immunoblot for p62 in Fig. 5C shows a convincing increase following DRAIC knockout, so the statement on p.8 should be revised.

Answer: We have revised the statement to say: Consistent with DRAIC decrease being associated with a decrease in autophagic flux, and despite a decrease in p62 mRNA, the level of P62 protein is increased in three of the DRAIC KO prostate cancer cells (Fig. 5C, KO1, KO2, KO4 compared to WT) and unchanged in the other two.

3) On p.8/Fig.5 the authors make a case that increased DRAIC levels increase lysosomal degradation of autophagosome core proteins LC3 II / p62 (resulting in decreased protein levels of both), while simultaneously increasing gene expression of LC3B and p62 (causing increased mRNA levels). The data for DRAIC overexpression fit this logic fairly well (even though I think more work is needed to fully support this claim), but I am finding it difficult to reconcile the DRAIC knockout data with this scenario - here, loss of DRAIC results in increased protein levels to decreased autophagy, but also decreased gene expression. To fully support this argument, rescue experiments would be needed using FoxO3a knockout/overexpression.

Answer: Note that the mRNA level is not always correlated with protein expression. This is particularly true for LC3 and p62, whose protein levels are significantly affected by the extent of fusion of autophagosomes and lysosomes and subsequent degradation in autophagolysosomes. Thus, although the mRNA of these genes is decreased in DRAIC KO cells (Fig. 5E), the proteins are increased (Fig. 5C) because of decrease of autophagic flux (and decrease of degradation in the autophagolysosomes).

The overexpression of FoxO3a in the DRAIC KO cells will not restore mRNA levels of LC3 or p62, because we show in Fig. 4H that FoxO3 phosphorylation by AMPK is suppressed by DRAIC KO. This phosphorylation is important for the induction of LC3 or p62 mRNA by FoxO3.

FoxO3 knockout or knockdown in DRAIC OE cells should decrease LC3B or p62 mRNA in Fig. 5D, but it is already known from the Literature that FoxO3a is necessary for inducing LC3B or p62 mRNA. Cell Metab. 2007 Dec;6(6):458-71. doi: 10.1016/j.cmet.2007.11.001.PMID: 18054315.

4) Similarly, the data supporting increased autophagy following DRAIC overexpression (Fig. 5F/G) are a bit weak and lack controls (is the LC3B-GFP overlapping with endogenous LC3B and autophagosomes? Was the transfection efficiency comparable? Is there fusion with lysosomes?). In the absence of stronger data, the authors should temper their claims that DRAIC increases autophagy.

Answer: LC3B of fusion protein LC3B-GFP is known to overlap with the p62 puncta (similar to endogenous LC3B). This result is in Fig. 4A of the citation that we have now added (Proc Natl Acad Sci U S A. 2016 Nov 22;113(47): E7490-E7499. doi: 10.1073/pnas.1615455113. Epub 2016 Oct 17)

To support our hypothesis that DRAIC OE induces more autophagy compared to empty vector, we used Bafilomycin A1 in Figure 5B to inhibit the autophagosome and lysosome fusion. We see the accumulation of more LC3B upon treatment with Bafilomycin A1 in the DRAIC OE cells (compared to EV containing U251 cells), consistent with the idea that autophagosome-lysosome fusion is increased by DRAIC OE.

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5) No information is provided on animal numbers used in this study. How many mice were used per cohort? Were male and female mice used? Authors should follow ARRIVE guidelines in reporting animal experiments. The method for calculating tumor volume needs to be specified.

Answer: We have included the details about the animal study in methods section of our modified manuscript .

6) Student's T-test is inappropriate for comparisons of more than two groups (i.e. all experiments using DRAIC knockout cells) - for these experiments a Kruskal Wallis test or ANOVA should be used. Did the authors test for normal distribution of their data? This may affect statistical testing and should be taken into consideration.

Answer: We have now modified our statistical calculation and included in the statistical analysis section in our modified manuscript.

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Minor Comments:

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7) Authors mention that DRAIC expression is undetectable in immortalized astrocytes and GBM cancer stem cells (Fig. S1). What is the source of these cells and how were they cultured?

Answer: The immortalized astrocytes and GBM stem cells and their culture conditions is now described.

8) The immunoblot in Fig. 3D could be replaced with a slightly lower exposure to make the difference between WT and DRAIC KO more obvious.

Answer: We have now replaced the immunoblot with lower exposure.

9) Some immunoblots in Fig. 3 (panel E, p-S6K and S6K; panel H, actin) are not of the best quality and an effort should be made to replace them.

Answer: We have now replaced the immunoblot p-S6K as reviewer mentioned.

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10) Why are different loading controls used in Fig. 3 (a-Tubulin v actin)?

Answer: We use multiple loading control to make sure that we are not underestimating or overestimating changes in the experimental protein because of unexpected changes in the loading controls.

11) Compared to other blot images in the same figure (e.g. Fig. 3E), the bands for p-mTOR and mTOR in Fig. 3F look compressed and should be shown appropriately sized.

Answer: We have modified the Figure as reviewer suggested.

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12) The layout of Fig. 4 is somewhat confusing. I would suggest organizing this according to DRAIC overexpression in A172 and U373 cells versus DRAIC knockout in LNCaP cells. Each immunoblot should be clearly labelled with the corresponding cell line, and it should be clearly explained why p-FoxO3a was tested in U251 cells, rather than A172/U373 as in the rest of the figure.

Answer: We thank the reviewer for the constructive criticism. We have labeled all the cell lines in the Figure as reviewer suggested. We have now systematically alternated the prostate cancer cells (for KO) and the GBM cells (for OE), as we looked at each relevant marker. We have now included the western blot for p-FoxO3a from another glioblastoma cell line U373. Please find the modified Figure 4K for p-FoxO3a.

13) Labelling of immunoblot in Fig. 5B is confusing and should be improved.

Answer: We have modified the Fig. 5B to make the label clearer.

14) Changes in GLUT1 expression (Fig. 7A) should be validated on the protein level.

Answer: We have included the immunoblot for GLUT1 from DRAIC KO cells in Figure 7B. GLUT1 protein is increased upon DRAIC KO.

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Reviewer #1 (Significance (Required)):

The authors describe a novel link between the lncRNA DRAIC and AMPK activation through inhibition of NF-kB-mediated regulation of GLUT1. This study extends their previous work on DRAIC inhibition of NF-kB in prostate cancer (Saha et al. Cancer Res 2020). There is one study describing DRAIC effects on growth and invasion in glioma cell lines (Li et al. Eur Rev Med Pharmacol Sci 2020), but the work presented by Saha and colleagues contains stronger experimental data and a more detailed and previously undescribed mechanism.

The current study presents a mechanistic advance that increases the understanding of tumor growth and protein synthesis in cancer cells. The data presented in the study are not supported by in vivo experiments (other than suppression of tumor growth by DRAIC overexpression), validation in human tissue and/or primary patient-derived human glioblastoma cells, or even substantial rescue experiments. This limits the influence of the work on the field. I'm also not sure how transferable findings from DRAIC knockout in prostate cancer cell lines are to glioma, although the results are mostly complementary to the data from glioma cell lines. This is particularly relevant to the proposed mechanism of GLUT1 regulation by NF-kB, as the bulk of experimental data in Figures 6 and 7 was generated in prostate cancer cell lines and is only poorly validated in glioma cells. The study results will be most relevant for researchers investigating cell signaling pathways and autophagy in cancer.

Answer: We like to thank reviewer for the positive comments on our study. The DRAIC KO experiments of Fig. 6 and 7 cannot be done in glioma cells, because as we show if Supp. Fig. S1, there are no glioma cells or GSC that express DRAIC to levels comparable to LnCaP. We have shown that GLUT1 mRNA decreases in the glioma cells when DRAIC is overexpressed (Supp. Fig. S4. We also show in Fig. 7G that AMP levels increase when DRAIC is overexpressed in glioma cells.__

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

In this manuscript, the authors describe DRAIC as a lncRNA downregulated in prostate cancer. They postulate that DRAIC expression surpasses invasion, migration and growth. Mechanistically, the authors show that DRAIC activates AMPK by suppressing NFkB target gene TOR and indirectly impacting translation and autophagy. Collectively the observation is interesting and robust. However, I have several technical requests, particularly regarding the mechanistic part of the paper.

Answer: We appreciate the positive feedback. We have addressed the reviewer’s concerns in our modified manuscript.

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Major Comments:

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1) The authors should rescue Ko phenotypes by over expressing DRAIC to consider potential off target effects.

Answer: DRAIC OE alone is sufficient to have exactly the opposite effect as DRAIC KO in protein translation (Fig. 3C-F), so DRAIC OE will rescue the effect of DRAIC KO. We make a similar argument for all the phenotypes, including mTOR, S6K and ULK1(S757) phosphorylation (Fig. 3G-J), AMPK and FoxO3a phosphorylation (Fig. 4B-C; J-L), autophagic flux (Fig. 5B, C) and effects on LC3B and p62 mRNAs (Fig. 5D, E). The same is true for our published phenotypes of DRAIC KO on invasion, migration and NF-kB activity (Saha, Cancer Research, 2020)

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2) The blots showing TOR and ULK1 phosphorylation need to be repeated. This is an important part of the paper and I feel that these blots are hard to interpret. p-S6K typically run a bit higher in gels. there may be a technical problem.

Answer: We are not sure which specific blots the reviewer is referring to, and it is possible that the blots the other reviewers pointed to are the ones under question. We have changed those blots so that the results are clear.

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3) GLUT1-related results are interesting, but the authors should provide genetic evidence that the effects are mediated by GLUT1. How do we know that glucose uptake is indeed upregulated upon knockout?

Answer: In Fig. 7 C-F we show that the effects of DRAIC KO on invasion, protein translation, AMP levels and AMPK activity are reversed by the GLUT1 inhibitor Bay-876. This is a cleaner result than using siRNA to knockdown GLUT1. siRNAs can have off-target activity and sometimes cannot decrease a protein sufficiently below the threshold necessary to see reversal of action.

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Minor Comments:

4) The figures need to be updated. FOnts are all different, lots of unaligned graphs, quality of the blots are poor.

Answer: We have updated the Figures and changed fonts as reviewer mentioned.

Reviewer #2 (Significance (Required)):

The observation is interesting, but the mechanism is incompletely understood. This is a nice addition to the literature, even without the mechanism.

Answer: We want to thank the reviewer for the constructive criticism.

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

**Summary:**

Shaha and colleagues present a study demonstrating the tumor suppressive role of DRAIC, a long non-coding RNA transcript, through transmission of the signal from IKK/NF-kB to the AMPK/mTOR pathway via regulation of GLUT1 expression. The inhibition of mTOR by this pathway results in the reduction of protein translation, cellular invasion and activation of autophagy. Several diseases and models as well as multiple genetic and pharmacological manipulations were used to investigate the mechanisms at play. The manuscript is well written and the experiments are well designed. The conclusions are supported by the results. The following major and minor comments should be addressed:

Answer: We appreciate the reviewer for the positive comments on our study.

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Major Comments:

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1) In addition to reporting the effect of DRAIC overexpression on tumor volume, the authors should present survival studies with one or more models.__

Answer: __We thought of doing the survival study in our glioblastoma model but unfortunately, the tumor growth is very rapid (exceeding the size permitted by our IACUC in 2-3 weeks). The animal ethics welfare committee did not allow us to keep the mice for a longer time to perform the survival study.

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2) Since the authors study metabolic energy sensor pathways, related to glycolysis, it would be important to perform some of the key experiments in physiological level of glucose: e.g., pmTOR, pAMPK, LC3-II expression level in DRAIC overexpressing and deficient cells.

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Answer: The concentration of glucose in plasma is 1G/L, while that of the RPMI medium is 2G/L. We do not think we are too far from the physiological levels of glucose.

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3) In addition to RT-PCR data, GLUT1 protein levels should be investigated in the different DRAIC expressing cells.

Answer: We have incorporated the GLUT1 protein expression data from DRAIC KO cells in Figure 7B and DRAIC overexpressing cells in supplementary Figure 4G-H. The blots from the same gels were split into different panels, the loading control GAPDH remain same in Figure 4K and Supplementary Figure S4H.

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4) The effect of DRAIC on GLUT1 expression is also measured in condition of glucose saturation, which does not reflect disease state. The decrease of GLUT1 in response to DRAIC overexpression and the increased GLUT1 level in DRAIC deficient cells should be investigated in physiological levels of glucose.

Answer Same as above. We are near physiological levels of glucose.

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__Minor Comments:

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5) All the data are generated with established cell lines (e.g., U87) but more clinically relevant models, such as patient-derived primary cells like the ones used in Fig. S1, could be used to replicate some of the key findings.

Answer: As we showed in Fig. S1 that DRAIC is not expressed in glioma stem cells, and so knockout experiments are not possible. We believe that the knockout experiments are the most relevant to this paper because they do not run the risk of artefacts from overexpression of an RNA far beyond physiological levels.

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6) Also please provide further details about the patient-derived cells from Fig. S1.

Answer: We have mentioned the details of the cell lines in our modified manuscript.

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7) The statistical analysis section states that the number of measurements is indicated however I don't see the sample size of the experiments.

Answer: We have now incorporated the number the experiments in our modified text.

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Reviewer #3 (Significance (Required)): The study reports a new model of regulation of tumor via long non-coding RNA. This article adds to the growing literature The topic and content of the article is relevant and significant to the field of tumor research but the significance and impact could be enhanced with the use of more physiologically relevant models and conditions as pointed in the major comments.

Answer: We want to thank the reviewer for the positive feedback on our study.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Shaha and colleagues present a study demonstrating the tumor suppressive role of DRAIC, a long non-coding RNA transcript, through transmission of the signal from IKK/NF-kB to the AMPK/mTOR pathway via regulation of GLUT1 expression. The inhibition of mTOR by this pathway results in the reduction of protein translation, cellular invasion and activation of autophagy. Several diseases and models as well as multiple genetic and pharmacological manipulations were used to investigate the mechanisms at play. The manuscript is well written and the experiments are well designed. The conclusions are supported by the results. The following major and minor comments should be addressed:

Major comments:

1. In addition to reporting the effect of DRAIC overexpression on tumor volume, the authors should present survival studies with one or more models.
2. Since the authors study metabolic energy sensor pathways, related to glycolysis, it would be important to perform some of the key experiments in physiological level of glucose: e.g., pmTOR, pAMPK, LC3-II expression level in DRAIC overexpressing and deficient cells.
3. In addition to RT-PCR data, GLUT1 protein levels should be investigated in the different DRAIC expressing cells.
4. The effect of DRAIC on GLUT1 expression is also measured in condition of glucose saturation, which does not reflect disease state. The decrease of GLUT1 in response to DRAIC overexpression and the increased GLUT1 level in DRAIC deficient cells should be investigated in physiological levels of glucose.

Minor comments:

1. All the data are generated with established cell lines (e.g., U87) but more clinically relevant models, such as patient-derived primary cells like the ones used in Fig. S1, could be used to replicate some of the key findings.
2. Also please provide further details about the patient-derived cells from Fig. S1.
3. The statistical analysis section states that the number of measurements is indicated however I don't see the sample size of the experiments.

Significance

The study reports a new model of regulation of tumor via long non-coding RNA. This article adds to the growing literature The topic and content of the article is relevant and significant to the field of tumor research but the significance and impact could be enhanced with the use of more physiologically relevant models and conditions as pointed in the major comments.

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, the authors describe DRAIC as a lncRNA downregulated in prostate cancer. They postulate that DRAIC expression surpasses invasion, migration and growth. Mechanistically, the authors show that DRAIC activates AMPK by suprressing NFkB target gene TOR and indirectly impacting translation and autophagy. Collectively the observation is interesting and robust. However, I have several technical requests, particularly regarding the mechanistic part of the paper.

• The authors should rescue Ko phenotypes by over expressing DRAIC to consider potential off target effects.
• The blots showing TOR and ULK1 phosphorylation need to be repeated. This is an important part of the paper and I feel that these blots are hard to interpret. p-S6K typically run a bit higher in gels. there may be a technical problem.
• GLUT1-related results are interesting but the authors should provide genetic evidence that the effects are mediated by GLUT1. How do we know that glucose uptake is indeed upregulated upon knockout?

Minor:

The figures need to be updated. FOnts are all different, lots of unaligned graphs, quality of the blots are poor.

Significance

The observation is interesting, but the mechanism is incompletely understood. This is a nice addition to the literature, even without the mechanism.

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Referee #1

Evidence, reproducibility and clarity

Saha and colleagues investigated the functions of the long non-coding RNA (lncRNA) DRAIC in malignant glioma. They find that DRAIC expression decreases cell migration/invasion and tumorsphere/colony formation in vitro, and tumor growth in vivo using established cell lines. Mechanistically, DRAIC is known to inhibit NF-kB signaling and the authors demonstrate that DRAIC activates AMPK leading to repression of mTOR, which decreases protein synthesis and increases autophagy. This is a solid study highlighting a potentially interesting pathway of tumor growth and invasion in brain tumors.

Major comments:

• It is unclear whether the presented values (mean +/- SD) in the histograms refer to repeat measurements (in which case n = 1) or independent experiments (n>1). The number of replicate experiments is not stated in the methods or figure legends. This must be included.
• I don't think the immunoblot for p62 in Fig. 5C shows a convincing increase following DRAIC knockout, so the statement on p.8 should be revised.
• On p.8/Fig.5 the authors make a case that increased DRAIC levels increase lysosomal degradation of autophagosome core proteins LC3 II / p62 (resulting in decreased protein levels of both), while simultaneously increasing gene expression of LC3B and p62 (causing increased mRNA levels). The data for DRAIC overexpression fit this logic fairly well (even though I think more work is needed to fully support this claim), but I am finding it difficult to reconcile the DRAIC knockout data with this scenario - here, loss of DRAIC results in increased protein levels to decreased autophagy, but also decreased gene expression. To fully support this argument, rescue experiments would be needed using FoxO3a knockout/overexpression.
• Similarly, the data supporting increased autophagy following DRAIC overexpression (Fig. 5F/G) are a bit weak and lack controls (is the LC3B-GFP overlapping with endogenous LC3B and autophagosomes? Was the transfection efficiency comparable? Is there fusion with lysosomes?). In the absence of stronger data, the authors should temper their claims that DRAIC increases autophagy.
• No information is provided on animal numbers used in this study. How many mice were used per cohort? Were male and female mice used? Authors should follow ARRIVE guidelines in reporting animal experiments. The method for calculating tumor volume needs to be specified.
• Student's T-test is inappropriate for comparisons of more than two groups (i.e. all experiments using DRAIC knockout cells) - for these experiments a Kruskal Wallis test or ANOVA should be used. Did the authors test for normal distribution of their data? This may affect statistical testing and should be taken into consideration.

Minor comments:

• Authors mention that DRAIC expression is undetectable in immortalized astrocytes and GBM cancer stem cells (Fig. S1). What is the source of these cells and how were they cultured?
• The immunoblot in Fig. 3D could be replaced with a slightly lower exposure to make the difference between WT and DRAIC KO more obvious.
• Some immunoblots in Fig. 3 (panel E, p-S6K and S6K; panel H, actin) are not of the best quality and an effort should be made to replace them.
• Why are different loading controls used in Fig. 3 (a-Tubulin v actin)?
• Compared to other blot images in the same figure (e.g. Fig. 3E), the bands for p-mTOR and mTOR in Fig. 3F look compressed and should be shown appropriately sized.
• The layout of Fig. 4 is somewhat confusing. I would suggest organizing this according to DRAIC overexpression in A172 and U373 cells versus DRAIC knockout in LNCaP cells. Each immunoblot should be clearly labelled with the corresponding cell line, and it should be clearly explained why p-FoxO3a was tested in U251 cells, rather than A172/U373 as in the rest of the figure.
• Labelling of immunoblot in Fig. 5B is confusing and should be improved.
• Changes in GLUT1 expression (Fig. 7A) should be validated on the protein level.

Significance

The authors describe a novel link between the lncRNA DRAIC and AMPK activation through inhibition of NF-kB-mediated regulation of GLUT1. This study extends their previous work on DRAIC inhibition of NF-kB in prostate cancer (Saha et al. Cancer Res 2020). There is one study describing DRAIC effects on growth and invasion in glioma cell lines (Li et al. Eur Rev Med Pharmacol Sci 2020), but the work presented by Saha and colleagues contains stronger experimental data and a more detailed and previously undescribed mechanism.

The current study presents a mechanistic advance that increases the understanding of tumor growth and protein synthesis in cancer cells. The data presented in the study are not supported by in vivo experiments (other than suppression of tumor growth by DRAIC overexpression), validation in human tissue and/or primary patient-derived human glioblastoma cells, or even substantial rescue experiments. This limits the influence of the work on the field. I'm also not sure how transferable findings from DRAIC knockout in prostate cancer cell lines are to glioma, although the results are mostly complementary to the data from glioma cell lines. This is particularly relevant to the proposed mechanism of GLUT1 regulation by NF-kB, as the bulk of experimental data in Figures 6 and 7 was generated in prostate cancer cell lines and is only poorly validated in glioma cells. The study results will be most relevant for researchers investigating cell signaling pathways and autophagy in cancer.

Reviewer keywords:

neurooncology, cancer stem cells, signaling pathways in cancer

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Reply to the reviewers

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

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Referee #2

Evidence, reproducibility and clarity

Summary:

In the study, the authors found that cancer cells including breast, bladder, lung, neuroglioma, and prostate display an increase of Lipid Droplet (LD) after 6 Gy x-ray (Fig.1). And, the cells containing high LDs showed more radioresistance than the cells with low LDs after irradiated with a dose Gy x-ray (Fig.2). Ferritin Heavy Chain (FTH1), the main intracellular iron storage protein, is found to be upregulated after 6 Gy exposure or in the LDs high cells. FTH1 knockdown decreases LD accumulation and increases radiation sensitivity (Fig.3). Overexpression of FTH1 or DFO (an iron chelator agent) treatment in shFTH1 cells rescue the LD accumulation and cancer radioresistance (Fig.4)

Major comments:

1. The conclusion has some conflict with some publication (PMC5928893) which shows fatty acid oxidation not LDs lead to cancer radioresistance. So the authors should rule out this possibility through knockdown of CPT1.
2. Lipid droplets are dynamic in cells. The sorted cells (10% highest or lowest LD-expressing cells) in Fig.3 may not stand for subpopulation, so the authors should add exogenous lipids or cholesterol to test cancer cell radioresistance.
3. It is impossible to overexpress FTH1 in shFTH1 cells (the stable shRNA will target all mRNA of FTH1) (Fig.4 and methods section: cell culture and FTH1 Reconstitution).
4. The relationship between the free cytoplasmic iron and LD accumulation is not so convincing. Add exogenous iron to test LD accumulation.

Minor comments:

1. Remove fig.1C which is not related to the conclusion;
2. Fig.2 label error: LD520 should be LD540;
3. Fig.3, A: change loading control HSC70 which not so stable in the cells; D: add quantification of LD number.
4. Fig.4, A: change loading control HSC70, and repeat western of MCF7 shFTH1/pcDNA3
5. Line 111, "...that general ROS levels resulted not altered..." should be "...that general ROS levels were not altered..."
6. Fig.S4 legend: "Figure S2" should be "Figure S4".

Significance

The FTH1 affects Lipid Droplets is novel (some results in this study have published: radiation led to LD accumulation (PMC5928893) and an increase of FTH1 (PMC4688087 and PMID:32937103).

The finding is helpful to improve radiation therapy which may combine with drugs targeting FTH1 or iron metabolism.

The researchers who worked in cancer treatment are interested in this finding.

My expertise is cancer lipid metabolism and cancer therapy.

Referee Cross-commenting

I completely agree the comments from the other Reviewer. The authors need enhance the correlation between the lipid droplets genes (e.g., DGAT1/2, SOAT1, ACAT1) and the iron metabolism (e.g., FTH1), and improve data quality as suggested by reviewers.

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Referee #1

Evidence, reproducibility and clarity

In this work Tirinato and co-authors used different experimental approaches to trace correlations between cancer cell stemness, lipid droplets, iron homeostasis and radiation resistance. Their findings were acquired by using different cancer cell lines from different origin, and using diverse techniques, including cytometry, microscopy, clonogenic assays and shRNAi. In general, the manuscript is well written and organized. It is also easy to be followed and the authors managed to convince the readers about the importance of this important aspect of cancer metabolism. The fact that cell lipid droplets content might condition cancer survival to radiotherapy poses novelty and therefore it deserves attention in the delimitation of new anticancer therapies or protocols. There are however some issues that should be amended to improve the quality of the manuscript.

Major comments:

The main concern is that all the work is based on cancer cell lines. The use of some cell lines derived from clinical samples or analysis of the clinical data already deposited in bank webs could be useful to support their conclusions. This last could be easy. Exploration of this depositories could help the author to reinforce the correlation between the expression of the lipid droplets genes, as well as that related with the iron metabolism, with the radiotherapy efficacy in patients.

When analyzed in detail I have some comments regarding specific sections:

Lipid droplets detection images (Figs 1, 3 y 4). Why are the nuclei size look so different in both conditions? FTH1 expression. Fig 3A vs 3C and 4A. As depicted is difficult to have a clear picture of the variations in expression in FHT1 in the different cell lines. Why the authors evaluate the success of shRNAi by PCR if the wb works so well? Is there any correlation between the RR and the expression of FTH1 intra cell lines? What happen when FTH1 is downregulated in the rest of the cell lines? More than restore, the authors overexpressed FTH1, what is the result when FTH1 is overexpressed in the different cell lines?

Minor comments:

The correlation between nile red staining and ROS is not clear (Fig S2). The authors may try to graphic the ROS mfi of different subpopulations (lineal in X) vs the mfi of red nile (y, in log scale). Abreviation in introduction ER, is endoplasmic reticulum? A picture with a putative model could be helpful to summarize the findings.

Significance

As stated above the idea that lipid droplets and iron metabolism might be determinants in the cancer survival to radiotherapy poses novelty and therefore it deserves attention in the delimitation of new anticancer therapies or protocols.

Although I have experience in cancer lipid metabolism I am not an expert in the field of lipid droplets.

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Referee #4

Evidence, reproducibility and clarity

Summary:

In this study, the investigators describe two new Trypanosoma brucei brucei strains wheich have been subjected to minimal passage in rodents or in culture. In addition to characterizing some of the phenotypic and genetic characteristics of these isolates and lines derived from them the authors also characterize the changes in gene copy numbers occurring during in vitro passage. While this study illuminated the impact of in vitro culturing for adaptation that might have been overlooked for years in unicellular eukaryotes, the experimental design and method description make their conclusions less convincing.

Major comments:

My main discomfort with this work is that it provides just enough information to be interesting and perhaps important, but not enough to be definitive. Part of this is not the fault of the authors - especially in dealing with various "lab" strains and their genomes - over which they have no control in terms of where and how they were isolated, maintained and investigated. But the authors seem to add to this problem by not determining the clonality of their new strains, failing to provide a full genome sequence (although they seem to have the data they need - including long-read sequencing - to do so), and with often vague descriptions of the analyses they performed as part of this report. My biggest concern has to do with the population structure of the beginning parasites set and to what extent the cultivation - even though limited - is selecting for variants within an existing non-clonal population rather than showing evidence of evolution of a (mostly"??) clonal population. The authors note that they have not cloned so as to "minimize manipulation" - which is sensible. But the provided references (particularly Ref 21) use microsatellite markers to establish wide clonality in populations in individuals. Although the authors have not indicated it here, one would presume that that "evolution" being observed in their lines over time (in animal or in culture) have not altered the microsatellite signature of the lines (thus are still clonal based upon standards of Ref 21), but has resulted in genetically and biologically altered lines - i.e. clonal variants. It seems without knowing the complexity of the beginning populations (granted - not easy), it is hard to say whether the changes over time are due to selection of a subset of the initial population, genetic changes in an initially clonal population, or a combination of the two. Indeed, a selection process would explain the unexpected differences found in early sampled (low parasitemic but high stumpy) and late sampled (high parasitemic but low stumpy) lines as well as the erratic growth in culture. The authors seem to go back and forth on this issue but the key problem is that there is not enough information here to understand this clearly, and for that reason, the work, although considerable, adds mostly anecdotal observations but relatively little definitively. The authors attribute gene/chromosome ploidy changes under in vitro culturing as adaptation of parasites due to chromosome replication and/or segregation. But this conclusion can not be reached unless the populations were clonal at the beginning. Related to this point, it is surprising that there is not a comparison between the A and B lines of each isolate to each other (e.g. 65A/65B). If the isolates are clonal to begin with then one would expect little difference between the 2 harvested 1 day apart from different rats.

Overall the manuscript is difficult to follow and interpret, very light on details and Figure 2 is very confusing and is not particularly helped by the author's summary of them. A little more detail on how the experiment was done might help. It should be made clear and explicitly stated that the cultures were split (by how much/to what level - seems like it is 0.5 x 10e6?) once cultures reached what level (there doesn't appear to be any pattern there - so are they split every 24hrs unless they have declined?). Readers should not have to go to a previous publication to find these types of details needed to interpret the results. The text refers to "diluting them" but presumably a portion of the culture was reseeded/cultured - or was there a constant dilution of the culture over time? The authors refer to a "slowed growth" after 1-2 days, but 3 of the 4 cultures also declined again between 6 and 11 days. This is not consistent with the hypothesis that the rebound after 2-3 days was the result of an adaptation to a depleted component (if so, why would an additional "adaptation" be necessary?). 2E seems to have been handled differently from A-D. The growth curves in S2 fig do not exhibit the same drops as evident in the Fig 2 cultures.

A fundamental finding of this research is detecting the gene copy number variation from the sequencing results of the strains after in vitro passages. However, the description of how to processing the sequencing reads was used to determine the gene copy number is vague. The description lacks basic information like the analysis workflow, software packages, parameters, and other important details. These details are the keys to support their conclusion.

According to the authors, only one representative was considered for multicopy genes. But what is the criteria to call 'multicopy genes' here? How homologous these multicopy genes are set to be? The authors also mentioned that reads may also map to homologous genes, how is this issue dealt with during the analysis?

Overall, the study seems rushed and too preliminary; much of the writing is loose and lacking in details. The authors mention that they have long read nanopore sequence but don't appear to use it to appropriately address some of the questions (e.g. Line 195 "In the meantime..."). As a result, although the results are intriguing (but not totally surprising - this work uncovers a bit of the dirty little secret most scientist realize underlies the pathogen strains that we often present as stable entities while knowing that they have and are changing over time), the study presents more questions than it answers - as the authors seem to acknowledge in their final paragraph.

Minor comments:

Line 79-83 - statements require some references The strain "A/B" nomenclature comes into use in the manuscript (line 129 and Fig 1 legend) without defining what the difference between A and B is (apparently this is the samples from 2 different cultures (Line 187) or from different rats - but it takes a bit of work to figure that out. - 1C could be interpreted to mean that the 2 rats got infected by either the A or B lines - or that the resulting lines were named based on the rat they came from, or they are just different cultures of the same stabilate).

Line 141 "firmly confirmed" sounds a bit off - just "confirmed" or "firmly supported", perhaps better. Line 140/141 - S fig 2 a bit hard to understand - as is the remaining paragraphs on culture outcomes. As this is expressed as a ratio, could it not be argued that polyA+ enriches for (or ribo-minus "depletes" - using the author's treminology) short RNAs (or that ribo-minus enriches for longer)? It does seem likely that the relatively rare extra-long RNAs are underrepresented in the poly A+ method but not clear that this shows anything other than that the different techniques have different strengths/weaknesses.

Line 156 ."... had run out of a nutrient..."

Line 170: "several additional attempts ..." these were from the mouse blood "stabilates"?

Line 188 and 189, figure S1 is figure S2, not S1.

Line 192, comma is in the wrong place.

Line 225: do you mean 'figure 4E' instead of 'figure 4B'. It would be informative if the authors could provide a statistic comparison of frequency of copy number alteration between tandem repeated genes and those that are not in tandem to support their statement in line 293-294.

For the figures with chromosomes displayed, are these data points from the copy number of each unique gene? Or are they the average gene copy number per region? The chromosome number should be in order, Chr10 and Chr11 should be after Chr9, not the Chr1.

Significance

There is a lot of good information here, but it could be more clearly presented and better detailed. Placement in the literature is fine - although comparison to long-read sequenced T. brucei or T. cruzi genomes might deserve mention.

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Referee #3

Evidence, reproducibility and clarity

Summary

Most laboratory research using T. b. brucei has made use of two strains, the monomorphic Lister 427 strain and the pleomorphic EATRO1125 strain. These strains have been passaged in vitro for decades in separate laboratories, thus selecting for populations that differ from both the original isolate and between laboratories.

In this study, Mulindwa et al. describe the isolation of two new T. b. brucei strains from cattle in Uganda, MAK65 and MAK98, which show differences in virulence and propensity for stumpy form differentiation in rodents. To assess the effect of culture adaptation on trypanosomes, the researchers compared the gene copy number of the MAK65 and MAK98 isolates before and after culture adaptation. The study found that isolates cultured for as little as a week already demonstrated a broader gene copy number distribution than those that were not culture adapted. Broader gene copy number distributions, compared to the non-culture-adapted isolates, were also observed for a number of routinely-passaged Lister 427 and EATRO1125 cultures. The researchers observed reproducible increases in copy number for certain genes, such as those encoding histones, HSP70 and PFR proteins. The study postulated that changes in gene copy number observed across the genome increased bias towards rapid proliferation and stress tolerance upon culture adaptation.

Major comments

I have some concerns about the method that was used for the copy-number calculations. Firstly, I can imagine that a non-uniform distribution of the reads across the genome for any of the datasets could influence the results. Was this checked? Secondly, in the methods section lines 394/395 it is said that the modal RPKM for each dataset was 'adjusted slightly' to get a symmetrical distribution. Upon checking the modal RPKMs and the adjusted values used for the calculations, the adjusted values appear to have been adjusted to different extents between the different datasets to fit the authors assumptions. Do the authors believe that this could perhaps account for some of the subtle gene copy number changes observed (as is discussed in lines 287-290)? Next, why were the reads aligned 20 times? I think in general the method needs to be explained far more clearly so that the audience can understand what you did.

Minor comments

Additional experiments:

Would it be possible to generate a phylogenetic tree comparing these new isolates with the Lister 427, TREU927 and EATRO1125 isolates in circulation to get an idea of how these strains may have evolved? I this could add to the strength of the manuscript.

Title: Since no other unicellular eukaryotes are mentioned throughout the text, I think a more appropriate title for this study might be 'Adaptation of Trypanosoma brucei brucei bloodstream forms to in vitro culture results in gene copy-number changes.'

Line 59: There have been more recent studies than the referenced paper (Cross et al., 2014) that quote far higher numbers of alternative VSG genes or pseudogenes in the Lister 427 strain. In Müller et al (2018, doi: 10.1038/s41586-018-0619-8) over 2500 VSGs are quoted and in Cosentino et al (2021, doi: 10.1101/2021.04.13.439624) 2872 VSGs are identified.

Line 109/110: The dates of isolation written here, Feb 1st and July 30th 2016, are different to what is written under the Date of Isolation column in Supplementary text 1, table 1- 25/5/2016 and 30/7/2017. Do these two dates represent different things?

Line 233: Should Figure 5 A, B, C and E (rather than A, B, D and E) be referenced here to show all the initial, not cultured results?

Line 270/271: PIP39 does not promote differentiation to stumpy forms, but instead contributes towards the efficient differentiation of stumpies to procyclic forms (Szoor et al, 2010, doi: 10.1101/gad.570310).

Discussion:

It should be discussed in the text that changes in gene copy number have also been observed upon Leishmania culture adaptation (see, for e.g., Gerald Späth's work). This will strengthen the authors' conclusions. Furthermore, similar observations of aneuploidy and triploidy in some T. brucei Lister 427 strains have recently been reported in Cosentino et al (2021, doi: 10.1101/2021.04.13.439624). This could also be referenced somewhere in the text.

Line 364: I think the Nijuru et al (2005, doi: 10.1007/s00436-004-1267-5) paper would be the correct reference here since it describes the use of the ITS-1 PCR. Reference 13 is actually referring to another paper, I cannot find Nijuru et al in the references list.

Line 379: PAD staining should be referenced to be more informative and allow reproducibility.

Figure 1, Line 580: How many cells were counted for determining the % PAD positivity?

Figure 1, Line 581: Scale bar should be included in D.

Supplement Text 1:

I found Supplement Text 1 a little confusing for two reasons. Firstly, I was never sure if the tables or references being referenced were from the main text or from the supplement text 1, perhaps this could be made a little clearer to aid the reader. Secondly, the names of the isolates switched between, for e.g., MAK 65 and Tb065. For simplicity it could help to try and stick to one naming system.

It might be worth adding a sentence about why Tb236B was not followed up. Was this because it could not easily be distinguished from MAK65 by microsatellite analysis?

S3 Figure: Legend describes red bars but there are none in the figure.

Table 2: In the legend for Sheet 2, the cut-off for the increase is missing.

Significance

Though the T. b. brucei Lister 427 and EATRO1125 strains are used most commonly for laboratory-based research, they have been extensively passaged in vitro without characterisation of the changes that have occurred between them and the original isolate, or indeed, between laboratories.

Mulindwa et al. demonstrated that changes to gene copy number occur rapidly upon adaptation to culture of field isolates, and that different cultures of the same isolate can furthermore have different ploidies. This is an important advance and raises awareness a) that trypanosomes undergo changes upon laboratory adaptation, b) of the nature of some of these changes and c) that the changes can occur rapidly. Changes to gene copy number have also been shown to effect Leishmania donovani upon culture adaptation (Prieto Barja et al, 2017, doi: 10.1038/s41559-017-0361-x).

This study will be of interest to the trypanosome community in general, but particularly those who work on biology that we already know is impacted by prolonged in vitro passage-differentiation, virulence and antigenic variation.

Reviewer expertise: BSF differentiation, antigenic variation

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Referee #2

Evidence, reproducibility and clarity

Summary:

Trypanosoma brucei brucei is a protozoan parasite studied both because it is the cause of Human African Trypanosomiasis, and as a unicellular eukaryotic model organism. However, only few isolates are lab-adapted and most literature focuses on derivatives of Lister 427 and EATRO1125, which have both been cultured under in vitro conditions for several decades. In this manuscript, Mulindwa and colleagues describe and characterise two novel T. b. brucei strains (MAK65 and MAK98) isolated from the field with minimal passage through rodents. These new strains are clearly more closely related to trypanosomes in the field, compared to the aforementioned in vitro models. The authors investigate infection dynamics in murine models, in particular to assess differentiation capabilities of the parasites. Furthermore, optimisation of in vitro adaptation is also attempted successfully. Finally, the authors show, through genome sequencing, that adaptation of both strains to laboratory-based culture affects gene ploidy, and several examples of interest are discussed. A key conclusion is that cultured lines from the same original isolate can rapidly (in a matter of weeks) develop karyotype differences, which could carry implications for the study, as well as genetic manipulation of these organisms. The availability of novel laboratory strains for the study of T. b. brucei is a significant outcome for the trypanosome research community.

Major comments:

The key conclusions, in particular that adaptation to in vitro culture results in reproducible genomic alterations are convincing. In my opinion, this is a descriptive study, in that the underlying causes (and consequences) of the findings are not investigated further.

The authors state several hypotheses based on their results in the 'Outlook' section. The majority of these hypotheses are valid points to make, but I feel the following should be toned down: Lines 321-323 - These hypotheses are speculative, based on available data, and should be revised. There are likely many factors that will impact parasite growth in initial stages of culture adaptation, but most commonly there is a selection bottleneck effect that will impact initial growth.

Line 344 - With the data presented, it is difficult to say whether tissue distribution is different between the two trypanosome strains. I would suggest removing this claim, unless work was carried out to investigate differences in tissue distribution.

Changes in gene copy number is discussed, but one interesting aspect is whether this translates to changes at the protein level (e.g. are there increases in HSP70 mRNA or protein levels after in vitro adaptation?). Whilst not essential, carrying out qPCRs or Western blots to confirm this would enhance the impact of this study. If these experiments are not carried out, this point should be added to the discussion.

It would be very interesting to further analyse the genomics data to assess whether there are further changes to metabolic gene copy number, perhaps as a result of in vitro adaptation. These changes could be linked to the high levels of nutrients available in HMI-9 medium. Currently, only a pteridine transporter is mentioned, although the tables list several genes such as glycerol kinase and an arginine transporter. Are there changes in glucose transporter copy number after culturing these strains? Copy number of this array can often vary in field isolates.

The arginine transporter, AAT5 was previously shown to be an essential transporter (PMID: 28045943). Loss of copy numbers could reflect reduced need in a nutrient-rich environment such as in vitro culture medium. I feel this may be worth mentioning, but not investigating further.

More detail is required in the Materials and Methods section, in particular the "sequence analysis" section: Which Illumina kits were used? What tool/software was used for genome alignment? Which reference genome was used (if TREU 927, what version)? What parameters were used for tryprnaseq and DeSeqU1 (if default settings were used, please mention this)? What tool/software was used for gene copy analysis (i.e. the alignment that was restricted to 20 alignments)? How was modal RPKM value adjusted to obtain a symmetrical distribution? These details are essential for this work to be reproducible. In addition, they would help establish a pipeline that can be used to directly compare other novel field isolates.

In the RNA preparation section (line 372), please indicate replicate number for each sample group used for transcriptomics analysis, as well as parasitaemia or cell number used for extractions. In addition, if RNA extraction was carried out according to the Trifast manual, please state this.

The protocol for staining with PAD1 requires more detail (line 380). If this protocol is available in a previous study, it is sufficient to reference this.

The study mentions attempts to adapt cells using methylcellulose (line 179). It would be helpful for the reader to know what percentage (w/v) was attempted. In addition, did the authors consider using serum supplements from different host sources (e.g. goat or adult bovine?). If only FBS supplementation was attempted, it would be worthwhile mentioning this.

This reviewer is not an expert on statistical analysis of genomics data, but it may be of interest to carry out statistical analysis of the differences in copy number between non-culture-selected and culture-selected parasites (figure 6) to determine whether these differences are significant.

In my opinion, experiments are adequately replicated.

Minor comments:

Line 136 - What is the rationale of calculating Log2 fold changes of 65A/98A to compare to log2 fold change of ST/SL? Given the authors are in possession of normalised read count data for all 4 sample groups, it would also be interesting to show correlation between the individual datasets (e.g. 65A vs 98A, 65A vs ST, 65A vs SL, etc). Perhaps this would be more informative, and give an idea whether, for example, 65A is more similar to previously published datasets derived from stumpy or slender parasites.

Prior studies are referenced appropriately, with the following exception: Line 70 - note T. b. gambiense as an example of African trypanosome that does not undergo sexual recombination (reference PMID: 26809473)

To make the text and legends clearer, it would be beneficial to add commas to numbers >1,000. In addition, text would be more legible if spaces are added between numbers and units (e.g. 480 bp, instead of 480bp), with the exception of temperature and percentages. There are also inconsistencies in spacing around the multiplication symbol when stating cell densities (line 170: 1.5 x106/ml; line 176: 5x 105/ml) - please add spacing on both sides.

In figures 1A and 1B, y-axis numbering is not consistent (i.e. 1A is linear, 1B is logarithmic) I would recommend maintaining consistency between these two experiments.

In figure 1C, the 'A' and 'B' variants of the MAK strains are not defined in the legend, nor main text. It would be helpful for the reader to know what is meant by'98A' vs '98B'.

In figure 1E, statistical test used to generate R and R2 are not indicated.

In figure 2, please make y-axis scale consistent (e.g. 2A consistent with 2B, 2C consistent with 2D) as this makes it much easier for the reader to compare and contrast the data2.

In all figures showing gene copy number overviews (4, 5 and S3), the chromosomes not in order (i.e. chromosomes 10 and 11 should come after 9). I feel these figures would be improved by keeping chromosomes in increasing number, left to right.

In figures 5A and 5B, does the 'c' refer to culture or clone? This should be clarified. In addition, it is difficult to tell which colour represents which culture/clone in panel A in particular.

Line 308 - Could the authors explain why the translation factor eIF3c result (significant decrease in copy number) is an "odd" one?

In the legends for Tables 1 and 2 it is worth mentioning that the "Tb927" prefix has been removed for legibility.

Several genes/proteins are discussed in the study (e.g. HSP70, histones). However, the implications of these changes, and their potential significance, are not discussed. A paragraph on what these changes could mean for the biology of the parasite would be of interest.

As mentioned above, lack of PAD1 staining in MAK98 does not necessarily mean differences in tissue distribution compared to MAK65 and this sentence should be revised.

Can the authors comment on expected differences in drug sensitivity in these strains compared to Lister 427 and EATRO1125? For example, changes in pteridine transporter copy number, if reflected at the protein level, could impact anti-folate uptake.

A previous study investigating transcriptomics of culture-derived vs. in vivo derived T. brucei concluded that in vitro-cultured cells can be used as alternatives to animal-derived parasites (PMID: 29606092). How do the results presented here impact the findings of this previous study?

Significance

The availability of new strains to study African trypanosomes is significant. Furthermore, low passage number means that these strains more accurately reflect naturally occurring strains in the field, compared to the commonly used EATRO1125 and Lister 427, both of which have been adapted for laboratory use for decades.

The finding that adaptation to laboratory culture has a significant effect on gene copy number is also of importance, and suggests variation in karyotype between strains in different labs is common, even if they are derived from the same original isolate. The logical next step is to find out how these karyotype differences impact cell biology.

Few trypanosome strains are routinely used in a laboratory setting, in particular the monomorphic Lister 427 and the pleomorphic EATRO1125 (AnTat1.1). The adaptation of two novel strains with minimal passage and differing virulence is a significant outcome. In the past, adaptation involved growing parasites on feeder layer cells but addition of cysteine removes this requirement (PMID: 4045385). Comparison of culture- and animal-derived T. brucei has been carried out before (for example, PMID 29606092), and in addition, genomic analysis has previously suggested aneuploidy does not occur (PMID: 30256189). Therefore, the finding that adaptation to in vitro culture can result in changes in gene copy number is novel, and significant. It is unknown whether these changes are reflected at the mRNA or protein level and this is an important next step.

The main audience for these findings is the trypanosome research community, especially groups working on the genomics of African trypanosomes. In addition, the findings are of significance (as mentioned in the manuscript) to researchers designing genetic manipulation experiments with culture-derived trypanosomes.

The background of this reviewer is in biochemical parasitology, with work focusing on livestock trypanosomes (Trypanosoma congolense, Trypanosoma vivax). Key words: Metabolism, Metabolomics, Transcriptomics, Drug mode of action, Drug resistance.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The authors present an analysis of gene copy number in Trypanosoma brucei lines based on resequencing. The analysis includes 2 strains isolated very recently, which are sequenced straight from passage in animals and following culture in flasks. They then compare gene copy number in these strains to T. brucei lines commonly used in the lab and reference DNA probably isolated after multiple passages through animals, concluding that there are some genes that consistently change copy number on adaptation to culture. There is some overlap with the authors' previous work [PMID: 26715446] comparing isolates of T. b. rhodesiense (data also included here), but here they have the addition of lines pre and post adaptation to culture, and focus more on gene copy number over transcriptomic changes.

The question is a really interesting one: what are labs selecting for when they adapt these parasites to culture? The authors generate some useful data on this subject and suggest some of the additional analysis that could be done with these data in future in the Outlook section. However, I have 2 major comments about the analysis presented that I believe affect whether it can be used to support the conclusions made in this manuscript.

Major comments:

1) In the analysis, the authors' discuss variation in measured copy number as if representing genuine changes in gene number. However, variation can also result from differences in sequencing depth or be introduced during DNA isolation, processing, sequencing and/or mapping. In my opinion, this variation is not adequately dealt with in the analysis presented and as a result at least some of the subsequent interpretations are likely to be incorrect. While array copy numbers in particular would be expected to become more heterogeneous as a population ages, this is unlikely to affect the majority of diploid genes and should not greatly influence the distribution around copy number of 2. Moreover, where average diploid genes do change, they are expected to do so across whole chromosomes or large sections of chromosomes, which does not appear to be the pattern seen in S3 Fig. Moreover, heterogeneity in the population of chromosomes present in the culture resulting from changes in replication/segregation [as suggested in the text] would affect the spread of copy numbers between chromosomes, but not the distribution on each chromosome (as the genes are still linked even when the average chromosome copy number is non-integer). For example, for the distribution broadening seen in MAK98_cA (which appears to be broadening of the distribution at all positions on all chromosomes) to be the result of a biological process would require widespread and extreme fragmentation of the chromosomes. I would suggest this is very unlikely against the alternative of variation introduced during DNA processing, sequencing and/or mapping - and indeed, this is the likely the cause of for the majority of observed difference in the overall distribution around 2 copies.

This source of variation in copy number estimates needs to be accounted for in testing for differences as the certainty for each estimate will vary with sample preparation and also gene length. In addition, the distributions for Lister427 1313 and MAK98_cA probably suggest that these should be removed from the analysis.

2) The lines being analysed are not phylogenetically independent, meaning that similarity can reflect shared ancestry rather than selection. The 3 lines derived from Lister 427 are obviously expected to be very closely related, and the authors have previously shown that the 4 T. b. rhodesiense isolates are highly similar (suggesting recent common ancestor) [PMID: 26715446]. In addition, although EATRO1125 was derived independently, this too might be more closely related to 427 than to the new lines. As such, copy number in each cannot be taken as an independent measurement. I'm afraid I think this makes the statistical approach taken to identify 'reproducible' gene copy number changes invalid. For example, the monooxygenase Tb927.9.1400 in Fig. 6E is present as 2 copies in T. b. rhodesiense isolates and 3 copies in EATRO1125/Lister427 lines, but this probably only represents a single difference between 2 clades, not 8 independent measurements. On adaptation to culture this gene is unchanged in MAK98 (excluding cA) and goes from 2→3 in MAK65. This last observation is interesting (especially if the 4 MAK65 lines are completely independent, which wasn't clear from the text), but whether such change is statistically significant across the whole genome needs testing.

Notwithstanding the above, unique to this study are the copy number estimates for the same populations pre and post adaptation. I think if the authors could apply a method that tests for directional changes in these while accounting for effects of gene size and the heterogeneity due to sequencing discussed above, then an analysis of which of these were observed in both MAK65 and MAK98 - and which also shared by Lister427/EATRO1125 - would be very interesting and potentially very informative, but I don't think the current analysis is sufficient to support the claims made. The authors may want to consult a statistician, but I suspect biological replicates of the sequencing samples will be required.

Minor comments:

The concept of "gene ploidy" is rather unhelpful and I would suggest that the authors make a more strict distinction between changes in ploidy (caused by duplication/loss of chromosomes) and changes in gene copy number (predominantly due to array expansion/contraction). For example, a gene with copy number of 4 can exist as 2 copies on each of 2 homologous diploid chromosomes - and an expansion of one array that increases the copy number to 5 is not a change in ploidy.

The purpose of Fig 1A/B is to compare the infections between 2 T.b. isolates (MAK65/98), but the parasitaemias are displayed on different scales and with different transforms making such comparison extremely difficult. Same for Fig 2A-D and F-G - text discusses growth rate, but can't be judged on linear scale.

Very difficult to see PAD1 staining when overlaid with transmitted light (Fig1D) - could this be separated?

Discussion is made comparing the proportion of cells with PAD1 staining, but it is unclear how many cells and replicates this is based on - percentages should be given with estimate of SEM, confidence intervals or similar.

I found naming of the cultures derived from MAK65 and MAK98 confusing and counter-intuitive. I believe the following to be correct:

MAK65 cA/cB are independent cultures with a total of 30 days in culture and the doubling time ~7h (after ~day 20) MAK65 cC/cD have only 17 days in culture but achieved a similar doubling time to cA/B after ~day 8. MAK98 cA has 33 days in culture, but grew slowly throughout (doubling time ~13h). MAK98 cB has 24 days in culture including a freezing step, but doubling time dropped to 10h after ~day 9. This really needs to be clearer in the manuscript and it would be extremely useful to have some indication of the number of generations each line has been through in culture. To me, it was confusing that MAK65 cA/cB have been longer in culture than cC/cD.

"Our observations on copy number are the tip of the iceberg: a survey of just a single ~10 kb region revealed selection for smaller insertions, deletions and point mutations (S4 Fig).". S4 Fig shows a region around a repeated HSP70 gene, with a large number of SNP/INDELs versus reference. As would be expected, some of these are haplotype-specific, but changes in the ratio of the sequencing reads between the haplotypes should not be presented as evidence for selection without a statistical test for enrichment.

The Supplemental data are an important resource here. They are too large to check extensively, but in trying to reproduce the copy number estimates for the unique genes, I found that the read counts for MAK98 and EATRO1125 in Sheet 5 to be identical except for some NA values in EATRO1125. This is presumably an error. I was also surprised that no genes have 0 count when DNA was mapped to 927 reference - except for DNA from 927 itself which is the one sample I'd expect not to have this behaviour. Is there an explanation for this?

Significance

Combined with evidence, reproducibility and clarity.

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Reply to the reviewers

Response to Reviewers

We thank the reviewers for their valuable and pertinent suggestions that helped us immensely in revising our manuscript. Listed below are the responses to reviewers’ comments where reviewer’s comments are highlighted in bold while the responses are in normal font.

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

As HIV-1 bnAbs show extensive somatic mutations and have several unique properties such as long CDR and ability to recognize glycopeptides, there is a possibility that some of them can cross-react with SARS-CoV-2 and a fraction of these could even neutralize the virus. To explore if this is true, Mishra et al from the Luthra Lab study investigated the cross-reactivity of existing HIV-1 neutralizing antibodies with purified and stabilized SARS-CoV-2 trimeric ectodomain S2P and receptor binding domain. Indeed, they find that antibodies that recognize the CD4 binding site and MPER region of HIV-envelope show fairly strong reactivity to SARS-CoV-2 RBD and S2P like the CoV-1 cross reactive antibody CR3022. They further go on to test neutralization using pseudoviruses decorated with SARS-CoV-2 spike and find that one of the several binding antibodies, N6 can indeed neutralize, though not potently as expected. They also assessed the cross-reactivity and neutralization of plasma from children with chronic HIV-1 infection to SARS-CoV-2 pseudoviruses. Some sample of plasma indeed showed neutralization of SARS-CoV-2 pseudoviruses. Based on these observations, the authors propose cross-reactive epitopes such as the N6 epitope can be used as a blueprint to engineer variant super-antibodies that can be effective against several viral pathogens including SARS-VoV-2. Overall, the study is well conducted and detailed with clarity.

Q1. The authors should include an authentic SARS-CoV-2 neutralizing antibodies in the study as a positive control. There are several RBD as well as NTD binding nAbs that have been identified. This will allow one to assess the extent of cross-reactivity and neutralization exhibited by HIV-1 bnAbs and gauge the extent of practical utility. CR3022 while important, is not enough.

R1. We have now included an Anti-RBD nAb, CC12.1, as positive control for both binding assay and neutralization assays (line numbers 73 – 77).

Q2. Some plasma of HIV-1 infected children showed neutralizing activity against SARS-CoV-2. However, the neutralization seen may not be solely due to HIV-1 bnAbs in plasma. Antibodies to other human coronaviruses likely contributes to this neutralization. The authors should also test reactivity/neutralization to 229E, NL63, OC43, HKU1, MERS spike/pseudoviruses to support their claim. In its absence, the authors should tone down their interpretation and provide alternate explanation.

R2. We have now performed neutralization assays with the plasma from all the HIV-1 infected children against all seven coronaviruses (SARS-CoV-1, SARS-CoV-2, HKU1, NL63, MERS, OC43, and 229E) (line numbers 150 – 160 and 551 – 564).

Q3. The authors start out with the following premise "Given the unique nature of HIV-1 bnAbs and their ability to recognize and/or accommodate viral glycans, we reasoned that the glycan shield of SARS-CoV-2 spike protein can be targeted by HIV-1 specific bnAbs". While HIV-1 envelope is heavily glycosylated, it is interesting to note that the cross-reactive HIV-1 bnAbs target RBD of SARS-CoV-2 spike protein which is not heavily glycosylated (2 sites). In absence of glycosylations-probing experiments, it is difficult to justify this premise. Rather the extensive SHM and self-reactivity (MPER-directed antibodies) could be the likely reason for cross-reactivity.

R3. We have discussed the polyreactivity and/or autoreactivity of HIV-1 bnAbs as one of the plausible reasons for the observed cross-reactivity with SARS-CoV-2 (line numbers 166 – 180).

Minor

Q4. The color profile of in figure 3 needs to be revised or symbols added to allow the curves to be distinguished from each other.

R4. We have changed the color profile of figure 3 and have added symbols as well (line number 527__). Figure 3 from the original manuscript is now figure 4 in the revised manuscript.

Reviewer #2 (Significance (Required))

This is a significant study that highlights the concept of developing a broadly cross-reactive antibodies that may be effective against multiple pathogens, a concept not usually ascribed to antibodies that are known for their specificity unlike drugs that can be repurposed. In-depth study of epitopes that can elicit multi-pathogen cross-reactive/neutralizing antibodies can on the other hand allow generation of antigenic templates that can be effective as vaccines. This study is suitable for broader audiences to disseminate above-mentioned concepts.

**Referee Cross-commenting**

Q1. There is a lot of evidence now that lenti-pseudoviruses are a good surrogate for identifying SARS-CoV-2 neutralizing antibodies. That said the authors may want to ensure the data is reproducible even with authentic SARS-CoV-2 viruses as reviewer 2 suggests to avoid surprises.

R1. We have now tested the ability of N6 to neutralize authentic SARS-CoV-2 virus using a cytopathic effect (CPE) based neutralization assay. N6 failed to show any reduction in CPE upto a concentration of 20 µg/ml (line numbers 135 – 141).

We have now discussed N6’s inability to neutralize authentic virus in discussion section (line numbers 181 – 187).

Q2. I also agree with the Reviewer 2 that structural analyses of the cross-neutralizing antibody with SARS-CoV-2 spike will strengthen the manuscript.

R2. While we do agree with both the reviewer’s suggestion that a structural analysis of N6 binding with SARS-CoV-2 will provide details into the exact nature of epitopes-paratopes that are responsible for the cross-reactivity observed, we believe it to be out of scope for the current work. The current work was designed to show the ability of polyreactive and somatically hypermutated antibodies generated in chronic HIV-1 infection to bind to new and emerging pathogens such as SARS-CoV-2. While only N6 showed neutralization of SARS-CoV-2, it is also noteworthy that several HIV-1 bnAbs showed significant binding to both purified RBD as well as surface expressed spike from SARS-CoV-2. As all these distinct bnAbs have different paratopes, a detailed structural analysis to understand the exact nature by which several distinct bnAbs cross-reacted with SARS-CoV-2 is not feasible. We are optimistic that our results will provide that impetus for future detailed structural studies (line numbers 194 – 198__).

Q3. Also, the authors may want to avoid 293T-ACE2 cells as their targets for carrying out neutralization assays. There is data now from Davide Corti's group that indicate ACE2 overexpression can significantly underestimate the neutralizing capacity of antibodies especially those that bind to RBDs (bioRxiv 2021.04.03.438258; doi: https://doi.org/10.1101/2021.04.03.438258). This may be particularly true for N6. A cell line like Vero E6 that endogenously express ACE2 would be the way to go. If the authors decide to use Lenti pseudoviruses as the first step in Vero cells, please make sure to use G89V mutated HIV gag-pol to avoid TRIM5alpha restriction.

R3. Based on reviewer’s suggestion, we have now performed neutralization assays for all bnAbs using both HEK293T/ACE2 cells and Vero-E6 cells. We have used an HIV-1 proviral backbone that does contain G89V mutation in the gag-pol region (line numbers 96 – 121).

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

The authors report that HIV-1 sp-bnMAb cross-neutralized SARS-CoV-2 pseudotyped virus in vitro. The finding that N6 can block HIV-based-SARS-CoV-2 pVs from entering 293T-ACE2 cells is intriguing and requires further investigation before the manuscript can be considered for publication.

Major comments

Q1. The authors must demonstrate that N6 is capable of neutralizing authentic SARS-CoV-2.

R1. We have now tested the ability of N6 to neutralize authentic SARS-CoV-2 virus using a cytopathic effect (CPE) based neutralization assay. N6 failed to show any reduction in CPE upto a concentration of 20 µg/ml (line numbers 135 – 141).

We have now discussed N6’s inability to neutralize authentic virus in discussion section (line numbers 181 – 187).

Q2. The authors should also demonstrate it works in another system i.e., recombinant VSV/SARS-CoV-2 (used by Naveenchandra Suryadevara et al., 2021 "Neutralizing and protective human monoclonal antibodies 1 recognizing the N2 terminal domain of the SARS-CoV-2 spike protein" https://doi.org/10.1016/j.cell.2021.03.029) which is a well validated system for SARS-CoV-2 neutralization activity.

R2. We have additionally used a MLV based pseudovirus system to further increase the robustness of neutralization assays. In addition, we have now used three distinct cell lines (HEK293T/ACE2, Vero-E6 and Huh7 cells) to exclude any bias in neutralization assays due to endogenous overexpression of ACE2 on HEK293T cells (line numbers 96 – 121).

Q3. Please, reference the HIV-1 based SARS pVs neutralization assay used in this work if it has been used earlier, either by your team or others. Details regarding the performance of this particular assay for SARS-CoV-2 Nab detection will aid the reader to understand its robustness and weaknesses.

R3. We have provided several references for both the HIV-1 lentiviral and MLV retroviral pseudovirus neutralization assays. Furthermore, we have added detailed results for optimization of neutralization assays that we performed (line numbers 96 – 121 and figure 3). In addition, we have discussed the limitation and weaknesses of pseudovirus neutralization assays in comparison to authentic virus neutralization assays (line numbers 183 – 187).

Q4. In the method section, a list of all controls used to guarantee neutralization assay performance must be included (i.e., positive and negative mAb controls, virus controls, etc.); additionally, it might be necessary to show raw data of all control results.

R4. We have provided the detail of all controls that were used to guarantee the robustness of neutralization assay in the methodology section of “neutralization assay.’ Furthermore, we have now added an entire section in the results titled, ‘Optimized conditions for neutralization of pseudotyped coronaviruses,’ where we now provide all the technical details that were optimized to ensure the robustness of the neutralization assay (line numbers 96 – 121).

Q5. Control description are missing in the method section for all or most of the assays (some descriptions are found in the result section, but definitely they should be found in M&M, for each technique).

R5. We have now provided the description for all the controls in methodology section too.

Q6. The authors draw some similarities between HIV-1 Env and SARS-CoV-2 Spike proteins from structure and glycosylation point of view, but the fact that N6 binds to ENV-CD4bs is beyond these pictures and for that reason a detailed structural analysis MUST be performed to show the mechanism of cross-neutralization and corroborate it exists.

R6. While we do agree with both the reviewer’s suggestion that a structural analysis of N6 binding with SARS-CoV-2 will provide details into the exact nature of epitopes-paratopes that are responsible for the cross-reactivity observed, we believe it to be out of scope for the current work. The current work was designed to show the ability of polyreactive and somatically hypermutated antibodies generated in chronic HIV-1 infection to bind to new and emerging pathogens such as SARS-CoV-2. While only N6 showed neutralization of SARS-CoV-2, it is also noteworthy that several HIV-1 bnAbs showed significant binding to both purified RBD as well as surface expressed spike from SARS-CoV-2. As all these distinct bnAbs have different paratopes, a detailed structural analysis to understand the exact nature by which several distinct bnAbs cross-reacted with SARS-CoV-2 is not feasible. We wish that our results will provide that impetus for future detailed structural studies (line numbers 194 – 198).

Q7. As the authors may realize, based on previous comments, any false positive results that could have biased the main conclusion of this article, must be excluded in order to claim such intriguing finding.

Wherever applicable, we have tried to use proper positive and negative control and have tried to tone down the inferences.

Q8. Regarding BINDING AND NEUTRALIZATION OF SARS-COV-2 WITH PLASMA FROM HIV-1 INFECTED PAEDIATRIC PATIENTS, how can you discard previous exposure to actual SARS-CoV-2 in such population, and/or previous infection with other common hu-CoVs, which has been shown to elicit cross reactive SARS-CoV-2 Abs.

R8. All the pediatric plasma samples used in the binding and neutralization assays were pre-pandemic (2013-2014). While we do agree that exposure to common endemic coronaviruses might have elicited some degree of cross-reactive SARS-CoV-2 antibodies, we have now performed neutralization assay for all seven coronaviruses (including common endemic coronaviruses HKU1, OC43 and 229E) using the plasma that showed SARS-CoV-2 neutralization (line numbers 150 – 160 and figure 6__).

Minor comments can be addressed once these main concerns are solved.

Reviewer #3 (Significance (Required)):

Q9. The fact that N6 (and plasma from HIV-1 infected patients) can neutralize SARS-CoV-2 is novel and intriguing; however, these observations must be supported with new experiments. Showing that N6 neutralizes authentic SARS-CoV-2 would be a key point to address. Moreover, the mechanism for such interaction should be explored using a detailed structural analysis.

R9. Please see response to query 6.

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Referee #2

Evidence, reproducibility and clarity

The authors report that HIV-1 sp-bnMAb cross-neutralized SARS-CoV-2 pseudotyped virus in vitro. The finding that N6 can block HIV-based-SARS-CoV-2 pVs from entering 293T-ACE2 cells is intriguing and requires further investigation before the manuscript can be considered for publication.

Major comments:

The authors must demonstrate that N6 is capable of neutralizing authentic SARS-CoV-2.

The authors should also demonstrate it works in another system i.e., recombinant VSV/SARS-CoV-2 (used by Naveenchandra Suryadevara et al., 2021 "Neutralizing and protective human monoclonal antibodies 1 recognizing the N2 terminal domain of the SARS-CoV-2 spike protein" https://doi.org/10.1016/j.cell.2021.03.029) which is a well validated system for SARS-CoV-2 neutralization activity.

Please, reference the HIV-1 based SARS pVs neutralization assay used in this work if it has been used earlier, either by your team or others. Details regarding the performance of this particular assay for SARS-CoV-2 Nab detection will aid the reader to understand its robustness and weaknesses.

In the method section, a list of all controls used to guarantee neut assay performance must be included (i.e., positive and negative mAb controls, virus controls, etc.); additionally, it might be necessary to show raw data of all control results.

Control description are missing in the method section for all or most of the assays (some descriptions are found in the result section, but definitely they should be found in M&M, for each technique).

The authors draw some similarities between HIV-1 Env and SARS-CoV-2 Spike proteins from structure and glycosylation point of view, but the fact that N6 binds to ENV-cd4bs is beyond these picture and for that reason a detailed structural analysis MUST be performed to show the mechanism of cross-neutralization and corroborate it exists.

As the authors may realize, based on previous comments, any false positive result that could biased the main conclusion of this article, must be excluded in order to claim such intriguing finding.

Regarding BINDING AND NEUTRALIZATION OF SARS-COV-2 WITH PLASMA FROM HIV-1 INFECTED PAEDIATRIC PATIENTS, how can you discard previous exposure to actual SARS-CoV-2 in such population, and/or previous infection with other common hu-CoVs, which has been shown to elicit cross reactive SARS-CoV-2 Abs.

Minor comments can be addressed once these main concerns are solved.

Significance

The fact that N6 (and plasma from HIV-1 infected patients) can neutralize SARS-CoV-2 is novel and intriguing; however, these observations must be supported with new experiments. Showing that N6 neutralizes authentic SARS-CoV-2 would be a key point to adress. Moreover, the mechanism for such interaction should be explored using a detailed structural analysis.

Referee Cross-commenting

I am happy to realize that there is an agreement in the major points observed by both Reviewers. These comments Must be answered properly by the authors now.

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Referee #1

Evidence, reproducibility and clarity

As HIV-1 bNabs show extensive somatic mutations and have several unique properties such as long CDR and ability to recognize glycopeptides, there is a possibility that some of them can cross-react with SARS-CoV-2 and a fraction of these could even neutralize the virus. To explore if this is true, Mishra et al from the Luthra Lab study investigated the cross-reactivity of existing HIV-1 neutralizing antibodies with purified and stabilized SARS-CoV-2 trimeric ectodomain S2P and receptor binding domain. Indeed they find that antibodies that recognize the CD4 binding site and MPER region of HIV-envelope show fairly strong reactivity to SARS-CoV-2 RBD and S2P like the CoV-1 cross reactive antibody CR3022. They further go on to test neutralization using pseudoviruses decorated with SARS-CoV-2 spike and find that one of the several binding antibodies, N6 can indeed neutralize, though not potently as expected. They also assessed the cross-reactivity and neutralization of plasma from children with chronic HIV-1 infection to SARS-CoV-2 pseudoviruses. Some sample of plasma indeed showed neutralization of SARS-CoV-2 pseudoviruses. Based on these observations, the authors propose cross-reactive epitopes such as the N6 epitope can be used as a blueprint to engineer variant super-antibodies that can be effective against several viral pathogens including SARS-VoV-2. Overall the study is well conducted and detailed with clarity.

1. The authors should include an authentic SARS-CoV-2 neutralizing antibodies in the study as a positive control. There are several RBD as well as NTD binding nABs that have been identified. This will allow one to assess the extent of cross-reactivity and neutralization exhibited by HIV-1 bNAbs and guage the extent of practical utility. CR3O22 while important, is not enough.
2. Some plasma of HIV-1 infected children showed neutralizing activity against SARS-CoV-2. However the neutralization seen may both be solely due to HIV-1 bnAbs in plasma. Antibodies antibodies to other human coronaviruses likely contributes to this neutralization. The authors should also test reactivity/neutralization to 229E, NL63, OC43, HKU1, MERS spike/peusdoviruses to support their claim. In its absence, the authors should tone down their interpretation and provide alternate explanation.
3. The authors start out with the following premise "Given the unique nature of HIV-1 bnAbs and their ability to recognize and/or accommodate viral glycans, we reasoned that the glycan shield of SARS-CoV-2 spike protein can be targeted by HIV-1 specific bnAbs". While HIV-1 envelope is heavily glycosylated it is interesting to note that the cross-reactive HIV-1 bnAbs target RBD of SARS-CoV-2 spike protein which is not heavily glycosylated (2 sites). In absence of glysosylation-probing experiments, it is difficult to justify this premise. Rather the extensive SHM and self-reactivity (MPER-directed antibodies) could be the likely reason for cross-reactivity.

Minor:

1. The color profile of in figure 3 needs to be revised or symbols added to allow the curves to be distinguished from each other.

Significance

This is a significant study that highlights the concept of developing a broadly cross-reactive antibodies that may be effective against multiple pathogens, a concept not usually ascribed to antibodies that are known for their specificity unlike drugs that can be repurposed. In-depth study of epitopes that can elicit multi-pathogen cross-reactive/neutralizing antibodies can on the other hand allow generation of antigenic templates that can be effective as vaccines. This study is suitable for broader audiences to disseminate above-mentioned concepts.

Referee Cross-commenting

1.There is a lot of evidence now that lentipseudoviruses are a good surrogate for identifying SARS-CoV-2 neutralizing antibodies. That said the authors may want to ensure the data is reproducible even with authentic SARS-CoV-2 viruses as reviewer 2 suggests to avoid surprises.

1. I also agree with the Reviewer 2 that structural analyses of the cross-neutralizing antibody with SARS-CoV-2 spike will strengthen the manuscript.
2. Also the authors may want to avoid 293T-ACE2 cells as their targets for carrying out neutralization assays. There is data now from Davide Corti's group that indicate ACE2 overexpression can significantly underestimate the neutralizing capacity of antibodies especially those that bind to RBDs (bioRxiv 2021.04.03.438258; doi: https://doi.org/10.1101/2021.04.03.438258). This may be particularly true for N6. A cell line like Vero E6 that endogenously express ACE2 would be the way to go. If the authors decide to use Lenti pseudoviruses as the first step in Vero cells, please make sure to use G89V mutated HIV gagpol to avoid TRIM5alpha restriction.

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Reply to the reviewers

Manuscript number: RC-2021-00733

Corresponding author(s): Haiyun Gan

Sara Monaco, PhD Managing Editor Review Commons

Dear Dr. Monaco,

We would like to thank all the reviewers for their insightful and constructive comments, which have helped us to improve our work. Please find below our responses to each of the concerns raised by the reviewers.

Sincerely

Haiyun

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

**Summary:**

• In this article, Li and colleagues demonstrate the utility of a novel proximity labeling-based strategy, which they term AMAPEX (antibody-mediated protein A APEX) for the proteomic characterization of the protein environment of specific histone modifications. They apply this new methodology in mouse embryonic fibroblasts for subsequent purification of biotinylated proteins and mass-spectrometric identification. **Major comments:**

• The authors present a high quality, descriptive manuscript that introduces a novel proximity labeling approach from a mostly technical point of view. The presentation of the data and methods are mostly clear, such that they should be easy to reproduce.

  We thank the reviewer for the positive view of our work.

• The biggest shortcoming of the study in its current form seems to be the lack of a proper assessment of the method's sensitivity AND -most importantly also- specificity. The authors do not validate potentially new interactors of the modified histones experimentally, which would highlight their technology as a discovery tool. Without the assessment of newly identified proteins, these could simply represent false positives, which would point towards an additional requirement for optimization of the experimental setups. In that light the newly identified proteins are merely potential histone interactors. Validation would require establishment or purchase of additional antibodies, or alternatively cloning and transfection of respective candidates, which will probably take an extra 2-3 months of work. While the study may be publishable in its current form, this validation seems a valuable investment to strengthen the preliminary conclusions.

  We agree with the reviewer that lack of validation of our approach is the major shortcoming of our study. As described in our manuscript, we could identify most of the histone interacting proteins found by other methods in the published data(Vermeulen et al, 2010; Villasenor et al, 2020) (fig. 2A). We also provide evidence that the H3K27me3 proteins (NSD1, Brd9) identified by H3K27me3 AMAPEX are co-localized with H3K27me3 at the same genomic regions (fig. 2C). In addition, as we observed the enrichment of splicing proteins of the H4K5ac interacting proteins, we purified nucleosomes and performed SF3B1 IP and confirmed the interaction of SF3B1 with H4K5ac. We also performed H3K27me3 mono-nucleosome IP and confirmed the interaction between H3K27me3 and NSD2. Together, these results suggested that AMAPEX is a reliable method to map histone proximal proteins. We have added the SF3B1/H4K5ac, H3K27me3/NSD2 binding data to the revised manuscript (Fig. 2E and Fig. 7C).

• With two biological replicates the study fulfills the minimum requirements for reproducibility, however, it would benefit from an additional replicate.

  We added extra replicates in the assays for H3K27me3 and H3K4me3 (Fig. 2A and Fig. 6A ).

**Minor point:**

• The sentences in lines 22f (and 44f) could be misunderstood. Please rephrase statements to be unambiguous that it specifies the proteomic surrounding of post-translationally modified proteins. The proximity labeling technologies may themselves be limited in identifying post-translationally modified proteins, as they label reactive side-chains that are often targeted by PTMs. Post-translationally modified lysine residues cannot be targeted by biotin ligase-based methodologies, as tyrosine phosphorylations cannot be assessed by peroxidase-based labeling.

  We agree that these sentences are confusing. We rephrased the sentence to “cannot be applied to identify proteins surrounding of post-translationally modified proteins” and “mapping the biomolecules that are proximal to post-translationally modified (PTM) proteins like histone modifications is complex” in our revised manuscript.

Reviewer #1 (Significance (Required)):

• From a technical point of view, most of the key conclusions of this paper are convincing. Assessing the proteomic environment of post-translationally modified proteins is of extremely high interest and the methodology seems broadly applicable to address such questions. It should be noted that proximity labeling has not originally been utilized to map protein-protein interactions, as the enzymatic activities available probe their surroundings, this could be an over-interpretation. Nonetheless, the "proteomic surrounding" of target proteins, which would also include hard to identify transient interactions is of high general interest for molecular biologists.

  Thanks so much, we have rephrased our sentences in the revised manuscript.

• Although it can be generally agreed that having to express an exogenous fusion protein is a limitation of current proximity labeling setups, the methodology presented here in turn has the limitation that it cannot be performed in living cells, which is a significant disadvantage.

  We agree that it is a disadvantage that our assay cannot be done in living cells, as most of the antibody-based assays have these limitations. However, we have successfully applied the AMAPEX to the cell samples under native conditions and we have included the results in the revised manuscript (Fig. 8)

Moreover, our method working under fixation conditions can be potentially applied to FFPE samples. We will add discussion to our revised manuscript.

• My lab is establishing and constantly improving various proximity labeling methodologies in combination with mass spectrometry for sub-organellar proteomics. While the technology is sound and well executed, I am not an expert in the biology of histone modifications and the proteins involved and cannot assess the novelty nor the actual value of the generated datasets. It appears to me though that additional validation by independent experiments would strengthen the manuscript. The authors describe the usefulness of the technology mainly in confirming known interactors of modified histones, however, it would be nice (for non-specialists) to explicitly state how high the coverage of the known interactors is and discuss why some of them might have been missed.

  Thanks for the suggestion. We have added the statement of the coverage of the known interactors in our revised manuscript and we also include discussions.

• The manuscript in its current form completely lacks a discussion of the presented data. Even if the focus will remain on the technical aspects, the findings should be properly discussed by comparison to other proteomics approaches studying histones. Ideally the data should also be discussed in the light of current proximity labeling technologies and potential future directions.

  We have added the discussion part to our revised manuscript.

• While I cannot assess the value for histone research, the manuscript will be very interesting for experts focusing on proximity labeling technologies and subcellular proteomics. Reviewer #2 (Evidence, reproducibility and clarity (Required)):

• This work describes interesting approach for mapping interactome of specifically modified histone protein using antibody-based APEX2. Although it contains interesting results and useful techniques for biological community, I found that it requires revision and addition for the publication.

• I could understand the reason using antibody-based proximity labeling approach for mapping the biomolecules that interact with post-translationally modified histone because it should be complicated to map it with exogenous protein expression approach. However, the introduction of antibody-conjugated APEX2 should require fixation and permeabilization steps that can usually compromise ultrastructure. The authors should comment whether these procedures can affect protein composition and structures of nucleosome in the Discussion part of this manuscript.

  We agree with the reviewer. To address the concerns raised here, we have performed AMPEX under native conditions (H3K37me3). Our results showed that AMPEX under native conditions can still identify most of the surrounding proteins identified with crosslink approach. We have added the data to the revised manuscript.

There are also advantages to perform the AMAEX with fixed samples, which may help capture the temporary events. We have added this to the discussion of the revised manuscript.

Formaldehyde crosslinking may cause undesired effect. For instance, the nuclear proteins/loci are not equally efficiently cross-linked; cross-linking may trigger the DNA damage response; crosslinking can also mask epitopes of some antibodies, affecting antibody/antigen binding. However, most of the approaches studying chromatin/nucleosome in cells requires crosslinking and permeabilization. We have included discussion about whether fixation and permeabilization affects protein composition and structures of nucleosome.

• For generation of biotin-phenoxyl radical, HRP-conjugated antibody can be utilized as shown in BAR method (Daniel Z Bar et al. Nat Methods, 2018, 15, 127-133). Since secondary-HRP antibody is commercially available, one cannot make an effort to express and purify pA-APEX2 for this approach. The authors should clearly explain why they selected APEX2 and what is an expected advance(s) using APEX2 in their approach

  We have compared pA-APEX2 with secondary-HRP and we found that pA-APEX2 can give better specificity as there more proteins identified by H3K27me3 mediated APEX2 located in the nuclear than that of HRP. We found that while only 33.31% of proteins identified by HRP based method locates in the nuclear, 69.4% of that identified by pA-APEX2 locates in nuclear, which implicates the increased specificity of the pA-APEX2 method compared to HRP conjugated 2nd antibody. We have added to data to revised manuscript (Fig. 3C, D).

• For the detection of the APEX-mediated biotinylated proteins, direct mass identification of tyrosine-modified peptides with chemical probes can tell the most correct information of the proximal proteins (see Lee SY et al. J. Am. Chem. Soc. 2017, 139, 3651-3662; Namrata D Udeshi et al. Nature Methods 2017, 14, 1167-1170). Thus, if the authors can obtain the biotin-modified peptide information from each antibody-conjugated APEX2, the quality of their interactome results should be much improved. If authors may be under the situation that cannot conduct further mass experiments, it might be required to check whether their important finding molecules (e.g. Arid2, Brd7, Nsd2) are really "biotinylated" by conducting Streptavidin-HRP western blot experiment after enrichment of those proteins by using primary antibody. If biotinylation is specifically conducted by pA-APEX2 with H3K27me3 antibody, the authors can observe SA-HRP blot signal on the enriched protein band on the membrane. Negative controls should be the samples omit pA-APEX2, H3K27me3 antibody, biotin-phenol, H2O2, respectively or using different PTM targeted primary antibody. This result can confirm that their findings are enriched from proximity-dependent biotinylation of APEX2, not from spurious binding events to the other biotinylated proteins or self-labeled bait proteins.

  We thank the reviewer’s suggestion. We agree that finding the biotinylated peptides of the discovered proteins in our experiments could make the results more convincing. A variable modification of biotin-phenol on tyrosine was added to the search setting of MaxQuant (version 1.6.10.43), which indeed led to the identification of very few (only ten) biotinylated peptides without the ones from Arid2, Brd7, or Nsd2. However, these results were not surprising. Since, we pulled down the low abundant target proteins based on the strong binding of biotin-streptavidin (Kd ≈ 10−14–10−16 M ) (Laitinen et al, 2007)., which is also unusually stable against heat, denaturants, extremes of pH and proteolytic enzymes(Wilchek et al, 2006). These were considered as the obvious advantages of the technology. Thereafter, to obtain the better peptide coverage, on-bead tryptic digestion was performed under a relatively mild condition (50mM HEPES pH8.0, 1μM CaCl2, and 2% ACN). Therefore, it is highly likely that, the biotinylated peptides were still trapped on the streptavidin beads but not detectable in the samples.

We indeed performed the Streptavidin-HRP western blot experiment and compared to the samples omit H3K27me3 antibody, H2O2, or samples of IgG, there is increased SA-HRP blot signal in the samples conducted by pA-APEX2 with H3K27me3 antibody (Supplementary Fig. 1F).

Reviewer #2 (Significance (Required)):

• For antibody-binding APEX2 strategy, this work is not the first one and the authors should mention the precedent work in the manuscript: Jisu Lee et al. Chem. Commun., 2015, 51, 10945-10948. And the author also commented the antibody-based proximity labeling mapping works including Daniel Z Bar et al. Nat Methods, 2018, 15, 127-133 in the manuscript.

  We are sorry for the oversight. We have rephrased our sentences in the revised manuscript. In addition, we have also compared our method with the one published in Nat Methods.

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

**Summary:**

• The manuscript by Li and coworkers describe an approach for detecting the PTM specific interacting proteomes through Proximity labeling strategy. In this method, cellular proteomes were first cross-linked by formaldehyde and then incubated with PTM specific antibodies. Afterwards, a protein A-APEX2 fusion protein was added to target the antibody and label the proteins in proximity with biotin. The biotinylated proteins can be enriched and subsequently identified by LC-MS/MS. The authors claim that these proteins are PTM-specific interacting proteins because they are close to the PTM-recognition antibody. They mainly applied the method to the histone marks and identified the interacting proteins for several histone modifications. They found that the identified proteins overlap partially with the published CHIP-seq data and drew complicated interaction networks based the proteomic data using the STRING database. **Major comments:**

• The APEX technology has been widely used for capturing subcellular proteome or proteome proximal to the target protein of interest. Here the authors aimed to use antibodies specific for detecting certain histone PTMs to guide the APEX enzyme for proximal labeling. While the idea is interesting, the actual applicability is rather limited as APEX does not enable identification of direct interactions. With the current methodology design, the reviewer did not see its advantage over a formaldehyde crosslinking followed by a conventional immunoprecipitation using the PTM-specific antibody. The authors are suggested to compare the head-to-head performance of these two approaches.

  We agree with the reviewer that APEX2 is for proximal labeling and we have rephrased our statement from “binding” or “interaction’ to “proximal”.

We have compared with the data of published work. (Vermeulen et al., 2010; Villasenor et al., 2020) (Fig. 2A) and we were able to identify most of the proteins identified in the published work. To improve our method, we performed AMPEX under native conditions and still got very robust labeling of nearby proteins, which is an improvement compared to the formaldehyde crosslinking followed by a conventional immunoprecipitation using the PTM-specific antibody method.

• From the methodological point of view, it is unclear why formaldehyde crosslinking is included here. Neither did the authors justify its necessity for the whole workflow. With treatment with 0.1% formaldehyde for proteome crosslinking, how about physiological status of the cells? Authors should assess survival of cells under formaldehyde treatment. After a long labeling time, will the labeling results still reflect the physiological status of the protein interaction networks in living cells?

  We agree with the reviewer regarding the role of crosslinking in our method. To this point, we have performed AMPEX under native conditions and still got very robust labeling of nearby proteins. On the other hand, crosslinking allows our method to be potentially used for FFPE samples.

• Overall, the manuscript lacks sufficient description on how the pipeline was optimized. For example, does it matter where the APEX2 is fused to protein A? The authors need to do a better job to present their optimization process.

  We found the activity is robust with current pA-APEX2. There is published data that there is only minimal effect on enzymatic activity when APEX2 is fused to Tn5 at different positions, which suggested APEX2’s enzymatic activity is unlikely to be affected by the location of fusion proteins.

• Crosslinking will result in high background in proximity labeling, in figure 1 D, the signal from IgG is obviously high. Strangely, the background signals completely disappeared in figure 2A. What are the differences between these two experiments? Similarly, in figure S4, the IgG lanes in A, B and C looked quite different from each other. The results suggested that the workflow might not be as robust as the authors have claimed.

  The high background of IgG in Fig. 4A in very possibly caused by the relatively less effective labeling by H3K4me3. As we responded before, we have included news results obtained under native conditions, which reduced background.

• The authors drew complicated interaction networks for each histone PTMs, however, a comparison between the networks for a modified histone versus the unmodified one is missing. Such data would be more valuable and informative as they can provide new clues on how the PTM mediates different interaction networks for cellular signaling.

  We have performed AMPEX with different Histone modification antibodies, while there are overlaps between these modifications, we could find proximal proteins specific identified by different histone modifications.

• The manuscript lacks the experimental validation of the identified interaction protein partners. The authors used the published CHIP-seq data, however, the cell lines of CHIP-seq are different from the one they used for APEX labeling. In this regard, an side-by-side comparison of this method with the CHIP-seq results should be performed, in terms of both purification efficiency and specificity.

  To validate our findings, we have purified nucleosomes and performed SF3B1 IP and confirmed the interaction of SF3B1 with H3K5ac. We have also performed mono-nucleosome H3K27me3 IP and confirmed the binding between H3K27me3 and NSD2. We have included these validated results in our revised manuscript.

• For the proteomic data, reproducibility calculation should use intensity ratio instead of intensity. The high dynamic of signal intensity will mislead the audience. P values between replicates should be calculated, a cutoff of

  We have calculated P values between replicates by t-test and added them in supplementary table s1.

**Minor comments:**

• Reference format should be checked. eg. redundant citation, incorrect format etc.

  We have checked our references for the errors and corrected them as many as we can find.

• Loading controls should be attached along with western blotting results (figure 1d, figure 2A).

  We have included the Ponceau S staining as loading controls of these western blotting results in our revised manuscript.

• This manuscript lacks clear conclusion or discussions. Subtitles are needed to delineate results. The overall logical organization of this manuscript should be strengthened. Currently, it is quite hard to follow and appreciate which part of the data is more technically novel and biologically significant.

  We have added subtitles and discussion in the revised manuscript.

• The title is too broad as the authors only showed the application on histone PTMs.

  We have changed our title to “Defining proximity proteomics of Histone modifications by antibody-mediated protein A-APEX2 labeling”.

• The introduction lacks proper description of other strategies for mapping PTM specific interactome, especially those by photo-affinity peptides or photo-affinity unnatural amino acids.

  We have added the other strategies for mapping PTM specific interactome in the introduction in the revised manuscript.

Reviewer #3 (Significance (Required)):

• It is more technical improvement by fusing protein A with APEX2 so that the proximity labeling can be guided around a specific PTM using the proper antibody. Researchers in the field of histone modifications will be interested in the current technique. The reviewer is with expertise in proteomics with focus on PTM analysis. References

Laitinen OH, Nordlund HR, Hytoenen VP, Kulomaa MSJTiB (2007) Brave new (strept)avidins in biotechnology. 25: 269-277

Vermeulen M, Eberl HC, Matarese F, Marks H, Denissov S, Butter F, Lee KK, Olsen JV, Hyman AA, Stunnenberg HGJC (2010) Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. 142: 967-980

Villasenor R, Pfaendler R, Ambrosi C, Butz S, Giuliani S, Bryan E, Sheahan TW, Gable AL, Schmolka N, Manzo M et al (2020) ChromID identifies the protein interactome at chromatin marks. Nat Biotechnol 38: 728-736

Wilchek M, Bayer EA, Livnah OJIL (2006) Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction. 103: 27-32

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Referee #3

Evidence, reproducibility and clarity

Summary:

The manuscript by Li and coworkers describe an approach for detecting the PTM specific interacting proteomes through Proximity labeling strategy. In this method, cellular proteomes were first cross-linked by formaldehyde and then incubated with PTM specific antibodies. Afterwards, a protein A-APEX2 fusion protein was added to target the antibody and label the proteins in proximity with biotin. The biotinylated proteins can be enriched and subsequently identified by LC-MS/MS. The authors claim that these proteins are PTM-specific interacting proteins because they are close to the PTM-recognition antibody. They mainly applied the method to the histone marks and identified the interacting proteins for several histone modifications. They found that the identified proteins overlap partially with the published CHIP-seq data and drew complicated interaction networks based the proteomic data using the STRING database.

Major comments:

1. The APEX technology has been widely used for capturing subcellular proteome or proteome proximal to the target protein of interest. Here the authors aimed to use antibodies specific for detecting certain histone PTMs to guide the APEX enzyme for proximal labeling. While the idea is interesting, the actual applicability is rather limited as APEX does not enable identification of direct interactions. With the current methodology design, the reviewer did not see its advantage over a formaldehyde crosslinking followed by a conventional immunoprecipitation using the PTM-specific antibody. The authors are suggested to compare the head-to-head performance of these two approaches.
2. From the methodological point of view, it is unclear why formaldehyde crosslinking is included here. Neither did the authors justify its necessity for the whole workflow. With treatment with 0.1% formaldehyde for proteome crosslinking, how about physiological status of the cells? Authors should assess survival of cells under formaldehyde treatment. After a long labeling time, will the labeling results still reflect the physiological status of the protein interaction networks in living cells?
3. Overall, the manuscript lacks sufficient description on how the pipeline was optimized. For example, does it matter where the APEX2 is fused to protein A? The authors need to do a better job to present their optimization process.
4. Crosslinking will result in high background in proximity labeling, in figure 1 D, the signal from IgG is obviously high. Strangely, the background signals completely disappeared in figure 2A. What are the differences between these two experiments? Similarly, in figure S4, the IgG lanes in A, B and C looked quite different from each other. The results suggested that the workflow might not be as robust as the authors have claimed.
5. The authors drew complicated interaction networks for each histone PTMs, however, a comparison between the networks for a modified histone versus the unmodified one is missing. Such data would be more valuable and informative as they can provide new clues on how the PTM mediates different interaction networks for cellular signaling.
6. The manuscript lacks the experimental validation of the identified interaction protein partners. The authors used the published CHIP-seq data, however, the cell lines of CHIP-seq are different from the one they used for APEX labeling. In this regard, an side-by-side comparison of this method with the CHIP-seq results should be performed, in terms of both purification efficiency and specificity.
7. For the proteomic data, reproducibility calculation should use intensity ratio instead of intensity. The high dynamic of signal intensity will mislead the audience. P values between replicates should be calculated, a cutoff of < 0.05 or < 0.01 is mostly used in proteome data.

Minor comments:

1. Reference format should be checked. eg. redundant citation, incorrect format etc.
2. Loading controls should be attached along with western blotting results (figure 1d, figure 2A).
3. This manuscript lacks clear conclusion or discussions. Subtitles are needed to delineate results. The overall logical organization of this manuscript should be strengthened. Currently, it is quite hard to follow and appreciate which part of the data is more technically novel and biologically significant.
4. The title is too broad as the authors only showed the application on histone PTMs.
5. The introduction lacks proper description of other strategies for mapping PTM specific interactome, especially those by photo-affinity peptides or photo-affinity unnatural amino acids.

Significance

It is more technical improvement by fusing protein A with APEX2 so that the proximity labeling can be guided around a specific PTM using the proper antibody. Researchers in the field of histone modifications will be interested in the current technique. The reviewer is with expertise in proteomics with focus on PTM analysis.

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

This work describes interesting approach for mapping interactome of specifically modified histone protein using antibody-based APEX2. Although it contains interesting results and useful techniques for biological community, I found that it requires revision and addition for the publication.

1. I could understand the reason using antibody-based proximity labeling approach for mapping the biomolecules that interact with post-translationally modified histone becuase it should be complicated to map it with exogenous protein expression approach. However, the introduction of antibody-conjugated APEX2 should require fixation and permeabilization steps that can usually compromise ultrastructure. The authors should comment whether these procedures can affect protein composition and structures of nucleosome in the Discussion part of this manuscript.

2. For generation of biotin-phenoxyl radical, HRP-conjugated antibody can be utilized as shown in BAR method (Daniel Z Bar et al. Nat Methods, 2018, 15, 127-133). Since secondary-HRP antibody is commercially available, one cannot make an effort to express and purify pA-APEX2 for this approach. The autrhos should clearly explain why they selected APEX2 and what is an expected advance(s) using APEX2 in their approach.

3. For the detection of the APEX-mediated biotinylated proteins, direct mass identification of tyrosine-modified peptides with chemical probes can tell the most correct information of the proximal proteins (see Lee SY et al. J. Am. Chem. Soc. 2017, 139, 3651-3662; Namrata D Udeshi et al. Nature Methods 2017, 14, 1167-1170). Thus, if the authors can obtain the biotin-modified peptide information from each antibody-conjugated APEX2, the quality of their interactome results should be much improved. If authors may be under the situation that cannot conduct further mass experiments, it might be required to check whether their important finding molecules (e.g. Arid2, Brd7, Nsd2) are really "biotinylated" by conducting Streptavidin-HRP western blot experiment after enrichment of those proteins by using primary antibody. If biotinylation is specifically conducted by pA-APEX2 with H3K27me3 antibody, the authors can observe SA-HRP blot signal on the enriched protein band on the membrane. Negative controls should be the samples omit pA-APEX2, H3K27me3 antibody, biotin-phenol, H2O2, respectively or using different PTM targeted primary antibody. This result can confirm that their findings are enriched from proximity-dependent biotinylation of APEX2, not from spurious binding events to the other biotinylated proteins or self-labeled bait proteins.

Significance

For antibody-binding APEX2 strategy, this work is not the first one and the authors should mention the precedent work in the manuscript: Jisu Lee et al. Chem. Commun., 2015, 51, 10945-10948. And the author also commented the antibody-based proximity labeling mapping works including Daniel Z Bar et al. Nat Methods, 2018, 15, 127-133 in the manuscript.

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

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary:

In this article, Li and colleagues demonstrate the utility of a novel proximity labeling-based strategy, which they term AMAPEX (antibody-mediated protein A APEX) for the proteomic characterization of the protein environment of specific histone modifications. They apply this new methodology in mouse embryonic fibroblasts for subsequent purification of biotinylated proteins and mass-spectrometric identification.

Major comments:

• The authors present a high quality, descriptive manuscript that introduces a novel proximity labeling approach from a mostly technical point of view. The presentation of the data and methods are mostly clear, such that they should be easy to reproduce.

• The biggest shortcoming of the study in its current form seems to be the lack of a proper assessment of the method's sensitivity AND -most importantly also- specificity. The authors do not validate potentially new interactors of the modified histones experimentally, which would highlight their technology as a discovery tool. Without the assessment of newly identified proteins, these could simply represent false positives, which would point towards an additional requirement for optimization of the experimental setups. In that light the newly identified proteins are merely potential histone interactors. Validation would require establishment or purchase of additional antibodies, or alternatively cloning and transfection of respective candidates, which will probably take an extra 2-3 months of work. While the study may be publishable in its current form, this validation seems a valuable investment to strengthen the preliminary conclusions.

• With two biological replicates the study fulfills the minimum requirements for reproducibility, however, it would benefit from an additional replicate.

Minor point:

• The sentences in lines 22f (and 44f) could be misunderstood. Please rephrase statements to be unambiguous that it specifies the proteomic surrounding of post-translationally modified proteins. The proximity labeling technologies may themselves be limited in identifying post-translationally modified proteins, as they label reactive side-chains that are often targeted by PTMs. Post-translationally modified lysine residues cannot be targeted by biotin ligase-based methodologies, as tyrosine phosphorylations cannot be assessed by peroxidase-based labeling.

Significance

• From a technical point of view, most of the key conclusions of this paper are convincing. Assessing the proteomic environment of post-translationally modified proteins is of extremely high interest and the methodology seems broadly applicable to address such questions. It should be noted that proximity labeling has not originally been utilized to map protein-protein interactions, as the enzymatic activities available probe their surroundings, this could be an over-interpretation. Nonetheless, the "proteomic surrounding" of target proteins, which would also include hard to identify transient interactions is of high general interest for molecular biologists.

• Although it can be generally agreed that having to express an exogenous fusion protein is a limitation of current proximity labeling setups, the methodology presented here in turn has the limitation that it cannot be performed in living cells, which is a significant disadvantage.

• My lab is establishing and constantly improving various proximity labeling methodologies in combination with mass spectrometry for sub-organellar proteomics. While the technology is sound and well executed, I am not an expert in the biology of histone modifications and the proteins involved and cannot assess the novelty nor the actual value of the generated datasets. It appears to me though that additional validation by independent experiments would strengthen the manuscript. The authors describe the usefulness of the technology mainly in confirming known interactors of modified histones, however, it would be nice (for non-specialists) to explicitly state how high the coverage of the known interactors is and discuss why some of them might have been missed.

• The manuscript in its current form completely lacks a discussion of the presented data. Even if the focus will remain on the technical aspects, the findings should be properly discussed by comparison to other proteomics approaches studying histones. Ideally the data should also be discussed in the light of current proximity labeling technologies and potential future directions.

• While I cannot assess the value for histone research, the manuscript will be very interesting for experts focusing on proximity labeling technologies and subcellular proteomics.

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Reply to the reviewers

Dear reviewers,

We thank all reviewers for and their appreciation of our work and even more so for their constructive comments and suggestions, which will significantly improve the quality of the manuscript. We were able to complete the revision and address all reviewer comments. Aside a more stringent discussion of the literature, and rewording of certain paragraphs for clarity, we also generated additional experimental data.

More importantly, to address the concern that we did not provide a positive marker for the intranuclear compartment, we present new images. We attempted to label gamma-Tubulin by generating new antibodies, GFP-tagged strains, and trying multiple commercial antibodies since the beginning of the project. Only recently we found an antibody providing a more specific signal at the expected location, although with some likely cross-reactivity with alpha- and beta-tubulin, and now show these data in the supplements. Additionally, we generated expansion microscopy samples stained with a fluorophore-coupled NHS-Ester, a bulk protein label. These data show that the centrosome contains an exceptionally protein dense hourglass-shaped region, which spans from the extranuclear to the intranuclear compartment, as revealed by centrin and tubulin co-staining. This fortifies our claims about the distinct nature of the intranuclear centrosome compartment containing the microtubule nucleation sites.

Further, we add images of 5-SiR-Hoechst, SPY555-Tubulin, Centrin1-GFP triple labelling live cells to demonstrate the specificity of the microtubule dye and to underline that we are indeed acquiring the dynamics from the first nuclear division on.

In terms of formatting we added line numbers and uploaded high quality figures separately. Due to the added data and panels we needed to split Fig. 1 into two separate figures, rewrote the figure legends and moved them to the end of the document.

Please find below a point-by-point response to the comments.

Best regards,

Julien Guizetti

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

The manuscript by Simon and collaborators addresses the dynamic changes of spindle and hemi-spindle microtubules occurring along schizogony in Plasmodium falciparum. The work explores the temporal correlation of the changes observed in intranuclear spindles with changes at the level of the centriolar plaque; the nuclear microtubule organizing center of these parasites, using centrin as a bona fide marker of the structure. The study shows that spindle microtubules organize from an intranuclear region, devoid of chromatin, distinct from the centrin region which had not been observed or described before. It further shows that centrin does not localize at the nuclear envelope, but it is actually extranuclear.

This work significantly expands on previous knowledge regarding the functional and spatial organization of the nucleus in P. falciparum, and the structure once defined as "an electron dense mass on the nuclear envelope." It uses state of the art microscopy approaches such as STED, UExM and CLEM, in combination with immunolabeling, dyes and parasites over expressing fluorescent protein fusions, to address these questions.

**Major comments:**

• Are the key conclusions convincing?

I find the manuscript successfully addresses the posed questions. The data presented supports the conclusions.

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

• 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.

No

• 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.

N/A

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

Yes

• Are the experiments adequately replicated and statistical analysis adequate?

Yes

**Minor comments:**

• Specific experimental issues that are easily addressable.

On the data shown in Figure 1, it is unclear to me what elements are taken into account to define "anaphase." Anaphase could be defined by using chromatin markers - such as CenH3- which have been identified in Plasmodium and the authors make use of in Figure 1F.

We acknowledge that the term anaphase is ill-defined here. Further it suggests a mitotic morphology analogous to the one observed in “classical” models (prophase, metaphase, anaphase,…), which is not fully appropriate. In line with the comments by Reviewer 3 we, therefore, decided to use the term “extended spindle” instead (Fig. 1 & 2). This better reflects the morphological criterion on which we based the stage definition.

• Are prior studies referenced appropriately?

The authors state that "with the exception of centrins and gamma tubulins" few canonical centrosome components are conserved in Plasmodium. These parasites are in fact able to assemble a more or less canonical centriole for microgamete basal body formation. Widely conserved centriolar components such as Sas6 are coded by the malaria genome, and have been characterized previously. This work is neither referenced nor discussed in the manuscript.

The reviewer is right to point out this omission. We were too much focussed on the blood stage centriolar plaques while writing this section, where centrioles are not observed. Of course centriole-like structures are relevant in other life cycle stages, such as microgametes, and should be discussed (line 104). Some previous attempts to endogenously tag Sas6 to verify its localization in blood stages were unfortunately not successful.

• Are the text and figures clear and accurate?

I find the timings shown in Figure 1A, with respect to the schematic quantification shown in Figure 1B, confusing. Shown as it is, one naturally correlates the images on Fig1A above with the cell cycle progression timing shown on Fig1B, below. However, by time 260min, for example, two somewhat adjacent centrin signals can be observed. Though this is defined as anaphase- by an unspecified criterium- this could very well be representative of metaphase. Nonetheless, the timing shown on Figure 1B for "anaphase" onset is 170min, which is inconsistent with the images above. I suggest that either, the quantification is shown in a different format (ex. bar plots) which could then better reflect the cell to cell variations observed (by use of error bars, for example) or that the figure explanation in the results section clarifies this issue.

We understand how this representation is misleading and have adjusted the figure and text accordingly. We modified the time stamps in Fig. 1A (now Fig. 1C) to the scale used in Fig. 1B (now Fig. 1D) i.e. collapse of the hemispindle is t=0 and explain this in the text (line 158). Since we feel that Fig. 1B (now Fig. 1D) is a good and compact visual representation of progression through the first division we kept the bar plots in the supplements (Fig. S1), but added a title clarifying that average duration between multiple movies are shown.

As presented, the data in Figure 1C is rather uninformative. A pattern could be more immediately extracted if dots corresponding to subsequent appearance of centrin dots in the same nucleus were connected to each other.

Concerning the appearance of the centrin signals we adopted the good suggestion by the reviewer and connected “paired” centrin signals by lines (Fig. 1E).

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

There are a number of edits required on the text. Row numbers would have been helpful in pointing these out. I point some edits below, but thorough revision of the manuscript for grammatical and synthetic errors would be beneficial.

• Cytokinetic segmeter - please replace with "segmented"

• Please refer to Figure 1D when appropriate - there is quite an extensive paragraph describing the results shown on this figure, but it is only referenced at the start.

• "..., as did the and the number of branches per nucleus,..." please rewrite as appropriate.

We apologize for not providing line numbers, but have corrected the addressed points and applied a grammatical check throughout the manuscript. We have added additional references to Figure 1D (now Fig. 2A) in the text.

Reviewer #1 (Significance (Required)):

This manuscript could be interested to a wide audience interested in cell cycle, cell division, cell organization and organelle positioning, infectious diseases and microscopy. However, the introduction assumes that readers are somewhat experts in the malaria field. I suggest the authors include a brief introduction of the malaria life cycle, and a schematic representation of the division mode. This will help non-experts follow the narrative more easily.

We are happy to read that the reviewer sees value of this study for a broader audience. Following the suggestion, we added a small schematic (Fig. 1A, lines 54, 62) highlighting the relevant steps of schizogony and expanded the introduction of the life cycle (line 46).

This work rectifies long-standing inconsistencies observed by different experimental approaches in the nuclear organization of malaria parasites during schizogony. However, what the functional consequences of the alternative modes of spindle organization in malaria could be, are not clearly stated or discussed. In this respect, as it stands, the manuscript is rather descriptive and lacks mechanistic insight. Nonetheless, the data presented are of superb quality, and the manuscript represents a tremendous leap in structural insight and imaging resolution for the field of malaria. I find the data is suitable for publication albeit minor adjustments are made (specially to Figure 1 and/or the description of the results shown in Figure 1, for consistency).

We agree that the value of this manuscript lies in the clarification of conflicting data, unprecedented structural insight, and providing a useful working model for the malaria parasite centrosome. Although this study is ultimately descriptive it forms the indispensable basis to generate more meaningful functional insight about centrosome biology and nuclear division. Some of the functional consequences worth considering are: i) The (at least) bipartite composition indicating that centrosome functionality is spatially spread throughout the nucleoplasm/cytoplasm boundary. ii) The delayed appearance of the centrin signal after tubulin signal allows the prediction that centrosome assembly is a staged process occurring over an elongated period of time. iii) The generally amorphous structure of the compartment predicts the involvement of yet to be uncovered matrix-like proteins harbouring microtubule nucleation sites. iv) Lastly, our model has important implications for the mechanism of centrosome duplication. In a centrosome containing centrioles (like in vertebrates), the duplication event can easily be explained by physical separation of the daughter and mother centrioles. Spindle pole body duplication in yeasts is achieved by de novo formation of a new one, which remains connected by a half bridge until it is split. The centriolar plaque organization revealed here suggests that we need an entirely new model of centrosome duplication (or splitting) to describe and understand this process in malaria parasites. We now address those points more explicitly in the discussion section (e.g. lines 375, 443, 467).

**Referee Cross-commenting**

I agree with all the other reviewer's comments. I'm glad the reviewers seem to be experts in the field of malaria cell division and have pointed out previous studies which were not appropriately referenced. I second those comments.

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

**Summary:**

The manuscript by Simon et al have used advance cell biology technology like STED, expansion and live cell imaging to decipher the configuration of microtubules, centrin and nuclear pore during unconventional cell division process in malaria parasite. They have shown the dynamics of centrin and its localisation with respect to centriolar plaque that is characteristic of these parasite cell during schizogony> They also implicate from their studies that there is extended intranuclear compartment which is devoid of chromatin

**Major Comments**

• Are the key conclusions convincing? Yes to some extent

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

*Some part are preliminary and speculative as there is no solid data supporting it. Please see below

• 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.

*Yes to substantiate their claim

• 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.

*They can do these quite quickly less than a month

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

*Yes

• Are the experiments adequately replicated and statistical analysis adequate?

*Yes

The authors present beautiful imaging and some in depth structure using tomography and CLEM to show the location of centrin which is generally considered the marker for centrosome or Microtubule organising centre in malaria parasite. These approaches are still not been applied in Plasmodium and hence very informative. Though they present some advance microscopy but a lot of these concept for hemispindle were shown earlier in many light and super resolution microscopy studies. Authors claim that they are first to show that there is space between centrin and nucleus but it has been show previously in centrin studies in Plasmodium berghei using super resolution microscopy (Roques et al 2019 Fig1 and supplementary videos1&2) as well as expansion microscopy recently by group of Brochet etal 2021.

We thank the reviewer for the appreciation of our work. We are, indeed, not the first to describe the gap between centrin and tubulin or the nucleus. We just aimed to reiterate this finding, also visible in our data, in order to transition to the analysis of nuclear pore positioning to clarify whether centrin is actually extranuclear. Nevertheless, we should have cited the Roques and Bertiaux et al. studies again in this context, which we have now rectified (line 252).

In addition the microtubule dynamics was also recently shown with Kinesin5 live cell imaging for schizogony in Plasmodium berghei (PMID: 33154955) which author have omitted in their manuscript.

We thank the reviewer for pointing out the Kinesin-5 study by Zeeshan et al., which we failed to cite and discuss. We now state the findings of this publication and put it into the context of our work (see also answer to next point). Microtubule associated proteins, such as the microtubule plus end tracking EB1 and the aforementioned Kinesin-5, are indeed useful markers to investigate microtubule dynamics leading to the interesting results shown by Zeeshan et al. Nevertheless, we want to point out that labelling microtubule associated proteins (MAPs) remains an approximation of the underlying microtubule organization. As the authors in Zeeshan et al. indicate by themselves, Kinesin-5 does not decorate axonemal microtubules or the membrane-associated microtubule structure formed during cytokinesis in very late schizont stages. Further, colocalization between alpha-tubulin and kinesin-5 in schizont-stage parasites is not complete indicating a preferential decoration of certain sections of the microtubule structures (possibly the microtubule ends), which could only be resolved by super-resolution microscopy. Using a live cell dye, such as SPY555-tubulin, which directly binds to microtubules will provide a uniform labelling of any microtubule species and hopefully prove useful to the field in the future. Lastly, we present time-lapse microscopy analysis of blood stage cells, contrary to single time point images of live cells, providing a quantified chronology of microtubule reorganization at single cell level (with time stamps). Therefore, we feel that our claim, although it should be relativized, is formally speaking accurate.

It is also important that authors give valid discussion about previous studies on hemispindle, microtubule dynamics with respect to schizogony (PMID: 18693242; PMID: 11606229; PMID: 33154955) rather than giving the impression that they have given this concept first time on hemispindle dynamics and centrin location during schizogony.

We agree that those studies should be discussed in more detail. We are grateful to the reviewer for pointing out the Fowler et al. 2001 (PMID: 11606229) study. They use an antibody against gamma-tubulin to demonstrate its presence at the apical pole of subpellicular microtubules (f-MAST) in the merozoite and cytokinetic stages (line 102). However, we were unable to reveal a specific gamma-tubulin staining using the antibody used by them in the preceding schizont stage. After trying many different commercial gamma-tubulin antibodies and attempting to generate our own we now finally observe a gamma tubulin localization at the poles of intranuclear spindles in schizont stage, although the only successful antibody still displays some background staining, possibly including cross-reactivity with alpha or beta-tubulin (Fig. S4, line 237).

The highly insightful study by Mahajan et al. 2008 (PMID: 18693242) indeed suggests that centrin localizes away from the DNA and demonstrate the distinct localization from tubulin. They, however, likely due to the resolution limit of their microscopy techniques, speculate that the centrin signal is embedded in the membrane, while we could show by super-resolution and nuclear pore staining that centrin is distinct from the membrane (now Fig. 2A; line 257). The work done by Zeeshan et al. 2020 (PMID: 33154955) nicely shows dynamics of kinesin-5 in nuclear division. In schizont stages Kinesin-5 signal elongates and splits alongside the mitotic spindle with which it overlaps for the most part. Colocalization with centrin is less strong although the authors note some overlap. Our data suggest that centrin and tubulin are clearly distinct. In male gametes the authors show nicely time-resolved data of kinesin spreading along the elongating spindle, although hemispindles are not observed at this stage. We introduce and discuss these findings (lines 123, 432).

The concept of bipartite centrosome is already been discussed in Toxoplasma and the claim by authors in Plasmodium presented here is not substantiated experimentally. They showed that centrin is part of outer region while they do not show with any marker for the inner region. It will be very helpful if the authors use gamma tubulin or MORN1 to show the location with respect to centrin and microtubule. In the absence of this localisation the claims are preliminary and speculative. If the centrosomal protein complex is not involved in microtubule nucleation, then how the nucleation is happening. What are the molecules present in this amorphous matrix? It will be great to check the location of gamma-tubulin or some inner centrosome molecules described in Toxoplasma that is deemed to be MTOC.

We share the opinion that our Plasmodium data should be compared to Toxoplasma, while still being assessed independently. Despite Toxoplasma belonging to the apicomplexan the conclusion that their centrosomes should be organized in a similar fashion is by no means self-evident considering for example their significant evolutionary distance. Actually, several noteworthy morphological differences have already been well documented. i) Toxoplasma MTOC does contain centrioles in the outer core which is coherent with the centrin and gamma-tubulin localization in this region. ii) Toxoplasma MTOC contains an additional nuclear membrane protrusion enclosing the inner core. iii) mitotic microtubules in Toxoplasma are thought to penetrate the nuclear membrane to connect to centromeres. iv) the inner and the outer core are both extranuclear and therefore not to be equated with the intranuclear compartments. We now expand a bit on the discussion of the aforementioned differences (line 382). Nevertheless, we thank the reviewer for making us realize that the term “bipartite” is a poor choice to describe the centriolar plaque organization in this context. Therefore, we replaced it in the abstract (line 29) and the main text (line 375).

We acknowledge the fact that it would be desirable to show a marker localizing to the intranuclear compartment, and not only through visualizing the microtubule nucleation complex (Fig. 4A-B) and the positioning of the microtubule ends in this region (Fig. 3A). Concerning MORN1 we found no indication in the published localization data that it is, like in Toxoplasma, associated with the nucleus in Plasmodium species, where it is only found associated with the budding complex (and we are currently unable to procure an antibody) (line 422). We have attempted gamma-tubulin visualization on many occasions throughout the project (transgenic parasite lines, commercial antibodies, self-made antibodies) and only recently found an antibody revealing some specific signal. Indeed, we found localization at the poles of the spindles i.e. the intranuclear compartment (line 237). Unfortunately, this “best-possible staining” still showed some unspecific spindle staining likely resulting from cross-reactivity with alpha- or beta-tubulin causing us to put these data into the supplements (Fig. S4).

We had more luck with attempting a “new” type of staining, recently used in Plasmodium (Bertiaux & Balestra et al. 2021) using a fluorophore-coupled NHS-Ester in expanded samples. This chemical unspecifically stains proteins and revealed that the centrosomal region contains an exceptionally protein dense “hourglass-shaped” structure (Fig. 3F-H). Since the outer part of this structure colocalizes with centrin and the inner part overlaps with microtubules we assume that the centrosomal complex stretches throughout the nucleo-cytoplasmic boundary and fills part of the intranuclear compartment (line 320). Especially the highly protein dense region at the neck of the “hourglass” seems very coherent with the nuclear membrane embedded electron dense region which can be seen in electron microscopy (e.g. Fig. 3E & 4B). We feel that this staining strongly supports the presence of this novel intranuclear compartment.

The expansion microscopy is very nice and some of it presented in supplementary can be moved to main section.

Thanks for sharing our enthusiasm about this imaging technique. We have now selected a representative image of a hemispindle and mitotic spindle stage nucleus imaged by U-ExM and added it to the main section (Fig. 2B, line 231).

The localisation CenH3 is bit puzzling as it has been shown that centromere/ kinetochore cluster and are present during early and mid schizogony. The various foci with respect to nuclei are not what has been seen previously. Please discuss the difference in these two findings.

The localization pattern can easily be explained by the increased resolution of STED nanoscopy used in this study. Previous studies (e.g. Hoeijmakers et al. 2012 and Zeeshan et al. 2020) used classical confocal microscopy. Under those imaging conditions the individual foci seen here can´t be resolved and would, in accordance with the other studies, appear as one cluster. We slightly modified the text for more clarity (line 247).

Reviewer #2 (Significance (Required)):

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

* This is more technical advancement on the subject of centrin by using STED, tomography and CLEM.

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

* This work has relevance relation to cell division during schizogony in asexual stages in par with Toxoplasma or in Apicomplexa in general

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

Working with Apicomplexa, Protist, cell division and mitosis.

• 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.

Working on Cell division in Plasmodium.

**Referee Cross-commenting**

I agree with the reviewers and some of the experiment suggested and the minor details have to be addressed. There are some loose ends and these suggestions will enhance clarity of the data. It is a very nice study and some of the comments suggested by reviewers will improve the manuscript. __

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

**Summary:**

The centrosome is the primary microtubule-organizing center (MTOC) in eukaryotic cells that nucleate spindle microtubules necessary for chromosome segregation. In most eukaryotic cells, the canonical centrosome is composed of centrioles surrounded by an electron-dense proteinaceous matrix named the pericentriolar matrix (PCM) competent for microtubule nucleation mitotic spindle assembly. Following the breakdown of the nuclear envelope breakdowns, the mitotic spindle microtubules gain access to the kinetochores of the condensed mitotic chromosomes. Once the mitotic spindle is fully developed, centrosomes are at opposite poles of the cells, and chromosomes are pulled toward opposite poles. Cell division completes with cytokinesis resulting in the active formation of two nuclei within two daughter cells. Interestingly, during its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division within an intact nuclear envelope. The multi-nucleated cell is then subjected to a single round of cytokinesis that produces dozen of daughter cells. We know about the Plasmodium centrosome is that it is made of an acentriolar structure embedded in the nuclear envelope and serves as MTOC during cell division. However, the biogenesis and regulation of the Plasmodium centrosome are poorly understood. Given the peculiarity of the cell division in Plasmodium parasites, understanding the molecular mechanisms that drive and regulate MTOC duplication and maturation could unveil novel targets for the treatment of malaria. In this study, Simon et al. successfully applied challenging and cutting-edge microscopy techniques to monitor the dynamic formation of the spindle microtubules and MTOC during Plasmodium intraerythrocytic mitosis. In addition, they remarkably combined stimulated emission depletion (STED) with ultrastructure expansion microscopy to define an uncharacterized intranuclear compartment devoid of chromatin as the nucleation site of nuclear microtubules. And lastly, the authors adapted an in-resin correlative light and electron microscopy (CLEM) approach to define the centriolar plaque position in a novel intranuclear compartment with centrosomal function.

**Major comments:**

1. In the methods section, it is stated that across this study, three different anti-tubulin antibodies (alpha-tubulin B-5-1-2, alpha-tubulin TAT1, beta-tubulin KMX1) were used, and two anti-centrin antibodies (TgCentrin1 and PfCentrin3) were used, one of which seems to have been generated in this study (anti-PfCentrin3). It is unclear in the figures or results section when each of these antibodies was used, and the authors should give a rationale for using multiple antibodies in combination.

To label microtubules we used the mouse anti-alpha-tubulin B-5-1-2 (Sigma, T5168) antibody throughout the study. Except for U-ExM were we added two additional primary antibodies against tubulin. Due to the expansion of the samples the antibody binding epitopes are stretched out in space. This causes a significant reduction of local epitope concentration (expansion factor 4.5 in all directions results in ~ 80-fold increase in the volume), which can reduce the signal intensity. Adding multiple antibodies binding different epitopes of tubulin can compensate for this dilution effect to some degree, as has been shown before by Gao et al. 2018. At the same time the expansion contributes to the accessibility of the usually densely packed tubulin epitopes within the microtubule polymer, which certainly adds to the success of U-ExM. What the respective contributions of those effects are is not clear, but we found superior signal-to-noise ratios when combining three tubulin antibodies instead of using one. The TgCentrin1 antibody was only used in Fig. 2C (now Fig. 3B) and validated the localization pattern of our new PfCentrin3 antibody we used in the other pictures. We now provide clearer description of antibody usage in the methods section and a new supplemental table.

The anti-PfCentrin3 antibody seems to have been generated for this study. If this is the case, the authors should provide evidence that this antibody binds to the recombinant PfCentrin3 it was raised against and binds PfCentrin3 in parasite lysates.

The anti-PfCentrin3 antibody was, indeed, produced for this study and we should have provided our western blot data right away. We now show the requested blot, which shows bands at the appropriate size in parasite lysate as well as for the recombinant protein, in the supplements (Fig. S2, line 178).

In the first paragraph of the Results section, the authors' remark of centrin foci that they are "...only detectable later (Mov. S2) or sometimes not at all." In Figure 1 A-C, it is implied that the first observed division is the first nuclear division of that parasite. Given that some nuclei do not have a visible centrin focus, it cannot be concluded with certainty that these parasites only contain a single nucleus and that this is their first division. The authors would need to include a quantifiable DNA stain to show this unequivocally to show a single nucleus. It has undergone DNA replication, similar to Klaus et al., 2021 BioRxiv paper. In the absence of a DNA stain, the authors should reword to clarify that this is the first observed division and speculate that it is the first division of that nucleus, but the authors should draw no firm conclusions about the first division.

Indeed the variability in protein levels that can result from exogenous expression can lead to some cells not showing clear Centrin1-GFP foci. Although this is a rare event we wanted to acknowledge this observation. The live cell microtubule staining using Spy555-Tubulin we use is, however, highly specific and sensitive and would stain any nucleus undergoing division including the first one. If there would be more than one nucleus in the observed cell it would unequivocally show two clearly separated tubulin signals (hemispindle or mitotic spindle). To illustrate this we added Fig. 1B (line 148) showing two live parasites stained with SPY555-Tubulin plus a Hoechst-based dye showing one or two nuclei alongside the corresponding tubulin signal. We modified the text to clarify how we stage the parasite for time-lapse acquisition (line 154). We already extensively experimented with state of the art fluorogenic live cell DNA dyes (e.g. from Spirochrome and the Johnsson group) to visualize the nuclei directly in time lapse microscopy, but even at minimal concentrations they all significantly inhibit mitotic progression. We also add this information in the main text (line 150).

In the first paragraph of the Results section, the authors write: " We quantified the duration of hemispindle, accumulation and anaphase stages ...." Anaphase spindle fibers means that the sister chromatids are separated. In the absence of a centromeric marker like NDC80, it doesn't seem easy to claim the anaphase stage. The authors should write " extended spindle." The authors might also consider using the term collapsed spindle instead of accumulation to reflect the dynamic of the intranuclear microtubules during the blood-stage replication. The same modification should be made for Figure 1B, so we read " hemispindle, collapsed spindle and extended spindle."

We thank the reviewer for this suggestion, which is very much in line with a comment by Reviewer 1 on the definition of anaphase. We acknowledge that the term is ill-defined here. Further, it suggest a mitotic morphology analogous to the one observed in “classical” models (prophase, metaphase, anaphase,…), which is not fully appropriate. Consequently, we decided to adapt the suggested terminology in Fig. 1 (and also new Fig. 2) and in the text (line 160).

Based on the evidence in this study, it cannot be stated unequivocally that the centrosome is entirely extranuclear, at least not as it is implied in Figure 3C. In Supplementary Figure 4, the microtubules appear to be extruding from a circular structure that may either be intranuclear or span the nuclear envelope. In Supplementary Figure 6, the structure pointed to as the centrosome appears to be embedded within the nuclear membrane with a top structure on the cytosolic side of the nuclear envelope. Thus, the best support for an extranuclear centrosome comes from the CLEM images. Still, it is noteworthy that the double membrane of the nuclear envelope is not visible on this slice in the region where the centrin fluorescence is found. Considering some of the fluorescence pixels for centrin are outside the parasite plasma membrane, and some of the Hoechst pixels are outside the nuclear envelope, this data does not show unequivocally that centrosomes are entirely extranuclear. However, this argument would be strengthened if the authors performed a proteinase K protection assay (or something similar) to determine if Centrin1 and Centrin3 are exposed to the cytosol. However, in the absence of that or further evidence, the authors should dampen their claims about the centrosome being exclusively extranuclear, as represented in the schematic in figure 3C.

We thank the reviewer for this comment, which highlights an issue in our communication of our working model of the centriolar plaque. At no point we intended to claim that the centrosome is exclusively extranuclear. Rather, centrins, which are currently the only reliable marker proteins, localize to a subcompartment of the extranuclear region of the centriolar plaque. Additionally, the centrosome clearly contains an intranuclear region. The composition of this intranuclear compartment is elusive, except that it harbors microtubule nucleation sites. Indeed, our model in Fig. 3C (now Fig. 4C) is misleading and not well annotated. The newly added NHS-Ester staining fortifies this claim (Fig. 3F-H. Consequently, we corrected our working model by adding an explicit figure labelling (now Fig. 4C).

We apologize for the misleading labelling in Fig. S6 (now Fig. S7). The green arrow was intended to point out the electron dense region associated with the nuclear membrane, which has been seen in previous studies, and was not intended to represent the entire extended centriolar plaque. If anything, this smaller region might provide the link between the intra and extranuclear compartments that the reviewer also identified in Suppl. Fig. 4 (now Fig. 2D). We modified the annotation of the Fig. S7 and Fig. 4A-B accordingly, labelling it the “electron dense region”. More importantly, we hope that our newly added data using NHS-Ester staining of protein dense regions (Fig. 3F-H) highlights the spread of the centrosome across the nucleo-/cytoplasmic boundary more clearly.

Considering whether centrin is actually extranuclear, we feel that the data shown in Fig. 2A (now 3A) is convincing. We have, however, added two panels of the relevant regions showing centrin localization respective to the nuclear pore and adjusted the contrast as we acknowledge the limited “visibility” within the unadjusted panels. The fact, that the centrin signal slightly overlaps with the nuclear envelope in CLEM images can be explained by the relatively poor resolution of the widefield microscope we had to use to image the sections. From the other super-resolution images in the manuscript, we know that the perimeter of the better resolved centrin signal is significantly smaller. Otherwise one had to assume from the CLEM data that centrin is also in the cytosol of the red blood cell and that DNA is localized outside the nucleus. On a similar note the fluorescence image is, contrary to the tomography image, a single slice since the thickness of the sample section (about 200nm) is significantly below the z-resolution (about 500nm) of a fluorescence microscope.

Throughout the study, the level of biological replication is unclear. The authors rigorously include all the data points for each of their graphs and the total number of images/videos quantified. And what needs to be added, in either the figure legends or a methods section, is the number of biological replicates for each of these measures came from.

We have added the number of replicas in the figure legends.

**Minor comments:**

STED is present as an acronym in the abstract and should be spelled out in full and clarified that it is a super-resolution microscopy technique.

We opted to remove STED from the abstract (leaving it at super-resolution, which includes expansion microscopy) to avoid disrupting the “flow” of the abstract and now spell out the acronym at the first mention in the introduction (line 127).

The second paragraph of the Results section states that ring and early trophozoite stage parasites do not express tubulin or centrin. Still, only an early trophozoite is shown in Supplementary Figure 2. Therefore, the authors should either include a similar image of a ring-stage parasite or remove ring-stage parasites from that statement.

We have removed the ring stages from the statement.

The second paragraph of the Results section contains the sentence, "At which point tubulin is reorganized into the bipolar microtubule array, which then forms the mitotic spindle cannot be resolved here." The authors are implying that the point at which tubulin is reorganized into the microtubule array, which goes on to form the mitotic spindle, cannot be resolved here. This is not particularly clear, though, and this sentence could be reworded for clarity.

We reformulated the sentence to clarify the point we failed to make with the previous wording (line 188).

The second paragraph of the Results section contains some statements about the results without referencing the figures that these statements come from. The authors should clarify this to make clear which figures each statement refers to.

We added more references to the appropriate figure throughout the paragraph (lines 188, 219, 223).

In the third paragraph of the introduction section, the authors write, " Centriolar plaques seem partially embedded in the nuclear membrane, but their positioning relative to the nuclear pore-like "fenestra" remains unclear." Unfortunately, the lack of reference did not allow me to understand if the authors state literature or comment on past published results.

We added the reference which was incorrectly positioned before the sentence instead of at the end (line 82).

the authors could add some references:

• Second section of the introduction: " the 8-28 nuclei are packaged into individual daughter cells, called merozoites ( Rudlaff et al. 2019 PMID: 31097714)

• Third section of the introduction: " The centrosome of P.falciparum is called centriolar plaque" ( Arnot et al. 2011, Sinden 1991a); " the nuclear pore-like "fenestra" remains unclear (Wall et al. 2018; Zeeshan et al. 2020).

• Fourth section of the introduction: " tubulin antibody staining are extensive structures measuring around 2-4um ( Ref?)

• When the authors introduce subpellicular microtubules of segmented schizonts, a reference to a study that shows these structures should be included.

• A previous study that shows the distinct structure of microtubule minus ends should be cited when this structure is described.

• Third section of the results, the authors should cite Bertiaux et al. 2021 with the Gambarotto et al. 2019 paper regarding U-ExM.

We apologize for missing some important references or putting them in the wrong position. We now added all the references or cite them again at the appropriate locations throughout the text.

Figure 1E shows hemispindle and mitotic spindle lengths of U-ExM expanded parasites, but the position within the figure and figure legend implies that these lengths were determined unexpanded parasites. Therefore, it should be stated in the figure legend that these measurements come from U-ExM expanded parasites. Moreover, I encourage the authors to include U-ExM images in the main figures. The images are beautiful, represent a significant technical achievement, and directly relate to Figure 1E. To the best of my knowledge, this is only the second study to perform expansion microscopy on Plasmodium and the first to use PFA-fixed parasites and a nuclear stain. It would be valuable for the Plasmodium and ExM communities to see this technical advancement represented in the main text.

We thank the reviewer for the appreciation of our ExM data and added it to Fig. 2B before the quantification of the microtubule length and number and added the information to the legend.

In the second paragraph of the Results section, the authors write, " but clearly display the microtubule cytoskeleton associated with the inner membrane complex." It would bring clarity to define in few words what the IMC is.

We included a short definition of the IMC (line 223).

The methods section details that the length of microtubules was determined by dividing the observed values by an expansion factor of 4.5. If the authors recorded the expansion factors of their gels, this data should be included, and how it was recorded should be stated in the methods. If not, the authors should include the rationale of using an expansion factor of 4.5 as this is slightly different from the previously published expansion factor of P. falciparum of 4.3.

We recorded the expansion factor by measuring the gel size pre and post expansion with a ruler and found a factor of 4.5 on average. We added this information in the methods (line 688).

There are several parasite lines used in this study, and some figures are not clear what parasite line was used. Could the authors please include the parasite lines in the figure legends of Figure 1 D-F, Figure 3, Supplementary Figures 1-2, and Supplementary Figures 4-7?

We added the parasite line information in the legends as requested.

Nuclear pore complexes, of which Nup313 is a component, can have cytoplasmic, integral, and nuclear-facing components. If it has been shown previously that PfNup313 is the homolog of Nup214 in vertebrates present on the cytosolic side of NPC, this should be stated. If not, then it should be clarified that it is unknown whether Nup313 faces the cytoplasm, nucleus, or is embedded in the NE, as this has implications for the colocalization of Nup313 and Centrin.

Nuclear pore proteins are very poorly conserved in P. falciparum and Nup313 has only been recently identified as such (Kehrer et al. 2018) mainly by the presence of FG-repeats (as for all the other newly defined proteins). The only related ortholog that can be found through BLAST search against humans, yeasts, and Arabidopsis is Nup100 from S. cerevisiae. ScNup100 is a central pore localizing protein but the sequence similarity to Nup313 is low. We are not aware of any findings showing relatedness to vertebrate Nup214, while sequence analysis rather indicates the absence of orthology. To clearly demonstrate the individual positioning of the few known Nups within the parasite´s nuclear pore complex would require a dedicated long-term project. However, due to the presence of FG-repeats one can assume that it is part of the central FG-Nups layer rather than of the intranuclear basket or the cytoplasmic filaments (line 255). Therefore it would localize more closely to the nuclear envelope than the latter. Either way, a clear gap between centrin and Nup313 signal can be identified and colocalization has not been observed. These data indicate that the exact position of Nup313 on the cytoplasmic, integral or nuclear-facing site is not decisive for the conclusions made in this study and our observations preclude scenarios where centrin is not extranuclear.

It seems from the image in Figure 2C that DRAQ5 and Hoechst have at least visually indistinguishable localizations. Have the authors taken any STED deconvolved images of nuclei stained with both Hoechst and DRAQ5? Considering the striking increase in detail of the Hoechst signal in STED deconvolved images, it may be informative both to this study and to people who work on chromatin organization what the chromatin staining looks like in the absence of bias towards chromatin state.

It would, indeed, be interesting to analyse chromatin organization by those means, but DRAQ5 is not a STED compatible dye, highly prone to bleaching, and therefore not suitable for such analysis. Being an infrared dye DRAQ5 is compared to the UV excited Hoechst also yielding a reduced spatial resolution, which is limited by the emitted wave length.

For the tomography and TEM images, the centrosome is indicated with an arrow, but it isn't entirely clear what that arrow is pointing to for some images. It would be clearer if the centrosome were outlined in green, like the NE, rather than just an arrow. This is particularly important for Supplementary Figure 4, where to my eye, it appears that the microtubules inside the chromatin-free region are coming directly out of a circular structure, which could be interpreted as the centriolar plaque.

The reviewer is right to point out the use of arrows for centrosome annotation. It was intended for orientation of readers to indicate the “likely position of the centriolar plaque” since a clear boundary around the centrosome can´t be defined. It would have been more precise to indicate that the arrow is pointing at the electron dense region associated with the nuclear membrane, which is of course only one of the sub-regions of the centrosome. This is particularly important since we want to emphasize the extended dimensions of the centrosome. Consequently, we modified the annotation to “electron dense region” in all concerned figures and corresponding legends.

The ordering of Figure 2A-C seems to imply that the DNA-free region was measured in the STED deconvolved images, but the methods imply that it was in the confocal images. The authors should clarify this in the figure legend or by rearranging B and C's order.

Hoechst signal was indeed acquired and measured in confocal mode and to avoid confusion we have changed the order of the figures (now Fig. 3B-C) as suggested.

The authors should provide some more detail on how the DNA-free zone was measured. For example, was it measured on single slices or maximum intensity projections? Was it measured from the middle, far, or near side of the centrin focus? Etc.

The measurement was carried out in the slice where the DNA-free zone was in focus. Depth was measured from below the centrin signal until the “bottom” of the DNA-free zone. We hoped that the little schematic above the figure would clarify this question, but acknowledge the need to more clearly explain the measurement method, which we now do in the corresponding figure legend (Fig. 3C).

The methods state that the mCherry signal in figure 2C was detected using a mCherry nanobody. This should be clarified in the figure legends as it currently seems as if we see endogenous mCherry fluorescence.

The visible signal is certainly a combination of the mCherry plus the “boosting” effect from the Atto594-coupled nanobody that we added. Clearly, this should be mentioned in the figure legend, which we now do.

The data in Supplementary Figure 4 seems vital to the interpretation of the study. Therefore, for clarity, I encourage the authors to include Supplementary Figure 4 in Figure 2.

We share the reviewers view on these data and moved them to the main figures (now Fig. 3D).

In the last sentence of the discussion, it is unclear what the authors mean by how the nuclear compartment "splits," could they please clarify?

We were referring to the event of centrosome duplication, which has to occur during nuclear division. In a structure without centrioles or a spindle pole body structure forming a half bridge we therefore need a new model to explain how the two poles of the spindle are formed. Potential modes are splitting or de novo assembly. This aspect, as also pointed out by other reviewer, warrants a bit more explanation, which can now be found in the discussion (line 468).

If the pArl-PfCentrin3-GFP plasmid or pDC2-cam-coCas9-U6.2-hDHFR have been published previously, the respective studies should be cited. If not, the study where the vector backbones were first established should be cited.

We have now cited the original studies publishing the vector backbones for the first time in the methods (lines 490, 501).

From the current text, it is not clear that the Nup313 tagged parasites also had a GlmS ribozyme. It is shown in Supplementary Figure 3, but the authors should clarify either in the text of the results, or figure legends, that this parasite line was Nup313_3xHA_GlmS

The Nup313-tagged line indeed has a glms ribozyme after the HA-tag, which we now mention in the figure legends.

In the plasmid constructs section of the methods, the authors list several primers by number but not by sequence. Instead, the authors should include the sequence and orientation of each of the primers mentioned in a table as supplementary data.

This is a good suggestion. We have generated a table at the end of the supplementary data file and on this occasion we also added tables of all the antibodies and dyes used in this study.

The authors should cite the study where the TgCentrin1 antibody was generated and provide the Rat anti-HA 3F10 antibody catalog number, as catalog numbers are provided for other commercial primary antibodies.

We now provide the missing catalog numbers in the supplemental data table.

There is an issue with the formatting of the journal-title in the Kukulski et al. reference.

Thank you for noticing this error, which we now corrected.

Reviewer #3 (Significance (Required)):

The genome of P. falciparum is fully sequenced; however, over 50% of encoded proteins are of unknown function, with many of these proteins unique to Plasmodium parasites. By identifying and characterizing essential biological processes, especially those divergent from human host cell processes, we will formulate ways to interfere with them by developing novel antimalarial drugs. The process of Plasmodium cell division differs from the classical cell cycle of its human host. In the study led by Caroline Simon, authors successfully utilized recent developments of super-resolution microscopies on expanded parasites to identify novel features of cell division machinery of the malaria blood-stage parasite.

Simon et al.'s work highlight the growing interest in the diversity of cell division mode of Apicomplexan parasites, which will likely contribute to a deeper understanding of the origin and functional role of the centrosome in eukaryotic life. In 2020, the Open Biology journal published a unique article collection named Focus on Centrosome Biology showcasing research that advanced our knowledge on centrosome function, evolution and abnormalities. In addition, the reported findings will interest research groups studying cell cycle regulation and evolution beyond the field of parasitology.

Our lab studies the peculiar cell cycle of Plasmodium falciparum to gain a functional understanding of mechanistic principles of nuclear envelope assembly and integrity during the cell division of the human malaria parasite.

**Referee Cross-commenting**

It is a wonderful study, and once all reviewer's comments are addressed, the manuscript should be in excellent shape for publication.

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

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary:

The centrosome is the primary microtubule-organizing center (MTOC) in eukaryotic cells that nucleate spindle microtubules necessary for chromosome segregation. In most eukaryotic cells, the canonical centrosome is composed of centrioles surrounded by an electron-dense proteinaceous matrix named the pericentriolar matrix (PCM) competent for microtubule nucleation mitotic spindle assembly. Following the breakdown of the nuclear envelope breakdowns, the mitotic spindle microtubules gain access to the kinetochores of the condensed mitotic chromosomes. Once the mitotic spindle is fully developed, centrosomes are at opposite poles of the cells, and chromosomes are pulled toward opposite poles. Cell division completes with cytokinesis resulting in the active formation of two nuclei within two daughter cells. Interestingly, during its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division within an intact nuclear envelope. The multi-nucleated cell is then subjected to a single round of cytokinesis that produces dozen of daughter cells. We know about the Plasmodium centrosome is that it is made of an acentriolar structure embedded in the nuclear envelope and serves as MTOC during cell division. However, the biogenesis and regulation of the Plasmodium centrosome are poorly understood. Given the peculiarity of the cell division in Plasmodium parasites, understanding the molecular mechanisms that drive and regulate MTOC duplication and maturation could unveil novel targets for the treatment of malaria. In this study, Simon et al. successfully applied challenging and cutting-edge microscopy techniques to monitor the dynamic formation of the spindle microtubules and MTOC during Plasmodium intraerythrocytic mitosis. In addition, they remarkably combined stimulated emission depletion (STED) with ultrastructure expansion microscopy to define an uncharacterized intranuclear compartment devoid of chromatin as the nucleation site of nuclear microtubules. And lastly, the authors adapted an in-resin correlative light and electron microscopy (CLEM) approach to define the centriolar plaque position in a novel intranuclear compartment with centrosomal function.

Major comments:

1. In the methods section, it is stated that across this study, three different anti-tubulin antibodies (alpha-tubulin B-5-1-2, alpha-tubulin TAT1, beta-tubulin KMX1) were used, and two anti-centrin antibodies (TgCentrin1 and PfCentrin3) were used, one of which seems to have been generated in this study (anti-PfCentrin3). It is unclear in the figures or results section when each of these antibodies was used, and the authors should give a rationale for using multiple antibodies in combination.
2. The anti-PfCentrin3 antibody seems to have been generated for this study. If this is the case, the authors should provide evidence that this antibody binds to the recombinant PfCentrin3 it was raised against and binds PfCentrin3 in parasite lysates.
3. In the first paragraph of the Results section, the authors' remark of centrin foci that they are "...only detectable later (Mov. S2) or sometimes not at all." In Figure 1 A-C, it is implied that the first observed division is the first nuclear division of that parasite. Given that some nuclei do not have a visible centrin focus, it cannot be concluded with certainty that these parasites only contain a single nucleus and that this is their first division. The authors would need to include a quantifiable DNA stain to show this unequivocally to show a single nucleus. It has undergone DNA replication, similar to Klaus et al., 2021 BioRxiv paper. In the absence of a DNA stain, the authors should reword to clarify that this is the first observed division and speculate that it is the first division of that nucleus, but the authors should draw no firm conclusions about the first division.
4. In the first paragraph of the Results section, the authors write: " We quantified the duration of hemispindle, accumulation and anaphase stages ...." Anaphase spindle fibers means that the sister chromatids are separated. In the absence of a centromeric marker like NDC80, it doesn't seem easy to claim the anaphase stage. The authors should write " extended spindle." The authors might also consider using the term collapsed spindle instead of accumulation to reflect the dynamic of the intranuclear microtubules during the blood-stage replication. The same modification should be made for Figure 1B, so we read " hemispindle, collapsed spindle and extended spindle."
5. Based on the evidence in this study, it cannot be stated unequivocally that the centrosome is entirely extranuclear, at least not as it is implied in Figure 3C. In Supplementary Figure 4, the microtubules appear to be extruding from a circular structure that may either be intranuclear or span the nuclear envelope. In Supplementary Figure 6, the structure pointed to as the centrosome appears to be embedded within the nuclear membrane with a top structure on the cytosolic side of the nuclear envelope. Thus, the best support for an extranuclear centrosome comes from the CLEM images. Still, it is noteworthy that the double membrane of the nuclear envelope is not visible on this slice in the region where the centrin fluorescence is found. Considering some of the fluorescence pixels for centrin are outside the parasite plasma membrane, and some of the Hoechst pixels are outside the nuclear envelope, this data does not show unequivocally that centrosomes are entirely extranuclear. However, this argument would be strengthened if the authors performed a proteinase K protection assay (or something similar) to determine if Centrin1 and Centrin3 are exposed to the cytosol. However, in the absence of that or further evidence, the authors should dampen their claims about the centrosome being exclusively extranuclear, as represented in the schematic in figure 3C.
6. Throughout the study, the level of biological replication is unclear. The authors rigorously include all the data points for each of their graphs and the total number of images/videos quantified. And what needs to be added, in either the figure legends or a methods section, is the number of biological replicates for each of these measures came from.

Minor comments:

1. STED is present as an acronym in the abstract and should be spelled out in full and clarified that it is a super-resolution microscopy technique.
2. The second paragraph of the Results section states that ring and early trophozoite stage parasites do not express tubulin or centrin. Still, only an early trophozoite is shown in Supplementary Figure 2. Therefore, the authors should either include a similar image of a ring-stage parasite or remove ring-stage parasites from that statement.
3. The second paragraph of the Results section contains the sentence, "At which point tubulin is reorganized into the bipolar microtubule array, which then forms the mitotic spindle cannot be resolved here." The authors are implying that the point at which tubulin is reorganized into the microtubule array, which goes on to form the mitotic spindle, cannot be resolved here. This is not particularly clear, though, and this sentence could be reworded for clarity.
4. The second paragraph of the Results section contains some statements about the results without referencing the figures that these statements come from. The authors should clarify this to make clear which figures each statement refers to.
5. In the third paragraph of the introduction section, the authors write, " Centriolar plaques seem partially embedded in the nuclear membrane, but their positioning relative to the nuclear pore-like "fenestra" remains unclear." Unfortunately, the lack of reference did not allow me to understand if the authors state literature or comment on past published results.
6. the authors could add some references: • Second section of the introduction: " the 8-28 nuclei are packaged into individual daughter cells, called merozoites ( Rudlaff et al. 2019 PMID: 31097714) • Third section of the introduction: " The centrosome of P.falciparum is called centriolar plaque" ( Arnot et al. 2011, Sinden 1991a); " the nuclear pore-like "fenestra" remains unclear (Wall et al. 2018; Zeeshan et al. 2020). • Fourth section of the introduction: " tubulin antibody staining are extensive structures measuring around 2-4um ( Ref?) • When the authors introduce subpellicular microtubules of segmented schizonts, a reference to a study that shows these structures should be included. • A previous study that shows the distinct structure of microtubule minus ends should be cited when this structure is described. • Third section of the results, the authors should cite Bertiaux et al. 2021 with the Gambarotto et al. 2019 paper regarding U-ExM.
7. Figure 1E shows hemispindle and mitotic spindle lengths of U-ExM expanded parasites, but the position within the figure and figure legend implies that these lengths were determined unexpanded parasites. Therefore, it should be stated in the figure legend that these measurements come from U-ExM expanded parasites. Moreover, I encourage the authors to include U-ExM images in the main figures. The images are beautiful, represent a significant technical achievement, and directly relate to Figure 1E. To the best of my knowledge, this is only the second study to perform expansion microscopy on Plasmodium and the first to use PFA-fixed parasites and a nuclear stain. It would be valuable for the Plasmodium and ExM communities to see this technical advancement represented in the main text.
8. In the second paragraph of the Results section, the authors write, " but clearly display the microtubule cytoskeleton associated with the inner membrane complex." It would bring clarity to define in few words what the IMC is.
9. The methods section details that the length of microtubules was determined by dividing the observed values by an expansion factor of 4.5. If the authors recorded the expansion factors of their gels, this data should be included, and how it was recorded should be stated in the methods. If not, the authors should include the rationale of using an expansion factor of 4.5 as this is slightly different from the previously published expansion factor of P. falciparum of 4.3.
10. There are several parasite lines used in this study, and some figures are not clear what parasite line was used. Could the authors please include the parasite lines in the figure legends of Figure 1 D-F, Figure 3, Supplementary Figures 1-2, and Supplementary Figures 4-7?
11. Nuclear pore complexes, of which Nup313 is a component, can have cytoplasmic, integral, and nuclear-facing components. If it has been shown previously that PfNup313 is the homolog of Nup214 in vertebrates present on the cytosolic side of NPC, this should be stated. If not, then it should be clarified that it is unknown whether Nup313 faces the cytoplasm, nucleus, or is embedded in the NE, as this has implications for the colocalization of Nup313 and Centrin.
12. It seems from the image in Figure 2C that DRAQ5 and Hoechst have at least visually indistinguishable localizations. Have the authors taken any STED deconvolved images of nuclei stained with both Hoechst and DRAQ5? Considering the striking increase in detail of the Hoechst signal in STED deconvolved images, it may be informative both to this study and to people who work on chromatin organization what the chromatin staining looks like in the absence of bias towards chromatin state.
13. For the tomography and TEM images, the centrosome is indicated with an arrow, but it isn't entirely clear what that arrow is pointing to for some images. It would be clearer if the centrosome were outlined in green, like the NE, rather than just an arrow. This is particularly important for Supplementary Figure 4, where to my eye, it appears that the microtubules inside the chromatin-free region are coming directly out of a circular structure, which could be interpreted as the centriolar plaque.
14. The ordering of Figure 2A-C seems to imply that the DNA-free region was measured in the STED deconvolved images, but the methods imply that it was in the confocal images. The authors should clarify this in the figure legend or by rearranging B and C's order.
15. The authors should provide some more detail on how the DNA-free zone was measured. For example, was it measured on single slices or maximum intensity projections? Was it measured from the middle, far, or near side of the centrin focus? Etc.
16. The methods state that the mCherry signal in figure 2C was detected using a mCherry nanobody. This should be clarified in the figure legends as it currently seems as if we see endogenous mCherry fluorescence.
17. The data in Supplementary Figure 4 seems vital to the interpretation of the study. Therefore, for clarity, I encourage the authors to include Supplementary Figure 4 in Figure 2.
18. In the last sentence of the discussion, it is unclear what the authors mean by how the nuclear compartment "splits," could they please clarify?
19. If the pArl-PfCentrin3-GFP plasmid or pDC2-cam-coCas9-U6.2-hDHFR have been published previously, the respective studies should be cited. If not, the study where the vector backbones were first established should be cited.
20. From the current text, it is not clear that the Nup313 tagged parasites also had a GlmS ribozyme. It is shown in Supplementary Figure 3, but the authors should clarify either in the text of the results, or figure legends, that this parasite line was Nup313_3xHA_GlmS
21. In the plasmid constructs section of the methods, the authors list several primers by number but not by sequence. Instead, the authors should include the sequence and orientation of each of the primers mentioned in a table as supplementary data.
22. The authors should cite the study where the TgCentrin1 antibody was generated and provide the Rat anti-HA 3F10 antibody catalog number, as catalog numbers are provided for other commercial primary antibodies. There is an issue with the formatting of the journal-title in the Kukulski et al. reference.

Significance

The genome of P. falciparum is fully sequenced; however, over 50% of encoded proteins are of unknown function, with many of these proteins unique to Plasmodium parasites. By identifying and characterizing essential biological processes, especially those divergent from human host cell processes, we will formulate ways to interfere with them by developing novel antimalarial drugs. The process of Plasmodium cell division differs from the classical cell cycle of its human host. In the study led by Caroline Simon, authors successfully utilized recent developments of super-resolution microscopies on expanded parasites to identify novel features of cell division machinery of the malaria blood-stage parasite.

Simon et al.'s work highlight the growing interest in the diversity of cell division mode of Apicomplexan parasites, which will likely contribute to a deeper understanding of the origin and functional role of the centrosome in eukaryotic life. In 2020, the Open Biology journal published a unique article collection named Focus on Centrosome Biology showcasing research that advanced our knowledge on centrosome function, evolution and abnormalities. In addition, the reported findings will interest research groups studying cell cycle regulation and evolution beyond the field of parasitology.

Our lab studies the peculiar cell cycle of Plasmodium falciparum to gain a functional understanding of mechanistic principles of nuclear envelope assembly and integrity during the cell division of the human malaria parasite.

Referee Cross-commenting

It is a wonderful study, and once all reviewer's comments are addressed, the manuscript should be in excellent shape for publication.

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

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary:

The manuscript by Simon et al have used advance cell biology technology like STED, expansion and live cell imaging to decipher the configuration of microtubules, centrin and nuclear pore during unconventional cell division process in malaria parasite. They have shown the dynamics of centrin and its localisation with respect to centriolar plaque that is characteristic of these parasite cell during schizogony> They also implicate from their studies that there is extended intranuclear compartment which is devoid of chromatin

Major Comments

• Are the key conclusions convincing? Yes to some extent
• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? *Some part are preliminary and speculative as there is no solid data supporting it. Please see below
• 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. *Yes to substantiate their claim
• 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. *They can do these quite quickly less than a month.
• Are the data and the methods presented in such a way that they can be reproduced? *Yes
• Are the experiments adequately replicated and statistical analysis adequate?

*Yes

The authors present beautiful imaging and some in depth structure using tomography and CLEM to show the location of centrin which is generally considered the marker for centrosome or Microtubule organising centre in malaria parasite. These approaches are still not been applied in Plasmodium and hence very informative. Though they present some advance microscopy but a lot of these concept for hemispindle were shown earlier in many light and super resolution microscopy studies. Authors claim that they are first to show that there is space between centrin and nucleus but it has been show previously in centrin studies in Plasmodium berghei using super resolution microscopy (Roques et al 2019 Fig1 and supplementary videos1&2) as well as expansion microscopy recently by group of Brochet etal 2021. In addition the microtubule dynamics was also recently shown with Kinesin5 live cell imaging for schizogony in Plasmodium berghei (PMID: 33154955) which author have omitted in their manuscript. It is also important that authors give valid discussion about previous studies on hemispindle, microtubule dynamics with respect to schizogony (PMID: 18693242; PMID: 11606229; PMID: 33154955) rather than giving the impression that they have given this concept first time on hemispindle dynamics and centrin location during schizogony. The concept of bipartite centrosome is already been discussed in Toxoplasma and the claim by authors in Plasmodium presented here is not substantiated experimentally. They showed that centrin is part of outer region while they do not show with any marker for the inner region. It will be very helpful if the authors use gamma tubulin or MORN1 to show the location with respect to centrin and microtubule. In the absence of this localisation the claims are preliminary and speculative. If the centrosomal protein complex is not involved in microtubule nucleation, then how the nucleation is happening. What are the molecules present in this amorphous matrix? It will be great to check the location of gamma-tubulin or some inner centrosome molecules described in Toxoplasma that is deemed to be MTOC.

Minor comments:

• Specific experimental issues that are easily addressable.
• Are prior studies referenced appropriately? Partially
• Are the text and figures clear and accurate? Yes
• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? To gamma tubulin and some reference, move expansion microscopy

Minor comments:

• Specific experimental issues that are easily addressable.
• Are prior studies referenced appropriately? Partially
• Are the text and figures clear and accurate? Yes
• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? To perform experiments with gamma tubulin and add some references, move expansion microscopy.

Minor comments:

• Specific experimental issues that are easily addressable.
• Are prior studies referenced appropriately? Partially
• Are the text and figures clear and accurate? Yes
• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? To gamma tubulin and some reference, move expansion microscopy

Minor comments:

• Specific experimental issues that are easily addressable.
• Are prior studies referenced appropriately? Partially
• Are the text and figures clear and accurate? Yes
• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? To gamma tubulin and some reference, move expansion microscopy

The expansion microscopy is very nice and some of it presented in supplementary can be moved to main section. The localisation CenH3 is bit puzzling as it has been shown that centromere/ kinetochore cluster and are present during early and mid schizogony. The various foci with respect to nuclei are not what has been seen previously. Please discuss the difference in these two findings.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
  • This is more technical advancement on the subject of centrin by using STED, tomography and CLEM.
  • Place the work in the context of the existing literature (provide references, where appropriate).
  • This work has relevance relation to cell division during schizogony in asexual stages in par with Toxoplasma or in Apicomplexa in general
  • State what audience might be interested in and influenced by the reported findings. Working with Apicomplexa, Protist, cell division and mitosis.
  • 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.

Working on Cell division in Plasmodium.

Referee Cross-commenting

I agree with the reviewers and some of the experiment suggested and the minor details have to be addressed. There are some loose ends and these suggestions will enhance clarity of the data. It is a very nice study and some of the comments suggested by reviewers will improve the manuscript.

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Simon and collaborators addresses the dynamic changes of spindle and hemi-spindle microtubules occurring along schizogony in Plasmodium falciparum. The work explores the temporal correlation of the changes observed in intranuclear spindles with changes at the level of the centriolar plaque; the nuclear microtubule organizing center of these parasites, using centrin as a bona fide marker of the structure. The study shows that spindle microtubules organize from an intranuclear region, devoid of chromatin, distinct from the centrin region which had not been observed or described before. It further shows that centrin does not localize at the nuclear envelope, but it is actually extranuclear.

This work significantly expands on previous knowledge regarding the functional and spatial organization of the nucleus in P. falciparum, and the structure once defined as "an electron dense mass on the nuclear envelope." It uses state of the art microscopy approaches such as STED, UExM and CLEM, in combination with immunolabeling, dyes and parasites over expressing fluorescent protein fusions, to address these questions.

Major comments:

• Are the key conclusions convincing? I find the manuscript successfully addresses the posed questions. The data presented supports the conclusions.
• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
• 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. No
• 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. N/A
• Are the data and the methods presented in such a way that they can be reproduced? Yes
• Are the experiments adequately replicated and statistical analysis adequate? Yes

Minor comments:

• Specific experimental issues that are easily addressable.

On the data shown in Figure 1, it is unclear to me what elements are taken into account to define "anaphase." Anaphase could be defined by using chromatin markers - such as CenH3- which have been identified in Plasmodium and the authors make use of in Figure 1F.

• Are prior studies referenced appropriately?

The authors state that "with the exception of centrins and gamma tubulins" few canonical centrosome components are conserved in Plasmodium. These parasites are in fact able to assemble a more or less canonical centriole for microgamete basal body formation. Widely conserved centriolar components such as Sas6 are coded by the malaria genome, and have been characterized previously. This work is neither referenced nor discussed in the manuscript.

• Are the text and figures clear and accurate?

I find the timings shown in Figure 1A, with respect to the schematic quantification shown in Figure 1B, confusing. Shown as it is, one naturally correlates the images on Fig1A above with the cell cycle progression timing shown on Fig1B, below. However, by time 260min, for example, two somewhat adjacent centrin signals can be observed. Though this is defined as anaphase- by an unspecified criterium- this could very well be representative of metaphase. Nonetheless, the timing shown on Figure 1B for "anaphase" onset is 170min, which is inconsistent with the images above. I suggest that either, the quantification is shown in a different format (ex. bar plots) which could then better reflect the cell to cell variations observed (by use of error bars, for example)or that the figure explanation in the results section clarifies this issue. As presented, the data in Figure 1C is rather uninformative. A pattern could be more immediately extracted if dots corresponding to subsequent appearance of centrin dots in the same nucleus were connected to each other.

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

There are a number of edits required on the text. Row numbers would have been helpful in pointing these out. I point some edits below, but thorough revision of the manuscript for grammatical and synthetic errors would be beneficial. • Cytokinetic segmeter - please replace with "segmented" • Please refer to Figure 1D when appropriate - there is quite an extensive paragraph describing the results shown on this figure, but it is only referenced at the start. • "..., as did the and the number of branches per nucleus,..." please rewrite as appropriate.

Significance

This manuscript could be interested to a wide audience interested in cell cycle, cell division, cell organization and organelle positioning, infectious diseases and microscopy. However, the introduction assumes that readers are somewhat experts in the malaria field. I suggest the authors include a brief introduction of the malaria life cycle, and a schematic representation of the division mode. This will help non-experts follow the narrative more easily.

This work rectifies long-standing inconsistencies observed by different experimental approaches in the nuclear organization of malaria parasites during schizogony. However, what the functional consequences of the alternative modes of spindle organization in malaria could be, are not clearly stated or discussed. In this respect, as it stands, the manuscript is rather descriptive and lacks mechanistic insight. Nonetheless, the data presented are of superb quality, and the manuscript represents a tremendous leap in structural insight and imaging resolution for the field of malaria. I find the data is suitable for publication albeit minor adjustments are made (specially to Figure 1 and/or the description of the results shown in Figure 1, for consistency).

Referee Cross-commenting

I agree with all the other reviewer's comments. I'm glad the reviewers seem to be experts in the field of malaria cell division and have pointed out previous studies which were not appropriately referenced. I second those comments.

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Reply to the reviewers

Reviewer 1

This paper proposes a noise-aware approach SCRaPL for modelling the associations of single cell multi-omic data. For gene expression, it uses Poisson-lognormal model. For DNAm data, it uses Binomial noise model which explicitly takes into account the average within the region. The Bayesian hierarchical framework employed by SCRaPL could achieve higher sensitivity and better robustness in identifying correlations, and also offer a template for the application of more complex analysis techniques to multi-omics data. The symbols of this paper are a little bit confusing, and I suggest authors to carefully check them.

We thank the reviewer for his/ her appreciation, and apologise for the confusion arising from the dense notation, which we will thoroughly revise.

1. The symbols used in this paper are messy. For example, "1" and "2" are subscripts in Eq.(2) but become superscripts in Figure 5. Besides, there are many symbols not explained such as mj, Hj, Ψ0, etc. Also, I don't know if x{j,i}^{(1)} , x{j,i}^{(2)} in Figure 5 are same with x{ij1} and x{ij2} in Eq.(3). There are many places mismatch, authors should check carefully.

1. Why the equations in Fig.5 are totally different with Section 4.2? For example, pj ∼Beta(αj ,βj ) in Fig.5 but ρj ∼ Beta−1,1 in Eq.(8).

We apologise for the notational confusion, this will be fully revised.

The paper involves a lot of hyper-parameters which doesn't demonstrate their selection. For example, c1, c2, d1, d2.

This is a good point. We will include a sensitivity analysis on the hyperparameters, justifying the choices on both simulated and real data.

In section4.8, I am confused about $ρ_j$ the experiment 2, 5, 8, 11. Why $ρ_j$ both represents ZI rate and correlation?

We apologise for the notational oversight, which will be rectified.

In Section 4.5, it is difficult to understand the sentence "for me threshold u". Besides, what is $r$ represent in Section 4.5?

We apologise for the confusing sentence. $r$ is the Pearson correlation coefficient, as explained at the start of 4.5

Why there is "(6a)Agreement between SCRaPL and Pearson" in Fig. 4?

This simply means that the panel shows a methylation/ expression scatterplot for a gene where estimation by SCRaPL and Pearson return both a significant association. We will expand the caption to explain further.

For Fig.1, I cannot see the text in the rectangle.

Apologies, we will improve the readability of the figures

I would like to see the efficiency analysis for SCRaPL.

As part of part of re-implementation in a more accessible programming language, we have preformed preliminary efficiency analysis for MCMC , demonstrating linear scalability. Results will appear in the revised manuscript.

Reviewer 2

The authors present a Bayesian model to determine noise-corrected correlation coefficients for gene expression (RNA) and DNA-methylation data at single-cell resolution. The authors present a series of simulation data and an example of matched multi-omics data, and compare their results with Pearson correlation. Noise modelling allows the model to determine gene-methylation correlation patterns more accurately. While the authors demonstrate a neat application on accurate quantification of correlation coefficients, I see a limited use of the model for the broader single-cell community. The authors may therefore improve their manuscript on several aspects.

We thank the reviewer for the encouraging words, and thank him/ her for the critical observations, which we have taken at heart, considerably broadening the scope of our paper to make it more attractive to a larger community.

- Abstract: please specify the omics layers that you are analyzing (RNA + DNA methylation) in the abstract

We acknowledge that, while SCRaPL is potentially general, in the first submission we focused only on RNA and DNA methylation. We have now decided to expand our analyses to include 10X data of simultaneous chromatin accessibility (ATAC-seq) and RNA.

- What is the benefit of using a Bayesian model formulation in this setting?

The benefit is twofold: a principled treatment of noise, and a quantification of the resulting uncertainty which allows for a meaningful way to compute Bayesian significance levels. We will expand the discussion of the relative merits of a Bayesian vs frequentist approach.

- Does it also apply to unmatched data?

In principle, given measurements with the same number of cells in all modalities, it is possible to apply SCRaPL. However, unless there is a natural pairing between different cells, the scaling of this approach will be quadratic in the number of cells, hence potentially expensive (although largely parallelizable). We will discuss this now, particularly in the light of applying SCRaPL in conjunction with other suites such as Seurat.

• Would SCRaPL allow for differential correlation testing?

At the moment, SCRaPL does not allow for differential correlation testing. Of course, one may run SCRaPL separately on two groups of cells and compare the resulting estimates, which would be informative. Nevertheless, extending SCRaPL to perform differential correlation testing (e.g. using Bayesian model selection) would be a non-trivial effort. We will add a comment on this issue to the discussion section.

• Figure 1: The graphical description of the model is rudimentary. I believe that the model description could profit from a graphical model representation of SCRaPL (as presented in figure 5).

We will redraw Fig. 1 and incorporate the graphical model from Fig 5.

- Simulated data: all experiments seem to have rather low cell numbers (max. 200) and genes (max. 300). Given that 10X Genomics is the most widely-used sequencing platform with approx. 10,000 cells and 3,000 (highly variable) genes per experiment, and given that the authors show a use-case with 9480 genes in 487 cells, it seems appropriate to extend the simulations and runtime estimates of the presented model to several thousands of cells and genes, respectively.

Thank you for this comment. The original simulation settings were designed with scMT data in mind, where indeed only a few hundred cells can be assayed at most. Partly because of this feedback, and also because of the request of implementing SCRaPL in a different language, we are working on a more scalable Tensorflow implementation which will be able to handle thousands of cells and genes in a matter of tens of minutes . The new simulated data will therefore extend into this regime with larger data sets.

- Figure 4: Please revise the figure legend as I did not understand the plotted results based on the description.

We will do so.

- Results section 2.5: Please formulate your whole argument about epigenetic regulators. I do not think that "For further information please refer to supplementary figure XYZ." Is an appropriate closing statement for a paragraph, nor does it motivate the reader to look at the supplementary figures (I did look at them and I do not see how they support the point made in the paragraph). Please elaborate and consider a "take home message" for the paragraph such that the reader is able to understand the benefit of SCRaPL without revisiting the original data publication.

Thank you for this pointer, we will take it on board in the full revision.

- Conclusion: The authors mention that SCRaPL would further offer a "template for the application of more complex analysis techniques (such as clustering, dimensionality reduction and network inference)". If that was the case, the authors should consider a comparison to other tools, which offer exactly that (e.g. Seurat's CCA or non-negative matrix factorization in LIGER). Further, the authors should set their work into context with tools like bindSC.

Thank you for the suggestion. As far as we can tell, all of these methods are thought for unmatched data, rather than multi-omics assays performed in the same cells. Having said that, it is in principle possible to “preprocess” data with SCRaPL and then feed to Seurat or other tools the latent means computed by SCRaPL. We will include an example of how this may be done in the revision.

- Implementation: Matlab is used in about 6% of the single-cell RNAseq tools (according to scrna-tools.org). To reach a larger scientific community, do the authors plan to provide an R or Python implementation of their model?

We are now implementing SCRaPL in Python using Tensorflow probability, hoping to achieve substantial speedups (see response to previous point).

Additional minor points about formatting by Reviewer 2 will all be addressed.

Reviewer 3

Maniatis et al propose a sound strategy to analyse single-cell multi-comic data sets. A key advance is to use bespoke error models for each of the omics data. These are integrated into a multivariate gaussian model. This method is a novel and, in my opinion, a valuable addition to the analyses of the growing multi-omics single-cell data sets.

We thank this reviewer for his/ her appreciation of our work.

- Authors make a convincing argument of the importance of principle methods and in particular to use noise models that tailored to the data at hand. To further support this, can authors elaborate on how results would be different from using commonly applied methods ? Eg those embedded in the Seurat, OSCA, and scanpy 'suites'? Authors compare to Pearson correlation-based methods but is not clear if that is the true state-of-the-art on those methods

As far as we know, volcano plots of p-value versus Pearson correlation are the most commonly employed approaches to assess correlations amongst different molecular modalities in single-cell multi-omics (see e.g. Argelaguet et al, Nature 2020). Seurat and other methods normally do not deal with single-cell multi-omics (i.e., multiple omics measured in the same cell), rather with multiple single-cell omics (different molecular modalities assayed in different cells). Nevertheless, it is possible to pre-apply SCRaPL to non-matched data and then use another suite; as an illustration, we will perform an analysis on scMT data using SCRaPL followed by Seurat.

- In the case study on mouse embryonic stem cells, authors excluded the chromatic accessibilty. Why not using it to more clearly show the value of the method?

We did use SCRaPL also on chromatin accessibility, however the signal was weaker and we did not include it in the manuscript, we will now present these results as supplementary material.

- It would also be great if authors would use a different single-cell multi-comic data sets, using other dat modalities, e.g. CITE-Seq data. If this not possible, at least they should elaborate on which omics SCRAPL can handle, what would be the noise models for different data types, etc.

We have started analysing a joint scATAC-scRNA- seq data set generated using the new 10X commercial platform, and will add the results of this analysis to the revised manuscript. We will also expand the description of the suitability for different data types.

*- As the authors acknowledge, computational burden is high, which presumably limits scalability. Are authors able to further explore this (scalability on Insilico data)? Or how complex is adopting the variational inference method suggested? I appreciate that the variational inference implementation might be out of the scope of this paper, though.

• It is a pity that the method is in Matlab. Nearly no-one in single-cell omics use Matlab. Our own lab is largely invested in this topic and we do not even have Matlab licenses. I strongly encourage authors to implement their method in e.g. R or python, ideally compatible with the broadly used 'suites' (Seurat, OSCA, and scanpy,...).*

We are addressed these two comments jointly by re-implementing SCRaPL in Tensorflow probability (Python based), which allow us to leverage powerful libraries for variational inference. We hope that this will lead to a substantial increase of scalability, providing the possibility of running on thousands of cells / genes in under one hour (results will appear in ).

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Referee #3

Evidence, reproducibility and clarity

Maniatis et al propose a sound strategy to analyse single-cell multi-comic data sets. A key advance is to use bespoke error models for each of the omics data. These are integrated into a multivariate gaussian model. This method is a novel and, in my opinion, a valuable addition to the analyses of the growing multi-omics single-cell data sets.

I have some comments below that I hope are helpful for the authors:

• Authors make a convincing argument of the importance of principle methods and in particular to use noise models that tailored to the data at hand. To further support this, can authors elaborate on how results would be different from using commonly applied methods ? Eg those embedded in the Seurat, OSCA, and scanpy 'suites'? Authors compare to Pearson correlation-based methods but is not clear if that is the true state-of-the-art on those methods
• In the case study on mouse embryonic stem cells, authors excluded the chromatic accessibilty. Why not using it to more clearly show the value of the method?
• It would also be great if authors would use a different single-cell multi-comic data sets, using other dat modalities, e.g. CITE-Seq data. If this not possible, at least they should elaborate on which omics SCRAPL can handle, what would be the noise models for different data types, etc.

Minor:

• As the authors acknowledge, computational burden is high, which presumably limits scalability. Are authors able to further explore this (scalability on Insilico data)? Or how complex is adopting the variational inference method suggested? I appreciate that the variational inference implementation might be out of the scope of this paper, though.
• It is a pity that the method is in Matlab. Nearly no-one in single-cell omics use Matlab. Our own lab is largely invested in this topic and we do not even have Matlab licenses. I strongly encourage authors to implement their method in e.g. R or python, ideally compatible with the broadly used 'suites' (Seurat, OSCA, and scanpy,...).

This also precludes checking software and reproducibility of results.

Significance

I think this is an important methodological development for the analysis of single-cell multi-comic data.

To my knowledge, it goes beyond existing methods and does so in a principled manner.

The audience is mostly bioinformaticians dealing with the analysis of this type of data, ie single-cell multi-omics.

My expertise is in the computational analysis of omics data, though less on the statistical fundaments of it. Hence, my group members and I are probable users of this method (if implemented in free software, as mentioned above).

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Referee #2

Evidence, reproducibility and clarity

Summary:

The authors present a Bayesian model to determine noise-corrected correlation coefficients for gene expression (RNA) and DNA-methylation data at single-cell resolution. The authors present a series of simulation data and an example of matched multi-omics data, and compare their results with Pearson correlation. Noise modelling allows the model to determine gene-methylation correlation patterns more accurately. While the authors demonstrate a neat application on accurate quantification of correlation coefficients, I see a limited use of the model for the broader single-cell community. The authors may therefore improve their manuscript on several aspects.

Major comments:

• Abstract: please specify the omics layers that you are analyzing (RNA + DNA methylation) in the abstract
• What is the benefit of using a Bayesian model formulation in this setting?
• Does it also apply to unmatched data?
• Would SCRaPL allow for differential correlation testing?
• Figure 1: The graphical description of the model is rudimentary. I believe that the model description could profit from a graphical model representation of SCRaPL (as presented in figure 5).
• Simulated data: all experiments seem to have rather low cell numbers (max. 200) and genes (max. 300). Given that 10X Genomics is the most widely-used sequencing platform with approx. 10,000 cells and 3,000 (highly variable) genes per experiment, and given that the authors show a use-case with 9480 genes in 487 cells, it seems appropriate to extend the simulations and runtime estimates of the presented model to several thousands of cells and genes, respectively.
• Figure 4: Please revise the figure legend as I did not understand the plotted results based on the description.
• Results section 2.5: Please formulate your whole argument about epigenetic regulators. I do not think that "For further information please refer to supplementary figure XYZ." Is an appropriate closing statement for a paragraph, nor does it motivate the reader to look at the supplementary figures (I did look at them and I do not see how they support the point made in the paragraph). Please elaborate and consider a "take home message" for the paragraph such that the reader is able to understand the benefit of SCRaPL without revisiting the original data publication.
• Conclusion: The authors mention that SCRaPL would further offer a "template for the application of more complex analysis techniques (such as clustering, dimensionality reduction and network inference)". If that was the case, the authors should consider a comparison to other tools, which offer exactly that (e.g. Seurat's CCA or non-negative matrix factorization in LIGER). Further, the authors should set their work into context with tools like bindSC.

Minor comments:

• Implementation: Matlab is used in about 6% of the single-cell RNAseq tools (according to scrna-tools.org). To reach a larger scientific community, do the authors plan to provide an R or Python implementation of their model?
• Fig. 2: Legends for mean, median and y=0 are hardly legible.
• Figure order: 6a is referenced before 4b and 4c (what about 4a?) - seems like a referencing issue as 6a is also listed in the figure legend of Figure 4.
• Figure 6: AIC histogram is difficult to make out behind the blue bars of the DIC histogram. Please adapt.

Reference:

Unbiased integration of single cell multi-omics data Jinzhuang Dou, Shaoheng Liang, Vakul Mohanty, Xuesen Cheng, Sangbae Kim, Jongsu Choi, Yumei Li, Katayoun Rezvani, Rui Chen, Ken Chen, bioRxiv, 2020 https://www.biorxiv.org/content/10.1101/2020.12.11.422014v1

Significance

Significance:

The use of a single-cell specific noise-model to infer accurate correlation coefficients for multi-omic analysis is a novel approach to assess information from DNA-methylation and RNA-sequencing data at single-cell resolution. As far as I am aware, methods like canonical correlation analysis (CCA), as used in Seurat, rely on the accuracy of Pearson correlation, yet, the authors of this manuscript made a convincing point on the devastating impact of noise from transcription and methylation levels on Pearson correlation.

Audience:

In order to address downstream analysis questions such as gene regulatory network inference, it is essential to have an accurate metric to assess the regulatory impact of methylation on gene expression at hand. However, an efficient implementation in a more common language (e.g. R, Python or C++) would be advisable to create a broader applicability of the model.

The reviewer's field of expertise: single-cell RNAsequencing, data analysis, data integration, Bayesian modelling

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Referee #1

Evidence, reproducibility and clarity

This paper proposes a noise-aware approach SCRaPL for modelling the associations of single cell multi-omic data. For gene expression, it uses Poisson-lognormal model. For DNAm data, it uses Binomial noise model which explicitly takes into account the average within the region. The Bayesian hierarchical framework employed by SCRaPL could achieve higher sensitivity and better robustness in identifying correlations, and also offer a template for the application of more complex analysis techniques to multi-omics data. The symbols of this paper are a little bit confusing, and I suggest authors to carefully check them. My comments are as following:

1. The symbols used in this paper are messy. For example, "1" and "2" are subscripts in Eq.(2) but become superscripts in Figure 5. Besides, there are many symbols not explained such as mj, Hj, Ψ0, etc. Also, I don't know if x{j,i}^{(1)} , x{j,i}^{(2)} in Figure 5 are same with x{ij1} and x{ij2} in Eq.(3). There are many places mismatch, authors should check carefully.
2. Why the equations in Fig.5 are totally different with Section 4.2? For example, pj ∼Beta(αj ,βj ) in Fig.5 but ρj ∼ Beta−1,1 in Eq.(8).
3. The paper involves a lot of hyper-parameters which doesn't demonstrate their selection. For example, c1, c2, d1, d2.
4. In section4.8, I am confused about $ρ_j$ the experiment 2, 5, 8, 11. Why $ρ_j$ both represents ZI rate and correlation?
5. In Section 4.5, it is difficult to understand the sentence "for me threshold u". Besides, what is $r$ represent in Section 4.5?
6. Why there is "(6a)Agreement between SCRaPL and Pearson" in Fig. 4?
7. For Fig.1, I cannot see the text in the rectangle.
8. I would like to see the efficiency analysis for SCRaPL.

Significance

Audience who interested in multi-omic data, single-cell rna, machine learning will be interested in this paper.

My field of expertise: machine learning, single-cell RNA

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Reply to the reviewers

First of all, we would like to thank the editor and all reviewers for the effort to evaluate our paper in this difficult era of COVID-19.

Reviewer #1

(Significance): Overall, this manuscript is very clear and easy to follow. The manuscript could be improved by making the following changes:

We thank the reviewer for the favorable comment and will revise the manuscript according to the suggestions.

Reviewer #2

(Evidence, reproducibility and clarity): The use of genetics is particularly impressive but the lack of major discoveries dampens the enthusiasm. Additional efforts to mechanistically define wave initiation and wave propagation would significantly improve the impact of the manuscript. Moreover, some of the conclusions are not fully supported by the data and require further experimentation and/or analysis.

We admit that marked redundancy of function among the EGFR ligands and their essential roles in cell growth prevent us from obtaining very clear results. Considering the importance of EGFR ligands in biology, we believe, our observation will give invaluable suggestions to whom wishes to clarify the roles played by EGFR-family protein in other biological contexts.

(Significance): While it is known that ADAM17 is critical to process EGFR ligands, the specific or redundant roles of different ligands remains an open question. The authors find that all ADAM17 ligands contribute to ERK signaling waves but may have specific contributions to other phenotypes. This work would be of interest to the signaling dynamics, epithelial and developmental biology communities.

We thank the reviewer for the favorable comment.

Reviewer #3

(Evidence, reproducibility and clarity): Overall, this study is carried out with a high degree of rigor and technical excellence, with clear reporting of experimental details and replication. The writing and figures are very clear, and there are no obvious technical problems. However, there are some areas in which the strength and clarity of the conclusions could be strengthened by relatively simple experiments.

We thank the reviewer for the favorable comment. We have already performed some of the experiments suggested by the reviewer. As the reviewer might have anticipated, co-culture with the wild type MDCK cells helps mutant cells to survive. We believe we could propose a clearer model in the revised paper.

(Significance): This study definitively establishes the role of 4 EGFR ligands in the generation of ERK activity waves in MDCK cells. While other studies, including some from the senior author's lab, have strongly indicated that EGFR autocrine signaling is important for these waves, this study goes further in comparing the roles of these ligands using knockouts to unambiguously establish the autocrine factors involved. Others who use this common experimental system (MDCK) to study epithelial dynamics will find this study of great interest. A wider audience of those who work on EGFR-mediated signaling will also find the data quite fascinating as an example of the complex relationship between ERK activation and its downstream effects. The technical excellence of the paper will make it a must-read for those in these fields. However, there are some factors that limit the scope of the significance. MDCK cells are an important experimental model system but differ in substantial ways from other epithelial cells, particularly in the expression of EGFR ligands. Because different ligands such as amphiregulin dominate in other systems (as noted by the authors, and PMID 27405981), the ability to extrapolate from these findings to other cell types is somewhat limited. Also, the paper avoids addressing the major question of how ERK waves relate to collective migration rate. From the data presented it is clear that this relationship is complex; for example, bath application of the ligands restores a high migration rate but not ERK waves. Given this lack of a clear relationship it is an understandable decision to leave this question for future work; however this does limit the conclusions that can be drawn from the study.

We completely agree with the reviewer’s view. It is uncertain to what extent the observation with MDCK cells can be generalized to other cell types. We also admit that the conclusion is not very simple because EGFR signaling is required for various cellular functions including cell survival and migration. Even though the gene editing becomes so easy, it is still labor consuming work to knock out many genes in a single cell line with extensive characterization. We believe the data shown in our work will provide a basis for the understanding of EGFR ligands.

Reviewer #1

For Fig 1F, 3 individual experiments should be conducted to confirm results.

We will follow the reviewer’s suggestion and repeat the experiment.

For Fig 1G, could the authors please show the original western blot data in full rather than just the densitometry graphs?

We did not show just for the sake of brevity. We are happy to will include the images as a supplementary data.

The authors should explain the origin/phenotype of MDCK cells for those who are not familiar with the cell line.

We will modify the text according to the reviewer’s suggestion.

The authors should give a future outlook/direction for future experimentation to further confirm redundancy in EGF ligands in the propagation of ERK activation waves.

We will discuss on the redundancy in other cell types based on available NGS data.

Some mention of the use of biosensors in the abstract and introduction is recommended as this is a major part of the experimental work.

We will refer to the biosensors in the abstract and introduction.

Reviewer #2

There are conflicts with some of the conclusions made about ligands. dEGFR cells have basal ERK activity as high as WT which argues against EGF being responsible for basal ERK activity. Further, basal ERK activity was not rescued by restoration of EGF in the 4KO-EGF cells. The authors should address this discrepancy.

We agree that some new questions have arisen from our observations. The discrepancy of the phenotypes between dEGFR cells and dEGF cells is an example. We are currently establishing dEGF cell lines, in which different genomic sequences of the EGF gene were targeted. We have already started to develop these cell lines and will obtain them within a month. The result will provide some clues to answer the questions. However, even if we could not solve the question, we believe, it is worth reporting observations that could not be easily understood, because such questions are often leading to another discovery.

Besides the ones genetically disrupted in this work, other EGFR ligands seem to play functional roles given that dEGFR cells less migration and fewer ERK waves than 4KO cells. The authors could test if other ligands are upregulated in 4KO cells to compensate. On a similar note determining whether ADAM17 deficient cells are more similar to 4KO cells or dEGFR cells could provide some insight.

According to the reviewer’s suggestion, we will conduct qPCR of growth factors in mutant cell lines to see the expression levels of seven EGFR ligands might have changed significantly. At the same time, as the reviewer suggested, we will establish ADAM17 knockout cell lines and compare the phenotype with those of cell lines deficient from EGFR ligand genes.

• The authors propose that Nrg1 is responsible for ERK waves in QKO, 4KO, dEGFR, and 4KO-EGF cells but are limited in testing this due to Nrg1 being essential in 4KO cells. First, Nrg1 should have been deleted in TKO cells to confirm that it is only essential in the absence of the four EGFR ligands. Additionally, Nrg1 could be knocked out in 4KO-EGF cells to demonstrate the claim that EGF-induced ADAM17 cleavage of Nrg1 is responsible for ERK waves.*

We do not think the deletion of Nrg1 in the TKO cells will abolish the ERK activation waves because EREG in TKO cells could transmit the waves. To overcome the problem of cell growth, we will try to obtain 5KO cells by Cre-induced deletion of NRG1 in 5KO-loxP-NRG1 cells, wherein EGF is supplied exogenously. We already had preliminary data suggesting that co-culture with wild type MDCK cells helps 5KO cells grow.

The authors state that ERK activation waves are important for collective migration and seek to understand the roles of each EGFR ligand, but despite measuring migration and properties of ERK activity, there is very little analysis or commentary on the relationship between the two. The ability of HB-EGF to restore migration without ERK waves suggests that waves are not required per se. It is interesting to note that with restoration of ligands, migration is higher than WT but ERK activity is lower.

We refrained from spending much space about the essential role of ERK activation waves in collective cell migration, because several papers have already described this issue.

Probably, we should have spent more space to emphasize that the collective cell migration is comprised of at least two different phenomena. The migration of leader cells and the follower cells. The ERK activation waves are essential for the follower cells but not the leader cells. In 4KO cells, both the leader cell and follower cell migrations are impaired. We showed that GFs expression restore the leader cell migration, but not the follower cells. We will revise the text to include this issue.

It is suggested that the total amount of EGFR ligands may be the primary determinant of migration, but deletion of TGFα alone causes a significant decrease in migration comparable to the DKO cells. TGFα has the lowest expression of the four ligands studied but is the only ligand to have a significant impact on migration in the single knockout context, which disagrees with that conclusion.

Each EGFR ligand has different affinity to EGFR, which makes it difficult to link the mRNA levels directly to the effect of each EGFR ligand. We will modify the discussion to include this argument.

Other:

Fig. S3B needs clarification that the WT (black) and 4KO (green) did not receive a stimulus.

We will follow the reviewer’s advice.

Reviewer #3:

The experiments in Fig. 5 are undertaken with the purpose of assessing whether NRG acts as an additional ligand that mediates the residual ERK waves in 4KO/QKO cells. However, this question is never addressed in the NRG/4KO cells. While it might be challenging due to the proliferative defect, it seems important to attempt this experiment in some way; measuring the ERK waves for these cells would establish whether all of the critical autocrine factors have been identified. Can the proliferation be rescued by application of high amounts of growth factors?

This question is similar to a question raised by reviewer #2.

To overcome the problem of cell growth, we will try to obtain 5KO cells by Cre-induced deletion of NRG1 in 5KO-loxP-NRG1 cells, wherein EGF is supplied exogenously.

The bath exposure to EGFR ligands shown in Fig. S3A is an important experiment, but it is surprising that ERK signaling is not maintained under these conditions. Is this due to depletion of the added ligands, perhaps locally? Or is the intermittent nature of paracrine signaling needed to maintain ERK activity? These possibilities could be distinguished by checking whether the added EGF or the other ligands are depleted after several hours, or by restimulating with a new bolus of ligand after several hours.

We thank the reviewer for this invaluable suggestion. We will conduct the experiments suggested by the reviewer.

The connection between ERK activity and migration is somewhat confusing. It would be helpful to show the dose sensitivity of migration to a MEK or ERK inhibitor. Are other pathways downstream of EGFR such as PI3K involved in the autocrine-mediated migration? This could also be established with the appropriate inhibitors.

We should have spent more space to emphasize that the collective cell migration is comprised of at least two different phenomena. The migration of leader cells and the follower cells. The ERK activation waves are essential for the follower cells but not the leader cells. In 4KO cells, both the leader cell and follower cell migrations are impaired. We showed that GFs expression restore the leader cell migration, but not the follower cells. We will emphasize this issue in the revised manuscript.

Reviewer #1:

Line 47 in Abstract should read "Aiming for" not "Aiming at".

We have corrected the mistake as suggested.

Some in the field call fluorescence lifetime microscopy "FLIM", you could adopt the same wording in your manuscript to attract more readers.

We have included FLIM according to the reviewer’s suggestion.

Reviewer #1 :

Figure 1D, the images should be presented using the same scale for both the EKAREV and EKARrEV constructs so that they can be directly compared.

Because the basal FRET/CFP ratio is significantly different between EKAREV-NLS and EKARrEV-NLS, the changes during mitosis become unclear if we applied the same scale. This figure is prepared to show the reactivity to Cdk1 during mitosis; therefore, we believe the current scale is better for presentation.

The names QKO and 4KO are a bit confusing. Could the authors please change the naming of the knockout cells so that readers understand that QKO and 4KO are two separate cell types? Perhaps instead of 4KO use FKO for "full knockout" or something similar. The 5KO line might also need to be named something else if you change to FKO.

We have discussed this issue with the co-authors, but could not reach a better idea. Instead of changing the names, we will include a detailed explanation for each cell line.

Reviewer #2:

The interpretation of the RA-SOS coculture experiments is confusing. Based on the author's reasoning, I would expect ADAM17 shedding in the RA-SOS cells to trigger signaling at the interface of both WT and 4KO cells but the 4KO should be unable to propagate the wave farther away from the interface. This does not seem to be the case. Do RA-SOS ADAM17KO cells still trigger waves of ERK signaling in the WT cells? Do ADAM17KO cells behave as the 4KO cells in this coculture system?

Probably, the reviewer misunderstood the method. The GF-less 4KO cells were co-cultured with wild type cells harboring the RA-SOS system. We will describe more in detail to avoid misunderstanding.

Finally, the growth curve in Fig. 5B indicates that 5KO-loxP-NRG1-CreERT2 cells are viable for about two days after Cre induction. The authors could perform a confinement release assay of these cells 1-1.5 days after Cre induction to look for further reduction of ERK waves and migration to demonstrate the role of Nrg1.

This experiment may not be necessary. It is clear that NRG1 is required for the survival of 4KO cells. The reason why cells are still alive 1 to 2 days after 4-OHT application is simply because NRG1 protein is remaining. The interpretation of the results would be difficult during the phase of NRG1 reduction.

In Fig. 1G, the normalization of all WT pERK samples to 1 artificially lowers the variance to zero when performing the T-test.

For the comparison of immunoblotting data derived from independent experiments, the signals must be normalized to the control. We believe the use of pERK/ERK of the wild type cells as the control is reasonable for this experiment.

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Referee #3

Evidence, reproducibility and clarity

This manuscript seeks to clarify the mechanisms that underlie traveling "waves" of ERK activity that occur in monolayers of migrating epithelial cells. A combination of live cell imaging with ERK activity biosensors and CRISPR-mediated knockouts for autocrine regulators are used to dissect the factors that make these waves possible. The authors utilize the MDCK cell line, which shows very prominent wave behavior, and they perform an impressive number of knockouts to eliminate the most abundant autocrine EGFR ligands. They also introduce a novel ERK FRET reporter, which is less sensitive to off-target phosphorylation by Cdk1. Analysis of ERK biosensor data from the knockouts shows that knockout of all four main EGFR ligands is needed to substantially reduce the amplitude of ERK waves, although it does not completely eliminate it. Re-expression of any of the four ligands, with the exception of HBEGF, restores strong ERK waves. Application of the same ligands in solution restores migration but not the ERK waves.

Overall, this study is carried out with a high degree of rigor and technical excellence, with clear reporting of experimental details and replication. The writing and figures are very clear, and there are no obvious technical problems. However, there are some areas in which the strength and clarity of the conclusions could be strengthened by relatively simple experiments.

Major:

1. The experiments in Fig. 5 are undertaken with the purpose of assessing whether NRG acts as an additional ligand that mediates the residual ERK waves in 4KO/QKO cells. However, this question is never addressed in the NRG/4KO cells. While it might be challenging due to the proliferative defect, it seems important to attempt this experiment in some way; measuring the ERK waves for these cells would establish whether all of the critical autocrine factors have been identified. Can the proliferation be rescued by application of high amounts of growth factors?
2. The bath exposure to EGFR ligands shown in Fig. S3A is an important experiment, but it is surprising that ERK signaling is not maintained under these conditions. Is this due to depletion of the added ligands, perhaps locally? Or is the intermittent nature of paracrine signaling needed to maintain ERK activity? These possibilities could be distinguished by checking whether the added EGF or the other ligands are depleted after several hours, or by restimulating with a new bolus of ligand after several hours.

Minor (I think this is an important point overall, but it is outside of the scope of the paper as defined by the authors, which is focused on the ERK waves rather than how the waves relate to migration):

1. The connection between ERK activity and migration is somewhat confusing. It would be helpful to show the dose sensitivity of migration to a MEK or ERK inhibitor. Are other pathways downstream of EGFR such as PI3K involved in the autocrine-mediated migration? This could also be established with the appropriate inhibitors.

Significance

This study definitively establishes the role of 4 EGFR ligands in the generation of ERK activity waves in MDCK cells. While other studies, including some from the senior author's lab, have strongly indicated that EGFR autocrine signaling is important for these waves, this study goes further in comparing the roles of these ligands using knockouts to unambiguously establish the autocrine factors involved. Others who use this common experimental system (MDCK) to study epithelial dynamics will find this study of great interest. A wider audience of those who work on EGFR-mediated signaling will also find the data quite fascinating as an example of the complex relationship between ERK activation and its downstream effects. The technical excellence of the paper will make it a must-read for those in these fields. However, there are some factors that limit the scope of the significance. MDCK cells are an important experimental model system but differ in substantial ways from other epithelial cells, particularly in the expression of EGFR ligands. Because different ligands such as amphiregulin dominate in other systems (as noted by the authors, and PMID 27405981), the ability to extrapolate from these findings to other cell types is somewhat limited. Also, the paper avoids addressing the major question of how ERK waves relate to collective migration rate. From the data presented it is clear that this relationship is complex; for example, bath application of the ligands restores a high migration rate but not ERK waves. Given this lack of a clear relationship it is an understandable decision to leave this question for future work; however this does limit the conclusions that can be drawn from the study.

Areas of expertise: growth factor signal transduction, biosensors, quantitative modeling

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Referee #2

Evidence, reproducibility and clarity

Lin et al. address the mechanisms underlying ERK signaling waves in epithelial cells. While it is known that ADAM17 is critical to process EGFR ligands, the specific or redundant roles of different ligands remains an open question. First the authors generate a modified ERK FRET sensor with reduced cross-reactivity to CDK1 in MDCK cells and systematically knockout EGF, HBEGF, TGF⍺ and EREG (the highest expressed ligands in MDCK cells). The authors use live cell imaging of ERK activity upon release from confinement and find that all ligands contribute to ERK signaling waves. While differences in basal signaling and other dynamic features are found in individual knockouts, only the quadruple KO cells show a significant decrease in ERK waves. To determine if the 4KO cells are defective in wave propagation (as opposed to wave initiation), the authors coculture 4KO cells with an inducible cell line and conclude that 4KO cells are unable to propagate waves. Individual EGFR ligands are then restored in 4KO cells, and EGF, TGFα, and EREG, but not HBEGF, can rescue ERK activity waves. Finally, the authors attempt to eliminate all ERK activation waves by deletion of Nrg1 but find that it is essential in 4KO cells. The paper is well-written and technically sound. The use of genetics is particularly impressive but the lack of major discoveries dampens the enthusiasm. Additional efforts to mechanistically define wave initiation and wave propagation would significantly improve the impact of the manuscript. Moreover, some of the conclusions are not fully supported by the data and require further experimentation and/or analysis.

1. There are conflicts with some of the conclusions made about ligands. dEGFR cells have basal ERK activity as high as WT which argues against EGF being responsible for basal ERK activity. Further, basal ERK activity was not rescued by restoration of EGF in the 4KO-EGF cells. The authors should address this discrepancy.
2. Besides the ones genetically disrupted in this work, other EGFR ligands seem to play functional roles given that dEGFR cells less migration and fewer ERK waves than 4KO cells. The authors could test if other ligands are upregulated in 4KO cells to compensate. On a similar note determining whether ADAM17 deficient cells are more similar to 4KO cells or dEGFR cells could provide some insight.
3. The interpretation of the RA-SOS coculture experiments is confusing. Based on the author's reasoning, I would expect ADAM17 shedding in the RA-SOS cells to trigger signaling at the interface of both WT and 4KO cells but the 4KO should be unable to propagate the wave farther away from the interface. This does not seem to be the case. Do RA-SOS ADAM17KO cells still trigger waves of ERK signaling in the WT cells? Do ADAM17KO cells behave as the 4KO cells in this coculture system?
4. The authors propose that Nrg1 is responsible for ERK waves in QKO, 4KO, dEGFR, and 4KO-EGF cells but are limited in testing this due to Nrg1 being essential in 4KO cells. First, Nrg1 should have been deleted in TKO cells to confirm that it is only essential in the absence of the four EGFR ligands. Additionally, Nrg1 could be knocked out in 4KO-EGF cells to demonstrate the claim that EGF-induced ADAM17 cleavage of Nrg1 is responsible for ERK waves. Finally, the growth curve in Fig. 5B indicates that 5KO-loxP-NRG1-CreERT2 cells are viable for about two days after Cre induction. The authors could perform a confinement release assay of these cells 1-1.5 days after Cre induction to look for further reduction of ERK waves and migration to demonstrate the role of Nrg1.
5. The authors state that ERK activation waves are important for collective migration and seek to understand the roles of each EGFR ligand, but despite measuring migration and properties of ERK activity, there is very little analysis or commentary on the relationship between the two. The ability of HB-EGF to restore migration without ERK waves suggests that waves are not required per se. It is interesting to note that with restoration of ligands, migration is higher than WT but ERK activity is lower.
6. It is suggested that the total amount of EGFR ligands may be the primary determinant of migration, but deletion of TGFα alone causes a significant decrease in migration comparable to the DKO cells. TGFα has the lowest expression of the four ligands studied but is the only ligand to have a significant impact on migration in the single knockout context, which disagrees with that conclusion. Other:
7. In Fig. 1G, the normalization of all WT pERK samples to 1 artificially lowers the variance to zero when performing the T-test.
8. Fig. S3B needs clarification that the WT (black) and 4KO (green) did not receive a stimulus.

Significance

While it is known that ADAM17 is critical to process EGFR ligands, the specific or redundant roles of different ligands remains an open question. The authors find that all ADAM17 ligands contribute to ERK signaling waves but may have specific contributions to other phenotypes. This work would be of interest to the signaling dynamics, epithelial and developmental biology communities.

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Referee #1

Evidence, reproducibility and clarity

see below for comments.

Significance

Overall, this manuscript is very clear and easy to follow. The manuscript could be improved by making the following changes:

• Line 47 in Abstract should read "Aiming for" not "Aiming at".
• Some mention of the use of biosensors in the abstract and introduction is recommended as this is a major part of the experimental work.
• The names QKO and 4KO are a bit confusing. Could the authors please change the naming of the knockout cells so that readers understand that QKO and 4KO are two separate cell types? Perhaps instead of 4KO use FKO for "full knockout" or something similar. The 5KO line might also need to be named something else if you change to FKO.
• Figure 1D, the images should be presented using the same scale for both the EKAREV and EKARrEV constructs so that they can be directly compared.
• Some in the field call fluorescence lifetime microscopy "FLIM", you could adopt the same wording in your manuscript to attract more readers.
• For Fig 1F, 3 individual experiments should be conducted to confirm results.
• For Fig 1G, could the authors please show the original western blot data in full rather than just the densitometry graphs?
• The authors should explain the origin/phenotype of MDCK cells for those who are not familiar with the cell line.
• The authors should give a future outlook/direction for future experimentation to further confirm redundancy in EGF ligands in the propagation of ERK activation waves.

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Reply to the reviewers

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

**Summary:**

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

In this paper the authors used a targeted approach to identify rare mutations in a cohort of glioma patients. Using this approach they identified a recurrent mutation in the TOP2A gene encoding for Topoisomerase 2A, and suggest that this mutation creates a more effective protein, binding DNA strongly and maybe more enzymatically active. RNAseq analysis of TOP2A WT and TOP2A mut tumor samples suggest different transcription patterns and points to possible splicing defects. The most recurrent variant (E9448Q) is described in depth and some experimental information shows this variant might be a gain-of-function mutation.

**Major comments:**

• Are the key conclusions convincing? The validation of both the methodology and the presence of never described TOP2A variations in HGG is done quite successfully. Interesting evidence about relevance of the most frequent mutation is provided. However, besides having computational and biochemistry assays performed, lack of details about in vitro experiment statistics (no p-values are provided in figures 4 and 5, neither sample size, repetitions) weakens the conclusions claimed by the authors about the properties of the mutated topoisomerase. Ad. In the revised version we provided more details about in vitro experiments, including statistics when is applicable, sample size and a number of repetitions. In the fig. 4 we show the results of two repetitions (so we can’t calculate statistics) but I would like to stress that we tested independently two fragments of the protein and the results were similar, so our conclusion was justified. However, we do agree with the reviewer that a statistical analysis of those biochemical tests is required. We already started to produce a new batch of recombinant proteins and we will add repetitions to reinforce our claims. We will provide statistical analysis details once all experiments are performed. __

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Claims about E948Q variant function should be revised. Data is not presented in a convincing way, plus there is ambiguous language used from the results ("We conclude that the E9448Q TOP2A protein is functional, and MIGHT have a higher activity than the WT protein") to the rest of the paper where they strongly support the claims about the TOP2A activity. Ad. We will provide more data on biochemical features of the TOP2A variant to confirm the impact of the E948Q substitution on enzyme activities, which would allow more strong conclusions. This will present our results in more convincing way. A language of the manuscript has been critically revised and modified (see a version with tracked changes).

• 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. In line with the presented data in the paper, additional experiments that show catalytic changes of the E9448Q variation must be added. It is shown that there are differences in the DNA binding capacity by EMSA compared to the WT form, however, the DNA supercoil relaxation activities is not that different, at least the way the results are presented. The authors suggest that TOP2A mutation is a driver mutation but no validation in vitro of this claim is shown. Can this mutation alone or in combination with e.g. tumor suppressors transform normal cells to cancer cells? Do cell lines expressing this mutation (compared to parental TOP2A wt expressing cells) display increased transcription? Increased invasion? Ad. In the revised version we moderated our conclusions and we do not state that the mutated TOP2A is an oncogenic driver. We suggest this mutation (and possibly other TOP2A mutations, as we analyzed the impact of other variants on the TOP2A protein function) contribute to gliomagenesis. This conclusion is based not only on the changes in biochemical properties, but also on the observation of the impact of the mutation of transcription and patient survival. We expanded the analysis of TOP2A mutations and expression levels on TCGA datasets and those new results support our conclusions about a pathogenic nature of TOP2A overexpression and mutations (the supplementary fig.4). We believe in such situation, there is no rationale to make a classical oncogenic driver experiment.

Due to a rarity of the TOP2A mutations it is impossible to find a patient derived cell line with such defect. We attempted to overexpress TOP2A in glioma cells but apparently there is some autoregulation preventing overexpression of this protein is cells with endogenous TOP2A expression. Therefore, we can’t verify if cell lines expressing this variant (compared to parental TOP2A wt expressing cells) have increased transcription. Moreover, such experiments are costly and require more time investment for substantial experiments

I would like to stress that modeling some events in cell cultures is difficult and we found in GBMs the link between the mutated TOP2A and increased transcription along with decrease of splicing factors expression.

We have attempted to make CRISPR/Cas9 mediated knock-in in glioma cells but without success. This is a difficult and time consuming procedure. Although in principle, we agree on the rationale for such experiment, we think that the current data are consistent and convincing. If reviewers find it necessary we may attempt to create glioma cell lines with TOP2A knock-out and overexpression of the mutated TOP2A gene and study it functionally, but it would require more time.

• 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. If the authors can complement the already presented in vitro experiments with additional ones supporting their hypothesis, this should be feasible. The authors can use patient derived glioma cells or glioma cell lines manipulated to express either the parental TOP2A wt enzyme or the identified mutated form. __Ad. Due to a rarity of the TOP2A mutations it is impossible to find a patient derived cell line with such defect. Our findings partly relied on frozen historical samples, so it is not possible to develop patient-derived cell lines. As mentioned above, we can create a TOP2A knock-out cell line and overexpress a wild type or mutated version but there is no certainty that TOP2A deficient cells would survive (this is an essential enzyme) and such manipulation would be feasible.__

• Are the data and the methods presented in such a way that they can be reproduced? Yes, the authors provide a quite detailed explanation of the methods implemented to reach each one of the results they are presenting.

• Are the experiments adequately replicated and statistical analysis adequate? No, there is no information about the statistical analysis or number of replicates in any of the in vitro experiments performed. This information should be added to the manuscript.

Ad. In the revised version the requested information was added where was possible and additional repetitions for biochemical experiments are currently in progress.

**Minor comments:**

• Specific experimental issues that are easily addressable.
• Are prior studies referenced appropriately? Yes, authors clearly address the state of the art regarding previous NGS methodologies and let us know the advantages and novelty of their approach.

• Are the text and figures clear and accurate? There are some discrepancies between the strength of the language used in different sections of the paper to refer the conclusions they can infer from the results they are showing. While they are all valid, authors should revise it. Ad. The text of the manuscript has been unified and revised.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? First of all, describe the statistical analysis used in every figure, include number of biological and technical replicates. I would also suggest to change the title or the scope of the discussion, there is too much focus on the TOP2A in the introduction, neglecting all the technical NGS work that actually lead to several new variants being described. This may be confusing when it collides with a conclusion that is heavily focused on the first half describing potential implications of at least another 3 proteins where genetic alterations were described. Given the fact there is not much experimental work that shows TOP2A mutations relevance in HGG or strong enough evidence of the variant's function I would suggest to change a bit the scope of the title. Ad. The description of the results and discussion have been revised to include additional data/discussion on technicalities and other finding not related to TOP2A. We performed additional computational analyses of TOP2A expression/mutations in the TCGA datasets. We believe that the planned experiments on genetically modified cell lines would provide additional support for our claims. We think that in the revised version a balance between landscape/NGS content and TOP2A content is well balanced.

Reviewer #1 (Significance (Required)):

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. The authors describe a methodology that proved to be sensitive and specific enough in order to allow them to detect rare genetic alterations in patient glioma samples. This information could be valuable to describe new driver mutations or infer in genetic pathway alterations that could be potential therapeutic targets. As the authors state at the beginning of the paper, given the poor therapeutical approaches existing for HGG currently, information of this kind could still be highly useful and provide a better outcome to a specific cohort of patients.

On a personal note, I think there is too much speculation about how TOP2A mutations could be interesting from a biological point of view (authors referred to evidence about implications of this mutation in other forms of cancer) but since no experimental validation is provided in glioma cells, it is difficult to conclude that this enzyme gain-of-function mutation could have a relevant role in HGG and thus make these variants a potential therapeutic target. There are no experiments conducted in glioma cells that express TOP2A variants, it would be interesting to see if it has an effect in the migratory/invasive phenotype like described in other cancer types or like it is suggested by analysis of the genetic pathways activated in the HGG patients samples harboring TOP2A mutation. In addition, there is no evidence of the TOP2A mutations possible role as a driver mutation, which is an interesting aspect that could be further explored from both a computational and an experimental approach.

Ad. As mentioned above, there is no glioma cells that express TOP2A variants and we are not convinced that such experiment will be feasible taking into account an essential role of TOP2A. We will attempt to perform experiments with CRISP/Cas9 knock-in cell lines and functional validation, but until now we did not accomplish knock-in in glioma cells. We will try to knock-out the endogenous TOP2A using CRISPR and express a TOP2A WT or E948Q variant from plasmids encoding these proteins, but we can’t predict if TOP2A KO cell would survive. If we manage to produce such cells, then we will investigate proliferation, migration and invasion of cells expressing TOP2A WT or mutated variant.

We do agree with the reviewer that our previous conclusions were too strong, and in the revised version we moderated our claims. We do not say that the mutated TOP2A is an oncogenic driver. We suggest this mutation (and possibly other TOP2A mutations, as we analyzed the impact of other variants on the TOP2A protein structure) contribute to gliomagenesis.

__Data on the Fig. 1A suggests that TOP2A has a mutational hotspot in the position E948Q in our dataset. In the revised version of the manuscript we have extended RNA-seq analysis of our datasets and TCGA PanCancer datasets to search for TOP2A mutations/ overexpression. We found that another computational prediction using CADD algorithm strongly confirms that TOP2A E948Q is in the top 1% of most deleterious variants in the human genome (CADD score >20). This results was added to Supplementary Table 2.__

• Place the work in the context of the existing literature (provide references, where appropriate). The quality of the paper is high and in line with other studies in the literature that perform genome and transcriptome analysis of tumor samples. It is only the experimental validation that is lacking data supporting the "in silico" findings. Ad. We would like to point that we provided the results of experimental, biochemical validation (2 assays) showing that the variant TOP2A proteins have different properties. The associations of transcriptional dysregulation in variant TOP2A bearing gliomas was not a in silico prediction but the result of the analysis of real tumor samples.

As stated above, we are ready to perform further biological validation if the editors find it necessary.

• State what audience might be interested in and influenced by the reported findings. Computational biologists are the right audience to target this paper. If additional experimental work further validating their initial bioinformatic findings is added to the manuscript then probably a wider population could be targeted.

Ad. As stated above, we are working now on providing more replicates of biochemical assays and we are ready to perform further biological validation if the editors find it necessary. I would like to stress that genome editing by knock-in is not always possible/feasible, and these type of experiments is time and money consuming.

• 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. Brain tumors, immunotherapy, cancer stem cells, tumor microenvironment, tumor heterogeneity. I do not have sufficient expertise to evaluate the bioinformatic analysis and software/programs used to analyze the NGS data.

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

By exon targeted resequencing of 664 genes frequently mutated in cancer, authors identify novel mutations associated to Glioma in a cohort of 182 Polish and Canadian samples. Most of these novel mutations have been identified as potential rare germline mutations, somatic mosaicism or loss-of-heterozygosity variants. Among them, authors focus on mutations associated to the TOP2A gene, which encodes one of the two Type II topoisomerases paralogs present in humans. By a limited number of in vitro experiments, authors conclude that TOP2A recurrent variant E948Q, displays increased binding to DNA and topoisomerase activity. Therefore, authors suggest that the TOP2A E948Q variant is a gain-of-function mutation.

**Major comments:**

• Authors show an interesting plethora of new exon mutations associated with High Grade Glioma. Nevertheless, the characterization of TOP2A E948Q variant, which is the main focus of the study, although very interesting and potentially clinically relevant, remains incomplete. Association of the TOP2A E948Q glioma variant with a gain-of-function mutation would require to improve the statistical power of the presented experiments (increase number of replicates). With the existing experimental evidence, the increased DNA binding and activity of the TOP2A E948Q variant should be considered as preliminary, especially in the case of 431-1193 aa fragment. I would consider mandatory to increase experimental replicates and to analyse statistical significance in the case of DNA binding experiments and DNA relaxation assays with the TOP2A 431-1193 aa fragment. A more detailed biochemical characterization should be performed. A titration of different amounts of protein should be included in these experiments, and at least two batches of purified proteins should be analysed. Decatenation assays should also be performed to characterize the activity of the mutant protein in more detail. Recapitulation of DNA binding and activity results with other TOP2A variants obtained in this study will significantly reinforce authors claims too. This improved biochemical characterization should not take longer than two months.

Ad. We would like to stress that while two replicates are presented, we were testing two forms of TOP2A proteins and the results were similar, confirming our conclusions. But we agree that additional replicates would strengthen our claims. Therefore, we are in the process of producing another batch of recombinant proteins to increase a number of replicates and calculate statistics for the biochemical assays (binding and relaxation assay). We will perform titration of different amounts of the protein using two batches of purified proteins.

The occurrence of other TOP2A variants is low (identified in only a single patient sample), therefore we will perform experimental validation only for E948Q. However, we performed additional computational analysis for other TOP2A variants showing the influence of the substitution on DNA binding by docking the DNA fragment into TOP2A binding pocket (Supplementary table 4).

• To increase the significance of the results, I would encourage authors to include experiments showing the functional impact of this TOP2A mutation in cells. The connection with transcriptomic alterations is merely correlative, and would be greatly strengthened by functional experiments in cellular models. To draw definitive conclusions regarding the changes in transcription, I would encourage authors to complement the results with experiments that point to the physiological impact of TOP2A variants within the cell. Overexpression of WT and E948Q variants in a cell model and transcriptomic analysis would be desirable, but validation in these experimental models of some of the target genes identified as deregulated in patients could suffice. These experiments could be accomplished in no more than 3-4 months.

Ad. We agree that the connection of the TOP2A mutation with transcriptomic alterations is correlative, and would be greatly strengthened by functional experiments in cellular models. If we develop a TOP2A E948Q knock-in cell line or TOP2A KO cell line with E948Q over-expression, we are planning to evaluate transcriptomic changes on selected genes by qPCR or whole transcriptome by RNAseq. We estimate that developing a stable CRISPR/Cas9 cell line may take up to 6 months.

We provided additional results showing that the connection of the TOP2A mutation with transcriptomic alterations may be due to different expression of splicing factors (Supplementary Fig. 6).

• Some of the methods are not presented with sufficient detail. Regarding the DNA and RNA sequencing experiments, I consider necessary to specify the DNA fragmentation method, reference for the indexed adapters and ligation and amplification procedures (ligase reference, number of PCR cycles, etc). It would be helpful to clarify or reference which are the "special oligonucleotide probes" that are mentioned. Finally, a reference for the "special beads" and final amplification number of cycles is needed. The sequence of primers used for TOP2A cloning and mutagenesis should be included. The reference for the "site mutagenesis kit" used is missing. When studying the survival rate of glioma patients depending of TOP2A expression levels, it should be clarified what is considered HIGH or LOW expression (i.e: which percentiles are used).

Ad. We expanded the description of methodological aspects of DNA and RNA sequencing experiments. This description was revised and more details are provided in the revised version. Regarding cloning and mutagenesis, we added a table with primer sequences (Supplementary Table 5). We did not use any kit for cloning and mutagenesis. Standard methods and primers with modified nucleotides were used.

__We have included information about the partitioned groups in the survival analyses in the figure 2 caption. “D - Kaplan-Meier overall survival curve for patients with high (> TOP2A mRNA median expression x 1.25) or low (- There is a major concern about how the experiments are replicated and about the statistical analysis, which is inexistent in some cases. Indeed, Figures 4 and 5 do not present any statistical analysis, it is therefore hard to draw any conclusion. In Figure 4b, the results for the 890-996 aa fragment looks qualitatively clear, but this is not the case for the 431-1193 aa fragment. More replicates and statistical analysis are mandatory, together with a protein titration. The replicates should be performed with at least two independent batches of protein purifications. The individual values of each experiment should be included in the graph to provide a better understanding of experimental variability. All this also applies to Figure 5.

Ad. We will increase a number of replicates for the binding and relaxation assay. We will perform a titration of different amounts of protein in these experiments using two batches of purified proteins.

**Minor comments:**

• The effect on transcription of co-occurrence of TOP2A mutations with other mutations could also be analysed with the already available data. Also, a more detailed analysis of genome-wide transcription could also be used to at least partially address the proposed hypotheses of increased transcriptional rate or splicing aberrations.

Ad. We don’t have enough samples with the TOP2A mutation to analyze the effect on transcription of co-occurrence of TOP2A with other mutations.

We addressed the hypothesis of increased transcriptional rate or splicing aberrations by performed additional analyses of RNA-seq data to confirm splicing aberrations. Indeed we found splicing machinery genes down-regulated in the E948Q TOP2A glioma samples (Supplementary Fig.6).

• There is no reference for the following argument "As the identified germline variants were exceptionally rare in the general population ... it is likely that these variants are pathogenic". I also find low number of references to support the suggested high frequency of altered genes in gliomas compare to other cancer types. I miss specific works relating TOP2 activity with transcriptional regulation.

Ad. The appropriate references are provided to back-up these statements.

• At several points in the text there are quantitative and comparative statements that should be backed up by the actual numbers (e.g. "The results of the targeted sequencing indicate a high frequency of altered genes", "The most altered gene was TP53, followed by IDH1...", "Other genes that were found to be frequently altered included KDM6B...", "These partial results combined with a low frequency of this variant in the Polish population suggest a somatic mutation"). The same thing applies to the co-occurrence of mutations, in which the percentage of co-occurrence and significance is not indicated. This lack of detail in the description is also observed in the description of the transcriptomic alterations in which no detail is provided regarding how many of the 105 analyzed samples correspond to low or high gliomas.

Ad. We apologize that the frequencies of mutated genes were not specified. This information is included in the main text of the revised version. We now provide a gnomAD frequency for all variants of interest, confirming the low frequency in the population (AF__ __

Regarding the total number of samples in the transcriptomic analysis, we provided an updated supplementary table covering also samples that were used for transcriptomic analyses (Supplementary Table 1).

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• For TOP2A mutation analysis, sometimes is not clear when the analysis is done with the 9 mutated samples and when with the 4 recurrent TOP2A E948Q variants. For example, in figure 2b and 2c analysis are done with 9 samples while the figure 2e is based on the 4 E948Q variants. At least this is what I have deduced from the main text, it should be clarified in the figure legend).

Ad. This information has been included in the captions of Figure 2B, 2C and 2E and now we specify how many samples were used in each analysis.

• Fig1. In figure 1b it would be interesting to color-code patients by glioma grade. This would also apply to Figure S1a, S1c, 2a, S3 and S4. In figure 1D it would be very informative to distinguish mutations that passed the quality control or not with different colors.

Ad. Following reviewer’s suggestions, we have added this information, and oncoplot figures derived from the germline analysis have a distinct color for each glioma grade. In the figure 1D, all of the presented mutations have passed a quality control in terms of quality of sequencing. One additional criterion that was used for all genomic results (except some of the TOP2A variants) was a criterion of 20% variant penetration (20% of reads in the position had to come from the alternative allele). We corrected the description in the Supplementary Table to “passed 20% penetration criterion”. The rationale behind this criterion for TOP2A variants was a fact that for one of the E948Q samples it was ~13% and we didn’t want to lose this sample from the analysis due to rarity of the mutation.

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• Fig2. In figure 2b and 2c the statistical significance of differences between TOP2A and the rest of genotypes should be included. Looking at Figures 2d and 2e it looks surprising how similar is the overall survival of HIGH TOP2A mRNA expression (500 days, fig 2d) with the overall survival of the TOP2A WT samples (400 days, fig 2e). Here a I would include a graph that summarizes the TOP2A mRNA expression levels of each group in fig 2d and 2e.

Ad. We agree that median overall survival is similar comparing patients with high TOP2A mRNA expression to TOP2A WT patients in our cohort. It is worth noting, however, that both datasets were produced using different library protocols, and the methodology is different, so it can’t be expected the levels to be equal. We think that adding two more graphs, as suggested, would add another layer of information to this section of the analysis. We have included two boxplots depicting TOP2A mRNA RPKMs, and it is clear now that the medians of High TOP2 mRNA and TOP2A mutant (E948Q) are more closely related, despite the fact that we only have a few patients with the mutation.

• Fig3. It would be interesting to include the same simulation for the rest of TOP2A mutations as supplementary figure.

Ad. We agree that the other TOP2A SNPs could potentially affect DNA binding. We focused on the recurrent mutation and did not analyze those occurring in a single patient. In the revised version we included predictions whether these variants could affect TOP2A DNA binding. For WT TOP2A and variants, we calculated the Gibbs free energy (ΔG). This information can be found in Supplementary Table 4. We have extended description in the Results section: “The TOP2A E948Q substitution may affect protein-DNA interactions”

• Fig4 and Fig5. Include statistical analysis and dots representing individual replicates.

Ad. For Fig 4 we have two replicates for two protein fragments, so we can’t present statistics now. As mentioned above we are preparing a new batch of proteins and will make more repetitions of EMSA and relaxation assays. For Fig 5. we have 3 replicates but despite a trend there is no statistical significance. We intent to make more replicates and a separate protein preparation. After including additional repetitions we will present the results as dots representing individual replicates.

• Fig6. In Figure 6d I would increase the size differences in the dots representing the gene counts, as it is not easily perceived with current parameters.

Ad. The dot size in Fig 6d did not reflect the true meaning. To make it easier to understand, we changed a plot type to a barplot, which now represents the number of differentially expressed genes involved in each pathway.

• FigS2. In figure S2B, it would be informative to establish which dots are significatively above or below the diagonal.

Ad. The purpose of this figure was to show which oncogenic signaling pathways from TCGA cohorts were affected in our cohort. The pathway's size is a variable that is used to normalize the calculation (shown in abscissa axis in S2B). RTK-RAS and NOTCH pathways contain hundreds of genes, whereas other pathways, such as the NRF2 oncogenic pathway, contains only a few. On the other hand, we counted how many genes in each pathway in our cohort were mutated (shown in ordinate axis, S2B). We used logarithms in both axes for visualization purposes, but this has no effect on the enrichment of these pathways, which is shown in the color-coded legend.

• FigS3. How were the samples shown selected from the total?

Ad. In this plot we show only somatic variants that were found in at least two different patients. We apologize that this information was missing, and we have added it to the figure's caption.

• FigS4. I would include a line with the TOP2A mutation to have an idea of how these mutations are distributed between groups.

Ad. Based on the feedback of the reviewer, this figure has been modified and improved. A new row has been added to the figure, displaying TOP2A mutations alongside other highly frequent mutations in other genes.

Reviewer #2 (Significance (Required)):

In this work authors have identified new mutations associated to gliomas by targeted exome sequencing using an important cohort of 182 samples. Among these new mutations epigenetic enzymes and modifiers are found. These results potentially increase the repertoire of putative molecular targets for future cancer therapies. Authors focus in mutations associated to TOP2A gene, that provides stronger DNA binding and DNA relaxation capacity in vitro. Although further characterization is needed, tumours harbouring this kind of mutations could show higher level of sensitivity to TOP2 drugs, providing potentially interesting clinical implications. Although the link between TOP2A expression and cancer prognosis is well established, the relevance of specific mutations in still largely unexplored.

On one hand this work brings novelties in the field of Glioma providing a series of putative new players in the development of this type of cancer. Audience interested in basic or clinical aspects of these tumours would be a good target for this work. On the other hand, this putative gain-of-function mutation of TOP2A represent an interesting aspect for the DNA topology and topoisomerases field. Although, as stated above a more detailed biochemical and functional characterization would be required to draw the attention of this audience-

Scientifically, I have experience in the DNA topology and topoisomerases field, 3D genome organization and gene regulation. I have no experience in Gliomas or any other clinical aspect of cancer, so it is difficult for me to properly establish the potential impact of the newly discovered mutations. Technically I have no capacity to critically evaluate the aspects related to the targeted exome sequencing and the suitability of the analysis performed for mutation identification.

**Referee Cross-commenting**

I fully agree with the comments of the other reviewer, which are perfectly aligned with my own regarding the preliminary nature of the conclusions about the biochemical and functional characterization of the TOP2A mutations.

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Referee #2

Evidence, reproducibility and clarity

By exon targeted resequencing of 664 genes frequently mutated in cancer, authors identify novel mutations associated to Glioma in a cohort of 182 Polish and Canadian samples. Most of these novel mutations have been identified as potential rare germline mutations, somatic mosaicism or loss-of-heterozygosity variants. Among them, authors focus on mutations associated to the TOP2A gene, which encodes one of the two Type II topoisomerases paralogs present in humans. By a limited number of in vitro experiments, authors conclude that TOP2A recurrent variant E948Q, displays increased binding to DNA and topoisomerase activity. Therefore, authors suggest that the TOP2A E948Q variant is a gain-of-function mutation.

Major comments:

• Authors show an interesting plethora of new exon mutations associated with High Grade Glioma. Nevertheless, the characterization of TOP2A E948Q variant, which is the main focus of the study, although very interesting and potentially clinically relevant, remains incomplete. Association of the TOP2A E948Q glioma variant with a gain-of-function mutation would require to improve the statistical power of the presented experiments (increase number of replicates). With the existing experimental evidence, the increased DNA binding and activity of the TOP2A E948Q variant should be considered as preliminary, especially in the case of 431-1193 aa fragment. I would consider mandatory to increase experimental replicates and to analyse statistical significance in the case of DNA binding experiments and DNA relaxation assays with the TOP2A 431-1193 aa fragment. A more detailed biochemical characterization should be performed. A titration of different amounts of protein should be included in these experiments, and at least two batches of purified proteins should be analysed. Decatenation assays should also be performed to characterize the activity of the mutant protein in more detail. Recapitulation of DNA binding and activity results with other TOP2A variants obtained in this study will significantly reinforce authors claims too. This improved biochemical characterization should not take longer than two months.
• To increase the significance of the results, I would encourage authors to include experiments showing the functional impact of this TOP2A mutation in cells. The connection with transcriptomic alterations is merely correlative, and would be greatly strengthened by functional experiments in cellular models. To draw definitive conclusions regarding the changes in transcription, I would encourage authors to complement the results with experiments that point to the physiological impact of TOP2A variants within the cell. Overexpression of WT and E948Q variants in a cell model and transcriptomic analysis would be desirable, but validation in these experimental models of some of the target genes identified as deregulated in patients could suffice. These experiments could be accomplished in no more than 3-4 months.
• Some of the methods are not presented with sufficient detail. Regarding the DNA and RNA sequencing experiments, I consider necessary to specify the DNA fragmentation method, reference for the indexed adapters and ligation and amplification procedures (ligase reference, number of PCR cycles, etc). It would be helpful to clarify or reference which are the "special oligonucleotide probes" that are mentioned. Finally, a reference for the "special beads" and final amplification number of cycles is needed. The sequence of primers used for TOP2A cloning and mutagenesis should be included. The reference for the "site mutagenesis kit" used is missing. When studying the survival rate of glioma patients depending of TOP2A expression levels, it should be clarified what is considered HIGH or LOW expression (i.e: which percentiles are used).
• There is a major concern about how the experiments are replicated and about the statistical analysis, which is inexistent in some cases. Indeed, Figures 4 and 5 do not present any statistical analysis, it is therefore hard to draw any conclusion. In Figure 4b, the results for the 890-996 aa fragment looks qualitatively clear, but this is not the case for the 431-1193 aa fragment. More replicates and statistical analysis are mandatory, together with a protein titration. The replicates should be performed with at least two independent batches of protein purifications. The individual values of each experiment should be included in the graph to provide a better understanding of experimental variability. All this also applies to Figure 5.

Minor comments:

• The effect on transcription of co-occurrence of TOP2A mutations with other mutations could also be analysed with the already available data. Also, a more detailed analysis of genome-wide transcription could also be used to at least partially address the proposed hypotheses of increased transcriptional rate or splicing aberrations.
• There is no reference for the following argument "As the identified germline variants were exceptionally rare in the general population ... it is likely that these variants are pathogenic". I also find low number of references to support the suggested high frequency of altered genes in gliomas compare to other cancer types. I miss specific works relating TOP2 activity with transcriptional regulation.
• At several points in the text there are quantitative and comparative statements that should be backed up by the actual numbers (e.g. "The results of the targeted sequencing indicate a high frequency of altered genes", "The most altered gene was TP53, followed by IDH1...", "Other genes that were found to be frequently altered included KDM6B...", "These partial results combined with a low frequency of this variant in the Polish population suggest a somatic mutation"). The same thing applies to the co-occurrence of mutations, in which the percentage of co-occurrence and significance is not indicated. This lack of detail in the description is also observed in the description of the transcriptomic alterations in which no detail is provided regarding how many of the 105 analyzed samples correspond to low or high gliomas.
• For TOP2A mutation analysis, sometimes is not clear when the analysis is done with the 9 mutated samples and when with the 4 recurrent TOP2A E948Q variants. For example, in figure 2b and 2c analysis are done with 9 samples while the figure 2e is based on the 4 E948Q variants. At least this is what I have deduced from the main text, it should be clarified in the figure legend).
• Fig1. In figure 1b it would be interesting to color-code patients by glioma grade. This would also apply to Figure S1a, S1c, 2a, S3 and S4. In figure 1D it would be very informative to distinguish mutations that passed the quality control or not with different colors.
• Fig2. In figure 2b and 2c the statistical significance of differences between TOP2A and the rest of genotypes should be included. Looking at Figures 2d and 2e it looks surprising how similar is the overall survival of HIGH TOP2A mRNA expression (500 days, fig 2d) with the overall survival of the TOP2A WT samples (400 days, fig 2e). Here a I would include a graph that summarizes the TOP2A mRNA expression levels of each group in fig 2d and 2e.
• Fig3. It would be interesting to include the same simulation for the rest of TOP2A mutations as supplementary figure.
• Fig4 and Fig5. Include statistical analysis and dots representing individual replicates.
• Fig6. In Figure 6d I would increase the size differences in the dots representing the gene counts, as it is not easily perceived with current parameters.
• FigS2. In figure S2B, it would be informative to establish which dots are significatively above or below the diagonal.
• FigS3. How were the samples shown selected from the total?
• FigS4. I would include a line with the TOP2A mutation to have an idea of how these mutations are distributed between groups.

Significance

In this work authors have identified new mutations associated to gliomas by targeted exome sequencing using an important cohort of 182 samples. Among these new mutations epigenetic enzymes and modifiers are found. These results potentially increase the repertoire of putative molecular targets for future cancer therapies. Authors focus in mutations associated to TOP2A gene, that provides stronger DNA binding and DNA relaxation capacity in vitro. Although further characterization is needed, tumours harbouring this kind of mutations could show higher level of sensitivity to TOP2 drugs, providing potentially interesting clinical implications. Although the link between TOP2A expression and cancer prognosis is well established, the relevance of specific mutations in still largely unexplored.

On one hand this work brings novelties in the field of Glioma providing a series of putative new players in the development of this type of cancer. Audience interested in basic or clinical aspects of these tumours would be a good target for this work. On the other hand, this putative gain-of-function mutation of TOP2A represent an interesting aspect for the DNA topology and topoisomerases field. Although, as stated above a more detailed biochemical and functional characterization would be required to draw the attention of this audience-

Scientifically, I have experience in the DNA topology and topoisomerases field, 3D genome organization and gene regulation. I have no experience in Gliomas or any other clinical aspect of cancer, so it is difficult for me to properly establish the potential impact of the newly discovered mutations. Technically I have no capacity to critically evaluate the aspects related to the targeted exome sequencing and the suitability of the analysis performed for mutation identification.

Referee Cross-commenting

I fully agree with the comments of the other reviewer, which are perfectly aligned with my own regarding the preliminary nature of the conclusions about the biochemical and functional characterization of the TOP2A mutations.

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 #1

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2. In this paper the authors used a targeted approach to identify rare mutations in a cohort of glioma patients. Using this approach they identified a recurrent mutation in the TOP2A gene encoding for Topoisomerase 2A, and suggest that this mutation creates a more effective protein, binding DNA strongly and maybe more enzymatically active. RNAseq analysis of TOP2Awt and TOP2Amut tumor samples suggest different transcription patterns and points to possible splicing defects. The most recurrent variant (E9448Q) is described in depth and some experimental information shows this variant might be a gain-of-function mutation.

Major comments:

• Are the key conclusions convincing? The validation of both the methodology and the presence of never described TOP2A variations in HGG is done quite successfully. Interesting evidence about relevance of the most frequent mutation is provided. However, besides having computational and biochemistry assays performed, lack of details about in vitro experiment statistics (no p-values are provided in figures 4 and 5, neither sample size, repetitions) weakens the conclusions claimed by the authors about the properties of the mutated topoisomerase.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Claims about E948Q variant function should be revised. Data is not presented in a convincing way, plus there is ambiguous language used from the results ("We conclude that the E9448Q TOP2A protein is functional, and MIGHT have a higher activity than the WT protein") to the rest of the paper where they strongly support the claims about the TOP2A activity.

• 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. In line with the presented data in the paper, additional experiments that show catalytic changes of the E9448Q variation must be added. It is shown that there are differences in the DNA binding capacity by EMSA compared to the WT form, however, the DNA supercoil relaxation activities is not that different, at least the way the results are presented. The authors suggest that TOP2A mutation is a driver mutation but no validation in vitro of this claim is shown. Can this mutation alone or in combination with e.g. tumor suppressors transform normal cells to cancer cells? Do cell lines expressing this mutation (compared to parental TOP2A wt expressing cells) display increased transcription? Increased invasion?

• 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. If the authors can complement the already presented in vitro experiments with additional ones supporting their hypothesis, this should be feasible. The authors can use patient derived glioma cells or glioma cell lines manipulated to express either the parental TOP2A wt enzyme or the identified mutated form.

• Are the data and the methods presented in such a way that they can be reproduced? Yes, the authors provide a quite detailed explanation of the methods implemented to reach each one of the results they are presenting.

• Are the experiments adequately replicated and statistical analysis adequate? No, there is no information about the statistical analysis or number of replicates in any of the in vitro experiments performed. This information should be added to the manuscript.

Minor comments:

• Specific experimental issues that are easily addressable.

• Are prior studies referenced appropriately? Yes, authors clearly address the state of the art regarding previous NGS methodologies and let us know the advantages and novelty of their approach.

• Are the text and figures clear and accurate? There are some discrepancies between the strength of the language used in different sections of the paper to refer the conclusions they can infer from the results they are showing. While they are all valid, authors should revise it.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? First of all, describe the statistical analysis used in every figure, include number of biological and technical replicates. I would also suggest to change the title or the scope of the discussion, there is too much focus on the TOP2A in the introduction, neglecting all the technical NGS work that actually lead to several new variants being described. This may be confusing when it collides with a conclusion that is heavily focused on the first half describing potential implications of at least another 3 proteins where genetic alterations were described. Given the fact there is not much experimental work that shows TOP2A mutations relevance in HGG or strong enough evidence of the variant's function I would suggest to change a bit the scope of the title.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. The authors describe a methodology that proved to be sensitive and specific enough in order to allow them to detect rare genetic alterations in patient glioma samples. This information could be valuable to describe new driver mutations or infer in genetic pathway alterations that could be potential therapeutic targets. As the authors state at the beginning of the paper, given the poor therapeutical approaches existing for HGG currently, information of this kind could still be highly useful and provide a better outcome to a specific cohort of patients.

On a personal note, I think there is too much speculation about how TOP2A mutations could be interesting from a biological point of view (authors referred to evidence about implications of this mutation in other forms of cancer) but since no experimental validation is provided in glioma cells, it is difficult to conclude that this enzyme gain-of-function mutation could have a relevant role in HGG and thus make these variants a potential therapeutic target. There are no experiments conducted in glioma cells that express TOP2A variants, it would be interesting to see if it has an effect in the migratory/invasive phenotype like described in other cancer types or like it is suggested by analysis of the genetic pathways activated in the HGG patients samples harboring TOP2A mutation. In addition, there is no evidence of the TOP2A mutations possible role as a driver mutation, which is an interesting aspect that could be further explored from both a computational and an experimental approach.

• Place the work in the context of the existing literature (provide references, where appropriate). The quality of the paper is high and in line with other studies in the literature that perform genome and transcriptome analysis of tumor samples. It is only the experimental validation that is lacking data supporting the "in silico" findings.

• State what audience might be interested in and influenced by the reported findings. Computational biologists are the right audience to target this paper. If additional experimental work further validating their initial bioinformatic findings is added to the manuscript then probably a wider population could be targeted.

• 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. Brain tumors, immunotherapy, cancer stem cells, tumor microenvironment, tumor heterogeneity. I do not have sufficient expertise to evaluate the bioinformatic analysis and software/programs used to analyze the NGS data.

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Reply to the reviewers

We are grateful for the constructive and highly supportive reviews provided by our Reviewers. We especially appreciate the efforts they have made to provide suggestions on how to make our revised manuscript even more robust. We have incorporated many of these suggestions into the revised manuscript that will post to Biorxiv and will be submitted to an affiliate journal. We have provided point-by-point responses to each Reviewer below each item (starting with Response: …), along with any changes made in response to that comment/suggestion (starting with In our revised manuscript, …).

Finally, we agree with all Reviewers that this work should be of broad interest to the molecular biology, cell biology, and parasitology communities. Our discovery that Plasmodium and two related genera have taken the unorthodox approach of duplicating their NOT1 protein, and that Plasmodium has dedicated it for its unique transmission strategy, is a fascinating adaptation of the use of this core eukaryotic complex. We believe that those that focus on diverse aspects of RNA biology, including RNA preservation/decay, the maternal to zygotic transition, translational repression, and beyond will find this work to be of interest and relevant to their own research questions.

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

The manuscript „The Plasmodium NOT1-G paralogue acts as an essential nexus for sexual stage maturation and parasite transmission" investigates the two forms of NOT1 in rodent malaria parasites. The authors found out that the original NOT1 is crucial for gametocyte induction as well as transmission to the mosquito, they therefore renamed it NOT1-G. The paralogous proteins, on the other hand, appears to be crucial for intraerythrocytic growth, since it cannot be knocked out. The authors then investigated NOT1-G in more detail, using standard phenotyping assays. They found a slightly increased gametocytemia and a minor effect on transmission to the mosquito.

Response: In our submitted manuscript, we do focus on PyNOT1-G because of the exciting role it has for both sexes of gametocytes, which results in a complete defect in transmission to mosquitoes. Our investigations of what domains of PyNOT1-G focused on the most likely suspect: the putative tristetraprolin-binding domain (TTPbd). It was through deletion of this domain that we observed only a minor defect in the prevalence of infection of mosquitoes, indicating that the portion of PyNOT1-G that is required for transmission lies elsewhere (in part or in total). It is also important to correct Reviewer 1’s statement regarding the other (perhaps canonical) PyNOT1. To our surprise, PyNOT1 could be deleted, but resulted in a parasite that has an extreme fitness cost and a very slow growth phenotype. This is in stark contrast to other eukaryotes, where NOT1 is essential.

Reviewer #1 (Significance (Required)):

If the authors are able to provide convincing data that NOT1-G is indeed important for gametocyte induction and transmission to the mosquito, then the report would be of high significance for the malaria and molecular cell biology fields.

Response**: We have in fact shown this and more in the originally submitted manuscript, and thus we are grateful that Reviewer 1 considers this work to be of high significance in a broad readership (molecular and cell biology, parasitology). In our revised manuscript, we have added text throughout to make these results even more apparent and clear for the reader.

My expertise: molecular cell biology of gametocytes, translational regulation, parasite transmission

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

**Summary**

The manuscript by Hart et al. builds upon a fascinating finding presented in a previous manuscript by the same authors, in which they show that CCR4 seems to be able to associate with two members of the NOT1 family. In this work, the authors first re-annotate the two NOT1 paralogs in Plasmodium yoelii and then perform an in depth characterization of the role of NOT1-G during gametocytogenesis and early mosquito development. Using gene knockout and different genetic crosses, the authors show that NOT1-G is essential for male gametocyte development and leads to an arrest of development in zygotes arising from female gametocytes. Using RNA-seq the authors show that NOT1-G leads to lower transcript abundances, leading to the hypothesis that NOT1-G might be involved in preserving mRNAs in a larger RNA-binding complex. Lastly, the authors characterize a NOT1-G defining TPP domain and find that it is not essential for either male/female phenotype observed for the whole gene KO.

Response**: We appreciate the concise and accurate summary of these findings.

**Major comments:**

• Are the key conclusions convincing?

  The phenotypic characterization of NOT1-G during gametocytogenesis / early mosquito development is nicely presented and the experiments are well performed. Because a duplication of NOT1 with possibly opposing roles of the paralogs is a very unique feature with broad implication on RNA metabolism, it would have been great to see two select experiments on the molecular level adding evidence that 1) NOT1/NOT1-G are mutually exclusive in a complex with CCR4/CAF1 and 2) NOT1-G acts post-transcriptionally in an antagonistic way to NOT1 (i.e. as a mRNA 'stabilizer' as proposed by the authors).

Response**: We agree that inclusion of those two aspects would make for a more complete story about these two NOT1 paralogues.

First, we also think that it is highly likely that NOT1 and NOT1-G are mutually exclusive, as in other eukaryotes NOT1 acts as a scaffold protein upon which effector proteins bind and bridging interactions are made. In our original manuscript, we did not include a mention of our previous attempts to address this question through colocalization and proteomic approaches, as they were largely unsuccessful. Specifically, we generated rabbit polyclonal antisera to PyNOT1-G’s tristetraprolin-binding domain but it did not pass our rigorous quality control (e.g. too much staining persisted in pynot1-g- parasites). Using both asexual and sexual blood stage parasites, we also attempted immunoprecipitation (with and without chemical crosslinking) and proximal labeling approaches via BioID and TurboID but all approaches did not produce rigorous results and thus we did not report them in our original manuscript. However, this question of whether the two NOT1 paralogues were mutually exclusive in complexes was also taken up by the Bozdech Laboratory in their 2020 preprint (Liu et al.) where they were able to capture the P. falciparum NOT1-G and NOT1 proteins (called Not1.1 and Not1.2 in that work). While their proteomic evidence showed that they could capture these bait proteins and that the NOT1 paralogues were not in the same complex, these results should be taken with a grain of salt: all mass spectrometry-based proteomic approaches are limited in that an absence of evidence does not mean that the protein is not present/interacting. Moreover, these efforts only identified a few other proteins that were already known to interact with the CAF1/CCR4/NOT complex, but even so, they did not use statistically rigorous methods in an attempt to quantify these results. In our revised our manuscript, we have included additional text to describe our unsuccessful efforts to do these capture proteomics experiments, and we have expanded our discussion of the Liu et al findings that provide some evidence in support of a mutually exclusive complex.

Second, we also hypothesize that PyNOT1-G acts post-transcriptionally to affect mRNA abundance and translation. However, it is important to emphasize that NOT1 proteins typically act as scaffolds, with the recruited effector proteins acting to hasten the degradation and/or to preserve associated transcripts. We believe that studying these effector proteins is the next important effort to undertake. In fact, we hypothesized that these antagonistic effector proteins would be analogous to TTP and ELAV/HuR-family proteins as are found in other eukaryotes, and that the critical interaction with PyNOT1-G would be via its putative TTP-binding domain. It was for that reason that we interrogated the TTP-binding domain itself, and were surprised that its deletion did not phenocopy the complete gene deletion. Ongoing work will be focused on identifying these antagonistic effector proteins that likely are expressed in a stage-enriched manner, and to define how they interact with PyNOT1-G in order to direct specific mRNAs to their fates. Additionally, it would be very important and exciting to directly test if PyNOT1 and PyNOT1-G are functionally opposed. However, this would be exceptionally challenging to study from a technical standpoint. While we were able to delete the pynot1 gene after many repeated attempts, these parasites are very sickly and grow very slowly. Because of this, we believe that assessing direct versus indirect effects of PyNOT1 in these cells would not be feasible or robust. Given this, comparing functions between PyNOT1 and PyNOT1-G could not be done in a conclusive manner.** In our revised manuscript, we have expanded our descriptions of the mechanisms by which we believe PyNOT1-G and its complex affects mRNA fates. In particular, we have expanded our Discussion section to incorporate the results that indicate that the TTP-binding domain is not required for the essential functions of PyNOT1-G.

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

  The authors describe the role of NOT1-G as 'preserving' mRNA. The lower abundance of many transcripts in the NOT1-G knockout suggest this, but experimental proof is not provided (see suggestions below). Maybe rephrase to 'putatively preserved/stabilized' or 'has a potentially stabilizing function'. The same is true for the mutually exclusive association of the two paralogs with CCR4/CAF1. The authors refer to a protein co-IP of CCR4 showing that CCR4 can interact with both NOT1 and NOT1-G, but a reciprocal experiment is lacking.

Response**: In our first publication on the deadenylase members of this complex, we also saw a similar effect on specific mRNAs when pyccr4-1 was deleted: the abundance of specific mRNAs went up in pyccr4-1- parasites. In that work and here in this manuscript, we have carefully decided to apply the word “preserved” to the fate of these mRNAs as it describes in a general way what is happening. In order to robustly state that mRNAs are stabilized by PyNOT1-G (directly or indirectly) would require additional experiments designed to test this (more description on this is provided on a response below). Second, as described above, we agree that doing a reciprocal IP for mass spectrometry-based proteomics would be ideal, we attempted four different approaches to do this to no avail. However, the composite proteomics data that is already available in the literature and via the Liu et al. preprint from the Bozdech Lab all indicate that these interactions occur, and perhaps that NOT1 and NOT1-G are mutually exclusive as expected. In our revised manuscript, we have provided further explanation in the Discussion for our use of the descriptor “preserve” instead of “stabilize”, and as noted above, and we have expanded our Discussion to more comprehensively define the interaction network depicted in Figure 7.

In both cases, the conclusions of the authors are very likely (e.g. downregulation of many genes as seen by RNA-seq), but the final experimental evidence is not provided and a network such as in Figure 7 is not fully supported. If the authors would like to maintain these statements, then they should be rephrased and made clear or the additional experimental evidence suggested below is necessary.

Response**: We hold that the published proteomic datasets do support such a network, with further support offered from the preliminary proteomic evidence from the Liu et al preprint. Therefore, we have not modified our manuscript beyond the additional text now provided in the Discussion as noted above.

• 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.

  The essential claim that NOT1-G is important for gametocytogenesis and early mosquito development is well presented and fully supported by the experiments. As for the role of NOT1-G in 'preserving' mRNA, an mRNA half-life experiment would be necessary (or the text should be adjusted as mentioned above). In a short-term in vitro culture, pynot1-g- and WT parasites could be treated with ActD and abundances of select transcripts are measured by RT-qPCR.

Response**: We appreciate that Reviewer 2 considers the rigor of our experiments to be high. Regarding the use of the term “preserve” vs “stabilize”, we agree that to shift from our more general descriptor (preserve) to one that has specific connotations (stabilize) would require additional experimentation. To correctly and most robustly make the claim of stabilization would require work on par with that done by Painter et al. (PMID: 29985403) that uses a thiol-containing nucleotide (4-TU) along with a yeast-derived fusion enzyme (yFCU) to convert it for use by Plasmodium. Previously we have shown that an associated deadenylase (PyCCR4-1) also acted to preserve mRNAs, and moreover that deletion of its gene resulted in no discernable effect upon the poly(A) tail or 3’ UTR of an mRNA that is bound by this complex (p28).

While understanding mRNA stability is an exciting area of study, this 4-TU labeling experiment alone warranted a standalone, high impact publication for Painter et al. As this has not been adapted for any rodent-infectious Plasmodium species to date, and as adaptation of this labeling approach took several years for Dr. Painter while in the Llinas Laboratory (personal communication), we believe this work is beyond the scope of this study. Moreover, the additional information that it would provide to understand NOT1-g functions (preserve vs stabilize) would be incremental beyond the major storyline presented in this manuscript. In our revised manuscript, we have added text to ensure that our choice of “preserve” is well defined and explained.

To support the idea that NOT-1 and NOT1-G associate in a mutually exclusive way or to just show that they act in distinct complexes despite their similar expression patterns, an IFA with a double stained NOT1/NOT-1G cell line could be performed. Alternatively, the authors could perform a protein co-IP using the already existing NOT1/NOT1-G-GFP cell line and show that the proteins don't interact with each other or even have certain distinct interaction partners.

Response**: We agree, and these studies were attempted but were unsuccessful (described in our responses above). In our revised manuscript, we have included this information as noted above.

• 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.

  All necessary cell lines for a NOT1/NOT1-G co-IP and the ActD experiment are already present. The authors already present a ring to schizont in vitro culture (for ActD) and also have substantial experience in protein co-IP and proteomics.

  I am not sure about the cost for a proteomics experiment at the author's institute and I don't want to make a guess on time investment given the still on-going COVID situation.

Response**: We agree that these experiments would be interesting, and would be costly to do at a transcriptome-wide scale and would require substantial time to conduct. We believe that the 4-TU approach noted above is the most rigorous, but is well beyond the scope of this study as it has not yet been adapted to rodent-infectious malaria parasites. As noted above, we have attempted four different proteomics approaches to provide reciprocal evidence for the complex composition which were unsuccessful. In our revised manuscript, we have added text to ensure that our choice of “preserve” is well defined and explained, and have noted the unsuccessful reciprocal proteomics approaches.

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

  The MM section is well structured and presented and the supplemental material includes all data.

Response**: Thank you. We want to ensure that our work is clearly described and can be reproduced with the information reported.

• Are the experiments adequately replicated and statistical analysis adequate?

  There is hardly any test of significance presented in the main text of the manuscript (e.g. Figure 3B and 4A). Please show the individual data points for these graphs and make sure the n= and the statistical test is described in the figure legend. If you use the term significant in the text, then just add the p-value behind it. This is also true for the RNA-seq data: Genes are sorted by fold-changes, leaving it unclear if these changes are significant. These data are however presented in Table S1 and could be incorporated in the main text.

Response**: We agree. In our revised manuscript, we have incorporated additional details about the statistical tests used, p-values for noteworthy comparisons, and have included more panels for our comparative RNA-seq datasets (heatmap, PCA, MA plots). We have also made adjustments to our plots to make individual data points more readily observed, especially when error bars may block them (e.g. Figure 3B). And as in the original submission, all of the pertinent values, including fold changes, statistics and more are provided in our comprehensive supplementary files. We have structured the Supplementary Tables to flow from one tab to the next with the filtering/threshold applied noted both in the tab name and in the README tab that is found first among the tabs.

**Minor comments:**

• Specific experimental issues that are easily addressable.

  One idea that is also not discussed but could be added is for example that NOT1-G itself doesn't even have a stabilizing effect itself, but act as a decoy for other components of the CCR4/Caf1 complex, keeping them from associating with NOT1. In the NOT1-G knockout, the decrease in RNA abundance might then be just a result of an 'overactivity' of CCR4/Caf1/NOT1.

Response**: This hypothesis proposed by Reviewer 2, that PyNOT1-G is acting as a decoy or a binding partner sponge, is certainly feasible. For this scenario to be effective, PyNOT1-G would need to be in excess of PyNOT1 and/or would need to be able to bind to the critical effector protein(s) better than does PyNOT1. However, our microscopy data, along with the transcriptomic data presented here and previously published proteomic data would indicate that these two gene products are in approximately balanced proportions and are similarly localized. This does not exclude the possibility that PyNOT1-G could act as a sponge for relevant binding partners. In our revised manuscript, we have raised this possibility as an alternate explanation for the phenotype in the Discussion section.

• Are prior studies referenced appropriately?

  Throughout the manuscript, the authors should make clear what results come from which organism. Just as an example, the genome wide KO screens were performed in P. berghei and P. falciparum, CCR4/CAF1 experiments were performed in P. yoelii, whereas the original DDX6 work was done in P. berghei.

Response**: We agree. In our revised manuscript, we have added additional text to further clarify what data comes from which Plasmodium species.

• Are the text and figures clear and accurate?

  The Introduction is a bit long and partially turns into a minireview of eukaryotic RNA degradation. In the main text on page 13, the authors introduce a model for proteins involved in translational repression. This in not fully accurate, since for many of the proteins in this network, an effect on translation has actually not been shown. This includes NOT1-G characterized in the present work that most likely has an effect on mRNA stability, but for which a role in regulating translation is not presented.

Response**: We believe the length and content of this Introduction is appropriate to provide the context that some readers outside of the parasitology field will need to appreciate these findings. Regarding designations for these proteins as being related to translational repression, we think that the ample proteomic evidence tying them to translationally repressive complexes warrants this. In our revised manuscript, we have made it more clear that these proteins themselves have not been directly implicated in translational repression.

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

  Overall the RNA-seq is underrepresented and Figure 5 could easily be expanded by adding several panels that would help the future reader getting a better idea of the data:

1. Summary graphs such as PCA/MDS plots of the different replicates and MA-plots (all of which can be easily generated in DESeq2)
2. Heatmaps comparing the expression patterns of pynot1-g-, pbdozi-, pbcith-, pyalba4- highlighting some key gametocyte genes mentioned in the text

3. Alternatively to 2., a simple Venn Diagram would already be very informative

   An informative representation might also be to sort the differentially expressed genes as predominant male and/or female. The P. berghei data by Yeoh et al (PMID: 28923023) could be a starting point.

Response**: We agree. In our revised manuscript, we have expanded Figure 5 to include additional plots that speak the rigor of these datasets. Specifically, we have added a comprehensive heatmap and PCA plots, as well as MA plots as recommended. We have chosen not to include a Venn diagram for the overlap of affected mRNAs across these transgenic parasite lines, as we hold that this information is best provided in the text (high level observations) and the Supplement (details).

Reviewer #2 (Significance (Required)):

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

Technically this manuscript builds on standard methods of the field that are well executed. There is no direct clinical advancement, although one might argue that a unique adaptation of the parasite could always be a novel therapeutic target. Conceptually this is great advancement for the parasitology field as it is, providing additional evidence for the importance of post-transcriptional regulation for parasite transmission. With the two experiments suggested above and the additional evidence gained from it, this manuscript could also gain great interest to readers outside the field by clearly showing how alternative ways to regulate RNA stability evolved.

Response**: We are grateful for your careful review of our work and for the recommendations that you provided. We have incorporated many of them into the revised manuscript to make it even more rigorous and comprehensive. We also appreciate hearing that this work would be of great interest to a broader community. We feel that this is already the case, as the duplication of NOT1 and the dedication of one paralogue to an essential function is exciting and novel among eukaryotes.

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

The work builds on the early reports of the particular RNA metabolism in gametocytes performed in the groups of Andy Waters. Since then, the authors themselves have published a great set of manuscripts extending our knowledge of the proteins involved in gametocytogenesis and nicely place the current work into this framework.

Response**: We appreciate this positive feedback. This is a fascinating topic to study.

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

The manuscript as it stands is particularly interesting for the parasitology and potentially the evolutionary biology field. For a broader readership for example in the RNA field, the possibly antagonistic roles and mutually exclusive association with CAF1/CCR4 are likely most interesting.

Response**: We agree that this should be interesting to readers beyond our own field, as the duplication and specialization of NOT1, and the finding that the “canonical” PyNOT1 can be deleted, are both of general interest to how eukaryotes have adapted and deployed a highly conserved and essential RNA metabolic complex.

**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.**

**Expertise:**

RNA biology, Plasmodium falciparum, Bioinformatics

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

In this manuscript, the authors investigate the requirement of two possible Not1 paralogs for the development of asexual blood stages and for the sexual transmission stages of Plasmodium yoelii. While Not1 is critical for asexual blood stages, its putative paralog, Not1G is important for the development of sexual transmission stages. In the absence of Not1G, male gametes are not formed while female gametes are formed and can be fertilised by wt male gametes. However, the resulting zygote cannot develop further into ookinete. The in vitro genetic cross assay to show this is elegant! A transcriptomic analysis further indicates that the transcriptomes of Not1G deficient parasites are significantly different from their WT counterpart.

Response**: We are thrilled that you found our evidence and approaches to be rigorous and compelling. Thank you.

**Major comments:**

The discussion section is very nice and the authors describe well what is speculative and should be further confirmed by additional experiments. However, I did find this was not the case in the results section where the authors are proposing conclusions that are not supported by the results. I think the reading of this manuscript would be much more enjoyable if the authors only describe the results shown and move all the discussions to the dedicated section. Below are some examples. The data presented in this manuscript is not showing a nexus, this is a suggestion based on the results of other articles, the word should thus be removed from the title (and kept for a future review!). The last two sentences of the localisation section should be moved to the discussion because they do refer to results not shown in this manuscript. The last sentence of the second paragraph of the zygote development section should also be moved to the discussion. For the transcriptomic analysis there is also no formal comparison with transcriptomes of other previously analysed mutants: the results of the comparisons should either be shown or not discussed in the result section. Finally, the discussions mentioning interactors of the complex should be removed from the result section and moved to the discussion unless the results are formally analysed.

Response**: We again thank you for the complement. In our original manuscript, we opted to provide some limited interpretations and context within the Results section in order to help guide readers along our train-of-thought and line-of-experimentation. While a more traditional split of keeping essentially all discussion and interpretation for the Discussion is a tried-and-true approach, we prefer this more narrative method and have opted to keep these short sections in the Results section.

I would strongly suggest the author the better present and describe their transcriptomic results. There is only one volcano plot indicating the overall defect in mixed gametocytes in the main figure. Apart from this, the results are only described in the main text or in supplementary tables. It is therefore difficult to understand the subtilities of the analysis. For example, the authors frequently mention dysregulated genes, but without specifying whether it is up or down-regulated in the mutant. To address this issue, I would suggest the authors to better describe their results in the figures. They could show the GO term enrichment analysis they mention and show how they assign GO term or transcripts to male and female parasites. It would also be nice to discuss some of the results a bit more in details. For example, it is not surprising to see a reduction in transcripts that are under the control of AP2-O in retort-arrested ookinetes as the parasite do not reach this stage. It is thus highly speculative to specifically link this observation with ALBA4 without further detailed analysis. On the other hand, it is more surprising to see a decrease in ap2g transcripts, while the authors observe an increased gametocytaemia. Could the authors comment this observation? It may also be nice to better present the comparison between gametocytes and schizonts to possibly speculate on the early requirement of Not1G in committed schizonts.

Response**: We (and Reviewer 2) agree. In our revised manuscript, we have expanded Figure 5 to include additional plots that speak the rigor of these datasets. Specifically, we have added a heatmap, and PCA and MA plots as recommended. We have chosen not to include a Venn diagrams for the overlap of affected mRNAs across these transgenic parasite lines for the reasons stated above in our response to Reviewer 2. Similarly, we have opted to keep the specifics of the GO Term analyses in the Supplement as we believe these should always be taken with a grain of salt (especially high level GO Terms, as many choose to report). Finally, we have expanded our discussion on our observation that pyapiap2-g transcript levels are lower in the pynot1-g- line, despite seeing a slight increase in gametocytemia.

The conclusion regarding the similar localisation of Not1 and Not1G with other members of the CAF1/CCR4/NOT complex is not really convincing for two reasons. First, there is not colocalization shown and, second, the distribution is not very peculiar so it is difficult to draw any conclusion with this level of resolution. The presence of alpha-tubulin in the nucleus of male gametocytes is also very surprising as it is rather nucleus-excluded in both P. falciparum and P. berghei, could the authors comment this peculiar localisation?

Response**: We agree and disagree here. First, we agree that no colocalization data is presented here to place NOT1-G within the limit of resolution of fluorescence microscopy. What we can (and do) state is that these proteins are all localized to cytosolic puncta, which matches what is observed for essentially all other studied eukaryotes. In further support of this, our published, quantitative proteomic data indicates that the bioinformatically predictable members of the CAF1/CCR4/NOT complex do associate as anticipated. In the same vein, the micrographs presented were not captured by confocal microscopy, and thus the apparent localization of alpha tubulin “in” the nucleus is most likely attributed to being above and/or below the nucleus. Taken together, we do feel that the combined evidence is convincing. As we have already made all of these points in the original manuscript, we have not adjusted the revised manuscript further.

One of my major frustration when reading this manuscript was that the authors are not trying to discriminate between an early role of Not1G during gametocytogenesis or later in gametogenesis. The fact that the transcriptomes of gametocytes and schizonts seem to show similarities suggests that the phenotype observed during both male gametogenesis or ookinete development are probably linked to early knock-on defects during gametocytogenesis. Could the authors test whether male gametocytes replicate DNA or female activate translation? These are of course non-essential experiments as the authors are careful with their conclusions and mention possible defects during both gametocytogenesis or gametogenesis. Addressing this question may however add significant insights into the requirement for Not1G.

Response**: We are sorry for the frustration. We wrote the manuscript so as to state what we feel we could robustly say, and where we are drawn to speculate, we made that speculation clear. As Reviewer 3 notes, we have not attempted to discriminate between functions that PyNOT1-G may be playing in different stages or substages of development because we do not believe the experiments allow that discrimination. While we could investigate finer and finer aspects of possible defects in both male and female gametocyte development, the most impactful take home messages remain the same. We continue to address questions related to translational repression and its release, and anticipate that PyNOT1-G will play a substantial and essential role in this. As Reviewer 3 noted, we have already discussed these possibilities in the original manuscript, and thus have not added anything further about this in our revised manuscript.

**Minor comments:**

Please use page and line numbering for your next submissions! Please describe what "bioinformatics" was used. I would show the nice localisation in oocyst and sporozoite in the main section. The conclusions drawn from the genetic cross seem to come from a single biological replicate, if this is the case please indicate it clearly.

Response**: We apologize for these oversights. In our revised manuscript, we have provided page and line numbering, have expanded on what bioinformatic processes were done in the manuscript, and have made it more clear that the genetic crosses come from multiple biological replicates (biological triplicate for the transmission-based genetic cross, biological duplicate for the in vitro culture genetic cross). However, we have opted to retain the oocyst and sporozoite IFA data in the Supplement, as the rest of the story is focused on blood stage and early mosquito stage.

Reviewer #3 (Significance (Required)):

This manuscript highlights the requirement of a Not1 paralog in the transmission stages of a Plasmodium parasite. More specifically it describes a new player in the control of RNA biology during this process where our knowledge is scarce. It will be a valuable manuscript for molecular parasitologists interested in transmission or RNA biology.

Response**: We agree and are grateful that our colleagues find this study to be a valuable addition in our efforts to understand how malaria parasites have adapted classic eukaryotic mechanisms to suit their purposes.

Our expertise is largely in molecular and cellular parasitology.

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

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

In this manuscript, the authors investigate the requirement of two possible Not1 paralogs for the development of asexual blood stages and for the sexual transmission stages of Plasmodium yoelii. While Not1 is critical for asexual blood stages, its putative paralog, Not1G is important for the development of sexual transmission stages. In the absence of Not1G, male gametes are not formed while female gametes are formed and can be fertilised by wt male gametes. However, the resulting zygote cannot develop further into ookinete. The in vitro genetic cross assay to show this is elegant! A transcriptomic analysis further indicates that the transcriptomes of Not1G deficient parasites are significantly different from their WT counterpart.

Major comments:

The discussion section is very nice and the authors describe well what is speculative and should be further confirmed by additional experiments. However, I did find this was not the case in the results section where the authors are proposing conclusions that are not supported by the results. I think the reading of this manuscript would be much more enjoyable if the authors only describe the results shown and move all the discussions to the dedicated section. Below are some examples. The data presented in this manuscript is not showing a nexus, this is a suggestion based on the results of other articles, the word should thus be removed from the title (and kept for a future review!). The last two sentences of the localisation section should be moved to the discussion because they do refer to results not shown in this manuscript. The last sentence of the second paragraph of the zygote development section should also be moved to the discussion. For the transcriptomic analysis there is also no formal comparison with transcriptomes of other previously analysed mutants: the results of the comparisons should either be shown or not discussed in the result section. Finally, the discussions mentioning interactors of the complex should be removed from the result section and moved to the discussion unless the results are formally analysed.

I would strongly suggest the author the better present and describe their transcriptomic results. There is only one volcano plot indicating the overall defect in mixed gametocytes in the main figure. Apart from this, the results are only described in the main text or in supplementary tables. It is therefore difficult to understand the subtilities of the analysis. For example, the authors frequently mention dysregulated genes, but without specifying whether it is up or down-regulated in the mutant. To address this issue, I would suggest the authors to better describe their results in the figures. They could show the GO term enrichment analysis they mention and show how they assign GO term or transcripts to male and female parasites. It would also be nice to discuss some of the results a bit more in details. For example, it is not surprising to see a reduction in transcripts that are under the control of AP2-O in retort-arrested ookinetes as the parasite do not reach this stage. It is thus highly speculative to specifically link this observation with ALBA4 without further detailed analysis. On the other hand, it is more surprising to see a decrease in ap2g transcripts, while the authors observe an increased gametocytaemia. Could the authors comment this observation? It may also be nice to better present the comparison between gametocytes and schizonts to possibly speculate on the early requirement of Not1G in committed schizonts.

The conclusion regarding the similar localisation of Not1 and Not1G with other members of the CAF1/CCR4/NOT complex is not really convincing for two reasons. First, there is not colocalization shown and, second, the distribution is not very peculiar so it is difficult to draw any conclusion with this level of resolution. The presence of alpha-tubulin in the nucleus of male gametocytes is also very surprising as it is rather nucleus-excluded in both P. falciparum and P. berghei, could the authors comment this peculiar localisation?

One of my major frustration when reading this manuscript was that the authors are not trying to discriminate between an early role of Not1G during gametocytogenesis or later in gametogenesis. The fact that the transcriptomes of gametocytes and schizonts seem to show similarities suggests that the phenotype observed during both male gametogenesis or ookinete development are probably linked to early knock-on defects during gametocytogenesis. Could the authors test whether male gametocytes replicate DNA or female activate translation? These are of course non-essential experiments as the authors are careful with their conclusions and mention possible defects during both gametocytogenesis or gametogenesis. Addressing this question may however add significant insights into the requirement for Not1G.

Minor comments:

Please use page and line numbering for your next submissions! Please describe what "bioinformatics" was used. I would show the nice localisation in oocyst and sporozoite in the main section. The conclusions drawn from the genetic cross seem to come from a single biological replicate, if this is the case please indicate it clearly.

Significance

This manuscript highlights the requirement of a Not1 paralog in the transmission stages of a Plasmodium parasite. More specifically it describes a new player in the control of RNA biology during this process where our knowledge is scarce. It will be a valuable manuscript for molecular parasitologists interested in transmission or RNA biology.

Our expertise is largely in molecular and cellular parasitology.

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

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary

The manuscript by Hart et al. builds upon a fascinating finding presented in a previous manuscript by the same authors, in which they show that CCR4 seems to be able to associate with two members of the NOT1 family. In this work, the authors first re-annotate the two NOT1 paralogs in Plasmodium yoelii and then perform an in depth characterization of the role of NOT1-G during gametocytogenesis and early mosquito development. Using gene knockout and different genetic crosses, the authors show that NOT1-G is essential for male gametocyte development and leads to an arrest of development in zygotes arising from female gametocytes. Using RNA-seq the authors show that NOT1-G leads to lower transcript abundances, leading to the hypothesis that NOT1-G might be involved in preserving mRNAs in a larger RNA-binding complex. Lastly, the authors characterize a NOT1-G defining TPP domain and find that it is not essential for either male/female phenotype observed for the whole gene KO.

Major comments:

• Are the key conclusions convincing?

The phenotypic characterization of NOT1-G during gametocytogenesis / early mosquito development is nicely presented and the experiments are well performed. Because a duplication of NOT1 with possibly opposing roles of the paralogs is a very unique feature with broad implication on RNA metabolism, it would have been great to see two select experiments on the molecular level adding evidence that 1) NOT1/NOT1-G are mutually exclusive in a complex with CCR4/CAF1 and 2) NOT1-G acts post-transcriptionally in an antagonistic way to NOT1 (i.e. as a mRNA 'stabilizer' as proposed by the authors).

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

The authors describe the role of NOT1-G as 'preserving' mRNA. The lower abundance of many transcripts in the NOT1-G knockout suggest this, but experimental proof is not provided (see suggestions below). Maybe rephrase to 'putatively preserved/stabilized' or 'has a potentially stabilizing function'. The same is true for the mutually exclusive association of the two paralogs with CCR4/CAF1. The authors refer to a protein co-IP of CCR4 showing that CCR4 can interact with both NOT1 and NOT1-G, but a reciprocal experiment is lacking.

In both cases, the conclusions of the authors are very likely (e.g. downregulation of many genes as seen by RNA-seq), but the final experimental evidence is not provided and a network such as in Figure 7 is not fully supported. If the authors would like to maintain these statements, then they should be rephrased and made clear or the additional experimental evidence suggested below is necessary.

• 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.

The essential claim that NOT1-G is important for gametocytogenesis and early mosquito development is well presented and fully supported by the experiments. As for the role of NOT1-G in 'preserving' mRNA, an mRNA half-life experiment would be necessary (or the text should be adjusted as mentioned above). In a short-term in vitro culture, pynot1-g- and WT parasites could be treated with ActD and abundances of select transcripts are measured by RT-qPCR.

To support the idea that NOT-1 and NOT1-G associate in a mutually exclusive way or to just show that they act in distinct complexes despite their similar expression patterns, an IFA with a double stained NOT1/NOT-1G cell line could be performed. Alternatively, the authors could perform a protein co-IP using the already existing NOT1/NOT1-G-GFP cell line and show that the proteins don't interact with each other or even have certain distinct interaction partners.

• 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.

All necessary cell lines for a NOT1/NOT1-G co-IP and the ActD experiment are already present. The authors already present a ring to schizont in vitro culture (for ActD) and also have substantial experience in protein co-IP and proteomics.

I am not sure about the cost for a proteomics experiment at the author's institute and I don't want to make a guess on time investment given the still on-going COVID situation.

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

The MM section is well structured and presented and the supplemental material includes all data.

• Are the experiments adequately replicated and statistical analysis adequate?

There is hardly any test of significance presented in the main text of the manuscript (e.g. Figure 3B and 4A). Please show the individual data points for these graphs and make sure the n= and the statistical test is described in the figure legend. If you use the term significant in the text, then just add the p-value behind it. This is also true for the RNA-seq data: Genes are sorted by fold-changes, leaving it unclear if these changes are significant. These data are however presented in Table S1 and could be incorporated in the main text.

Minor comments:

• Specific experimental issues that are easily addressable.

One idea that is also not discussed but could be added is for example that NOT1-G itself doesn't even have a stabilizing effect itself, but act as a decoy for other components of the CCR4/Caf1 complex, keeping them from associating with NOT1. In the NOT1-G knockout, the decrease in RNA abundance might then be just a result of an 'overactivity' of CCR4/Caf1/NOT1.

• Are prior studies referenced appropriately?

Throughout the manuscript, the authors should make clear what results come from which organism. Just as an example, the genome wide KO screens were performed in P. berghei and P. falciparum, CCR4/CAF1 experiments were performed in P. yoelii, whereas the original DDX6 work was done in P. berghei.

• Are the text and figures clear and accurate?

The Introduction is a bit long and partially turns into a minireview of eukaryotic RNA degradation. In the main text on page 13, the authors introduce a model for proteins involved in translational repression. This in not fully accurate, since for many of the proteins in this network, an effect on translation has actually not been shown. This includes NOT1-G characterized in the present work that most likely has an effect on mRNA stability, but for which a role in regulating translation is not presented.

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

Overall the RNA-seq is underrepresented and Figure 5 could easily be expanded by adding several panels that would help the future reader getting a better idea of the data:

1. Summary graphs such as PCA/MDS plots of the different replicates and MA-plots (all of which can be easily generated in DESeq2)
2. Heatmaps comparing the expression patterns of pynot1-g-, pbdozi-, pbcith-, pyalba4- highlighting some key gametocyte genes mentioned in the text
3. Alternatively to 2., a simple Venn Diagram would already be very informative

An informative representation might also be to sort the differentially expressed genes as predominant male and/or female. The P. berghei data by Yeoh et al (PMID: 28923023) could be a starting point.

Significance

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

Technically this manuscript builds on standard methods of the field that are well executed. There is no direct clinical advancement, although one might argue that a unique adaptation of the parasite could always be a novel therapeutic target. Conceptually this is great advancement for the parasitology field as it is, providing additional evidence for the importance of post-transcriptional regulation for parasite transmission. With the two experiments suggested above and the additional evidence gained from it, this manuscript could also gain great interest to readers outside the field by clearly showing how alternative ways to regulate RNA stability evolved.

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

The work builds on the early reports of the particular RNA metabolism in gametocytes performed in the groups of Andy Waters. Since then, the authors themselves have published a great set of manuscripts extending our knowledge of the proteins involved in gametocytogenesis and nicely place the current work into this framework.

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

The manuscript as it stands is particularly interesting for the parasitology and potentially the evolutionary biology field. For a broader readership for example in the RNA field, the possibly antagonistic roles and mutually exclusive association with CAF1/CCR4 are likely most interesting.

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.

Expertise:

RNA biology, Plasmodium falciparum, Bioinformatics

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Referee #1

Evidence, reproducibility and clarity

The manuscript „The Plasmodium NOT1-G paralogue acts as an essential nexus for sexual stage maturation and parasite transmission" investigates the two forms of NOT1 in rodent malaria parasites. The authors found out that the original NOT1 is crucial for gametocyte induction as well as transmission to the mosquito, they therefore renamed it NOT1-G. The paralogous proteins, on the other hand, appears to be crucial for intraerythrocytic growth, since it cannot be knocked out. The authors then investigated NOT1-G in more detail, using standard phenotyping assays. They found a slightly increased gametocytemia and a minor effect on transmission to the mosquito.

Significance

If the authors are able to provide convincing data that NOT1-G is indeed important for gametocyte induction and transmission to the mosquito, then the report would be of high significance for the malaria and molecular cell biology fields.

My expertise: molecular cell biology of gametocytes, translational regulation, parasite transmission

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Reply to the reviewers

We thank all four reviewers for their positive and constructive comments! We have carefully considered these comments and provided a point-by-point response below.

Reviewer #1 (Evidence, reproducibility and clarity):

This paper explores an interesting problem of SHP1/SHP2 preferences of inhibitory immunoreceptors. The author are quick to point out that many of their individual data points confirm published results at some level, but the power of the paper is in the parallel analysis of both PD1, which is strongly biased towards SHP2 and BTLA, which is biased towards SHP1. This gives them the opportunity to test the predictions of descriptive experiment by making simple mutated receptors with swapped ITIM or ITSM domains.

The work is very well done and generally the authors are quite careful and precise about the language used to describe results, in general.

The results are quite striking in that the find plenty of evidence for transient interaction of SHP1 with PD1 based on the biophysical measurements, but don't detect the interactions in pull down or in "in cell" microcluster recruitment experiments. In describing the pull-downs they discuss the issue of dissociation during washing potentially missing interactions that are taking place. I would prefer that the pull down is fine evidence for binding, but lack of pull down is not evidence for lack of binding. They should double check that this language is consistent. Also, unless something has changed in the microcluster binding experiments, this in situ recruitment of SHP2 to PD1 is only observed or a 2-3 minutes and then can't be detected, the situation for SHP2 becoming the same as it is for SHP1. If the kinetics are different in the cleaner systems that have now developed they should show this in a primary figure as this would be then different when what is reported previously.

We agree with the reviewer that pull down is evidence for binding. Indeed, in most, if not all of our assays, our results with pull down were consistent with those in the microcluster imaging. As suggested by the reviewer, we will check through the manuscript and ensure the language is accurate and consistent. In our recent study (Xu et al., JCB, 2020, PMID: 32437509), we conducted a side-by-side comparison of SHP2 and SHP1 recruitment kinetics to PD-1 in a similar system as the current study. Both microcluster imaging and co-IP assays showed that PD-1:SHP2 association lasted at least 10 minutes, whereas PD-1:SHP1 recruitment was nearly undetectable. The duration of PD- 1:SHP2 association was in good agreement with Takashi Saito’s finding in CD4+ mouse T cells (Yokosuka et al., JEM, 2012, PMID: 22641383). Regardless the somewhat different kinetics in different studies, SHP2 recruitment was transient, as pointed out by the reviewer. We believe that some other effectors contribute to PD-1 inhibitory signaling. In supportive of this notion, we recently found that PD-1 remains partially inhibitory in CD8+ T cells deficient in both SHP1 and SHP2 (Xu et al., JCB, 2020).

The gap in this study is lack of any functional analysis. The Jurkat model could be quite useful as they have a relatively clean system for asking if the transient binding of SHP1 to PD1 has any functional impact, which they have not yet followed through on. Does PD-1 recruited SHP2 have any impact on function after the 5 minutes? Furthermore, the authors need to keep in mind that mice deficient in SHP2 respond to anti-PD1 checkpoint therapies (Rota, G., Niogret, C., Dang, A. T., Barros, C. R., Fonta, N. P., Alfei, F., Morgado, L., Zehn, D., Birchmeier, W., Vivier, E., & Guarda, G. (2018). Shp-2 Is Dispensable for Establishing T Cell Exhaustion and for PD-1 Signaling In Vivo. Cell Rep, 23(1), 39-49. https://doi.org/10.1016/j.celrep.2018.03.026). This is an important issue to discuss in light the the very interesting binding analysis the authors have performed. But I think the functional analysis can be part of a future paper.

In our recent publication (Xu et al. JCB, 2020, PMID: 32437509), we found that deletion of SHP1 from Jurkat cells had little, if any effect on PD-1 mediated suppression of IL-2 production. As the reviewer alluded to, we did observe SHP2 dissociation from PD-1 after 10 minutes, so the question of whether and how PD-1:SHP2 complex influence T cell function in a longer term is a great one. We currently are pursuing a hypothesis that there is a SHP2-independent mechanism of PD-1 inhibitory function, and indeed, in our recent study (Xu et al. JCB, 2020, PMID: 32437509), we found that PD-1 retains its partial inhibitory function in SHP1/SHP2 double knockout murine primary T cells. These results are consistent with the in vivo data by Rota et al. cited by the reviewer. We will also briefly discuss this point in a revised manuscript.

I would suggest that the title be modified slightly from "SHP1/SHP2 discrimination" to "differential SHP1/SHP2 interaction" and leave discussion of discrimination until they have the functional data integrated over times that are relevant to T cell transcriptional regulation (1-2 hrs). The functional analysis can be in another paper, but it would be interesting to have a paragraph in the discussion raising the outstanding issues beyond stable binding detected by the pull-down and microcluster recruitment experiments- what are the implications for function. Could the transient interactions in the noise of the steady state and equilibrium measurements be functional?

We thank the reviewer for the suggestion, even though reviewer #3 felt that our current title is appropriate. We will be happy to change the title at the editors’ discretion.

I would summarise that the work is outstanding as biochemistry and biophysics and it should be published nearly as is. I'm suggesting minor revisions in that the changes are just to text, but I think this is important and somewhat nuanced aspect of the paper that will make it even more helpful to readers.

We appreciate the positive and insightful comments!

Reviewer #1 (Significance):

The authors generate a detailed descriptive data set about the component interaction of SHP1 and SHP2 SH2 domains with PD1 and BTLA intracellular domains. They then test hypotheses generated from the descriptive data set to better define the nature of the interactions and why PD1 recruits primarily SHP2, while BTLA mainly recruits SHP1. PD1 is a major driver or the cancer immunotherapy revolution and SHP2 is the major candidate for a signalling effector of PD1. This paper can become the reference paper for the specificity and engineering of this interaction, which will make it highly significant in a very active and still expanding field.

Referee Cross-commenting

I still feel that "discrimination" has a functional/activity connotation that is not addressed at all in this paper, but can be addressed. I'm happy to have the suggestion stand and let the authors decide. They need to live with it once its published. Another suggestion- the citations on regulation are mostly old. A good recent paper is Pádua, R. A. P., Sun, Y., Marko, I., Pitsawong, W., Stiller, J. B., Otten, R., & Kern, D. (2018). Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2. Nature Communications, 9(1),

1. https://doi.org/10.1038/s41467-018-06814-w .

We believe that some of the functional questions raised by this reviewer, including the SHP1 and SHP2 contribution in PD-1 signaling, was addressed in our recent publication (Xu et al., JCB, 2020). Using SHP1 KO and SHP2 KO T cells, we showed that PD-1 inhibitory function is contributed by SHP2, but very little if any by SHP1. Thus in the current study, we focus on the mechanism behind the striking SHP2 preference by PD-1. We thank this reviewer for suggesting this excellent reference. We will cite this reference in the revised manuscript.

Reviewer #2 (Evidence, reproducibility and clarity):

In this study, Xu and co-workers investigate the biophysical nature of the interaction between the structurally-related non-transmembrane PTPs Shp1 and Shp2 with the ITIM/ITSM-containing inhibitory receptors PD-1 and BTLA using cell-based, biochemical, biophysical and domain swapping assays. The primary aim being to better understand how these receptors discriminate between binding Shp1 and/or Shp2, and the orientation of Shp1 and Shp2 engagement. These are major unresolved questions in the field that the authors go some way to addressing in a methodical, rigorous, clear and concise manner. Findings are convincing, correlate well with previous findings and internally, and are complemented with excellent schematics, making it easy to comprehend.

Major comments

The authors focus primarily on binding affinities to explain differential binding of Shp1 and Shp2 by PD-1 and BTLA ITIMs and ITSMs, but this is only part of the story. Avidity, compartmentalization, stoichiometry of kinases, and relative abundance of Shp1 and Shp2 are also important aspects of the discriminatory mechanism that are not addressed. Competition assays would go some way to addressing the latter point and should be at least be considered and discussed.

We agree that various parameters mentioned by this reviewers, such as compartmentalization and relative expression levels would be a concern for purely cell-free assays such as SPR, however, we feel that our cell-based assays already integrate these parameters. This is also precisely the reason why we chose to examine the recruitments of Shp1/2 in a cellular context instead of a purely cell-free system.

Regarding the competition, we have confirmed our key results in both WT and SHP2 KO background, with or without the potential competition from endogenous SHP2, suggesting that competition might not be a dominant mechanism for the recruitment specificity we observed.

Similarly, authors do not address how distortion of the pY binding pocket of Shp1 and Shp2 nSH2 domains in the auto-inhibited conformation is released, allowing the domain to engage with phopho-ITIM/ITSM. Again, this should be at least discussed. Current binding studies do not address this issue.

We feel that the overall recruitment to the PD-1 microclusters as we observed in cells already integrate this auto-inhibition mechanism of Shp1 and Shp2, because we used full length proteins. We do agree with the reviewer that future studies are warranted to address the contributions of each mechanism, including auto-inhibition, concentration, competition, etc., to the overall recruitment. This might require careful and extensive biophysical analyses coupled with mathematical modeling.

Minor comments:

Phosphorylation should be indicated in schematic representations in Figures 3, 6 b, c.

We thank the reviewer for this advice, we will indicate phosphorylation in the revised figure 3.

Cellular and physiological significance should be further discussed, as well as broader implications of findings to other ITIM/ITSM-containing receptors in other lineages.

We will further discuss this as suggested.

Reviewer #2 (Significance)

Findings from this study advance our knowledge of how inhibitory checkpoint regulatory receptors discriminate between Shp1 and Shp2, which has important implications for understanding how the unique biochemical, cellular and physiological functions of these receptors and phosphatases are dictated. Indeed, findings lay the foundation for a universal mechanism, that may apply to all ITIM/ITSM receptors in other cell lineages, and perhaps novel ways of targeting these interactions therapeutically.

Compare to existing published knowledge

Although largely correlative with previous studies, findings from this study start to fill major gaps in our knowledge of these biochemical processes, in a highly rigorous, concise and clear manner. Findings from previous studies were more 'piecemeal', whereas this study consolidates and advances important nuances of these interactions. Moreover, it lays the foundation for further structural, physiological and therapeutic studies.

Audience

The immune receptor signaling community and beyond, including any lineage in which ITIM/ITSM-containing receptors play a major role in regulating cellular responses.

Your expertise

ITIM/ITSM-containing receptors, kinase-phosphatase molecular switches, cellular reactivity to extracellular matrix proteins

Referee Cross-commenting

Generally agree with reviewer's comments. Constructive overall and fair. Although I was thinking additional competition experiments, I do not think necessary. Over the top for this study. Hence, 1 month should suffice to revise accordingly.

We thank this reviewer for the excellent comments and understanding!

Reviewer #3 (Evidence, reproducibility and clarity):

Summary:

Inhibitory immune receptors containing ITIMs function through recruiting the phosphatases SHP-1 and SHP-2. SHP-1 and SHP-2 are remarkably similar yet have different roles in vivo. How can ITIM-containing immune receptors specifically recruit SHP-1 or SHP-2? In this paper, Xu et al ask how SHP-1 vs SHP-2 specificity is achieved. They use very thorough biochemical assays to measure the affinity of SHP-1 and SHP-2 for various ITIM/ITSMs and finally pin point some key amino acids that switch an ITIM/ITSM from SHP-2 to SHP-1 specificity. The in vitro biochemical assays are augmented by in cell assays that support their conclusions. Overall, this paper is an incredibly elegant and straight forward paper addressing how SHP-1/SHP-2 specificity is achieved.

Major Comments: none

Minor Comments:

• Could the western blots in Figure 1 be quantified as the western blots in other figures?

We will quantify the western blots in Figure 1 as suggested in the revised manuscript.

• The data that the y+1 reside is essential for SHP-1/2 specificity is very convincing. We are curious if the other residues of the ITIM/ITSM also contribute to this specificity, albeit less potently. The PD-1 G224A mutant is still less potent than the PD-1 BTLA ITIM swap, suggesting that while the y+1 position is most important, the other residues contribute some specificity. The authors also included data on a PD-1 variant with the BTLA ITIM A224G mutation (8f), which is slightly better at recruiting SHP-1 than the PD-1 ITIM. It may be worth mentioning this data in the text of the paper as well as displaying it in the figure.

The reviewer raised an excellent point, yes, our data does suggest that other pY-flanking residues within the ITIM also contribute to SHP1 binding. However, the pY+1 residue replacement produced the strongest effect as the reviewer noted. In the revised manuscript, we will acknowledge the potential contributions of other residues.

• A brief introduction to ITIM vs ITSM in the introduction of the paper may be helpful background for readers. For example, ITIM receptors are reasonably well known but how ITSM functionally differs is probably less well known.

We will rewrite the introduction about ITIM and ITSM for better clarity.

• Although not the major focus of the paper, broadening out this SHP-1/2 specificity to other immune receptors in the discussion is fascinating. (a) The authors find that a Valine, Leucine, or Isoleucine in place of the Alanine in y+1 is very close to equivalent, yet the A is highly conserved. The authors speculate that there may be an advantage to sub-maximal SHP-1 affinity because it is more easy to regulate. I think this is reasonable speculation but a little unsatisfying given the very small observed difference in SHP-1 binding. If the authors have additional thoughts, I would be interested to hear them. (b) The authors note that PD-1 is the only ITIM with a glycine in the Y+1 position. Are there other receptors that function primarily through SHP-2, and how might they achieve this specificity?

Response to a: Even though valine, leucine or isoleucine did not produce a striking enhancement in Shp1 recruitment over alanine, the differences were statistically significant. In fact, when we performed these point mutations at a BTLA ITIM background, valine, leucine or isoleucine markedly enhanced the SHP1 recruitment (see unpublished data below). We speculate that other pY-flanking residues in BTLA, as this reviewer alluded to above, creates an environment that amplifies the differences. The strong sensitivity on pY+1 residue, as observed in BTLA, might be true for other SHP1-recruiting receptors too. If they were to have leucine or isoleucine at the pY+1 position of ITIM, they may recruit too much SHP1 that presumably decreases the fitness/growth of the cells. We propose to show this unpublished data as a supplemental figure in the revised manuscript. We will also discuss the potential contributions of other pY-flanking residues as this reviewer suggested.

{{images cannot be rendered at this time in reply letters}}

Response to b: Among the several receptors that we tested, PD-1 is the only receptor that exhibited no recruitment of SHP1. The lack of SHP1 recruitment is also true for murine PD-1, which has a glutamate residue (charged) at Y+1 position. In addition, earlier work reported that PECAM1 also selectively recruits SHP2, but not SHP1. We have noted that PECAM1 contain a threonine (polar) at the pY+1 position of their ITIMs. Thus, their inability to recruit SHP1 is consistent with our model that a nonpolar residue at Y+1 position is required for strong SHP1 recruitment. We will discuss these points in the revised manuscript.

• Figure 9 b Val not Vla, Figure 3a - a legend for the color code may be nice (ie, 20-1000 nM) Thanks for catching this, we will fix the error in Figure 9b and provide the color code in Figure 3a in the revised manuscript.

Reviewer #3 (Significance):

Significance:

SHP-1 and SHP-2 play a critical role in regulating immune system function. In addition, the receptors recruiting these phosphatases (like PD-1) are important immunotherapy targets. Previously, the question of SHP-1/SHP-2 specificity has been primarily described for ITIM bearing receptors individually. Other studies have predicted consensus sequences for the tSH2 domains of SHP-1 or SHP-2, but not addressed the defining molecular characteristics of these consensus sites or how these could be combined on ITIM receptors to generate selectivity between these related phosphatases. This paper represents a significant step forward because it provides a unifying mechanism explaining how ITIM-bearing immune receptors specifically recruit SHP-1 or SHP-2. I expect this paper will be broadly interesting to biochemists, immunologists and cancer biologists.

Referee Cross-commenting

I generally think the other reviewers comments are reasonable and insightful. Together, they suggest no new experiments are necessary. As for the proposed title change, I prefer the authors title and find it to be justified given their data.

Reviewer #4 (Evidence, reproducibility and clarity):

In this manuscript, Xu and college performed an elaborate study to investigate the molecular basis of Shp1 and Shp2 discrimination by immune checkpoints PD-1 and BTLA. The paper is original, clear, and well written. I only have a few minor comments:

1. Please label the molecular weights to all the western blots/IPs results.

We will label the molecular weights to all the blots in the revised manuscript.

1. Please add scale bars to all the microscopy pictures.

We will add scale bars to all the microcopy images in the revised manuscript.

1. For the SPR data, please add the fitting curves.

We thank the reviewer for the suggestion. However, we did not use the fitting curve to calculate the Kd, we plotted the maximum response as a function of concentration to determine the Kd. This is another well accepted method for Kd calculation. In fact, some of the SPR curves fit poorly with the existing algorithm. Thus, showing the fitting curve might distract the readers.

Reviewer #4 (Significance):

The strength of this paper relies on the details they dissected by using a series of mutagenesis screening experiments, which should be interesting to cell biologists and cancer immunologists.

Referee Cross-commenting

I think the other reviewer's comments are insightful and constructive, the suggested experiments are necessary and will improve the paper.

We thank this reviewer for the positive comments!

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Referee #4

Evidence, reproducibility and clarity

In this manuscript, Xu and college performed an elaborate study to investigate the molecular basis of Shp1 and Shp2 discrimination by immune checkpoints PD-1 and BTLA. The paper is original, clear, and well written. I only have a few minor comments:

1. Please label the molecular weights to all the western blots/IPs results.
2. Please add scale bars to all the microscopy pictures.
3. For the SPR data, please add the fitting curves.

Significance

The strength of this paper relies on the details they dissected by using a series of mutagenesis screening experiments, which should be interesting to cell biologists and cancer immunologists.

Referee Cross-commenting

I think the other reviewer's comments are insightful and constructive, the suggested experiments are necessary and will improve the paper.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Inhibitory immune receptors containing ITIMs function through recruiting the phosphatases SHP-1 and SHP-2. SHP-1 and SHP-2 are remarkably similar yet have different roles in vivo. How can ITIM-containing immune receptors specifically recruit SHP-1 or SHP-2? In this paper, Xu et al ask how SHP-1 vs SHP-2 specificity is achieved. They use very thorough biochemical assays to measure the affinity of SHP-1 and SHP-2 for various ITIM/ITSMs and finally pin point some key amino acids that switch an ITIM/ITSM from SHP-2 to SHP-1 specificity. The in vitro biochemical assays are augmented by in cell assays that support their conclusions. Overall, this paper is an incredibly elegant and straight forward paper addressing how SHP-1/SHP-2 specificity is achieved.

Major Comments:

none

Minor Comments:

• Could the western blots in Figure 1 be quantified as the western blots in other figures?
• The data that the y+1 reside is essential for SHP-1/2 specificity is very convincing. We are curious if the other residues of the ITIM/ITSM also contribute to this specificity, albeit less potently. The PD-1 G224A mutant is still less potent than the PD-1 BTLA ITIM swap, suggesting that while the y+1 position is most important, the other residues contribute some specificity. The authors also included data on a PD-1 variant with the BTLA ITIM A224G mutation (8f), which is slightly better at recruiting SHP-1 than the PD-1 ITIM. It may be worth mentioning this data in the text of the paper as well as displaying it in the figure.
• A brief introduction to ITIM vs ITSM in the introduction of the paper may be helpful background for readers. For example, ITIM receptors are reasonably well known but how ITSM functionally differs is probably less well known.
• Although not the major focus of the paper, broadening out this SHP-1/2 specificity to other immune receptors in the discussion is fascinating. (a) The authors find that a Valine, Leucine, or Isoleucine in place of the Alanine in y+1 is very close to equivalent, yet the A is highly conserved. The authors speculate that there may be an advantage to sub-maximal SHP-1 affinity because it is more easy to regulate. I think this is reasonable speculation but a little unsatisfying given the very small observed difference in SHP-1 binding. If the authors have additional thoughts, I would be interested to hear them. (b) The authors note that PD-1 is the only ITIM with a glycine in the Y+1 position. Are there other receptors that function primarily through SHP-2, and how might they achieve this specificity?
• Figure 9 b Val not Vla, Figure 3a - a legend for the color code may be nice (ie, 20-1000 nM)

Significance

SHP-1 and SHP-2 play a critical role in regulating immune system function. In addition, the receptors recruiting these phosphatases (like PD-1) are important immunotherapy targets. Previously, the question of SHP-1/SHP-2 specificity has been primarily described for ITIM bearing receptors individually. Other studies have predicted consensus sequences for the tSH2 domains of SHP-1 or SHP-2, but not addressed the defining molecular characteristics of these consensus sites or how these could be combined on ITIM receptors to generate selectivity between these related phosphatases. This paper represents a significant step forward because it provides a unifying mechanism explaining how ITIM-bearing immune receptors specifically recruit SHP-1 or SHP-2. I expect this paper will be broadly interesting to biochemists, immunologists and cancer biologists.

Referee Cross-commenting

I generally think the other reviewers comments are reasonable and insightful. Together, they suggest no new experiments are necessary. As for the proposed title change, I prefer the authors title and find it to be justified given their data.

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Referee #2

Evidence, reproducibility and clarity

In this study, Xu and co-workers investigate the biophysical nature of the interaction between the structurally-related non-transmembrane PTPs Shp1 and Shp2 with the ITIM/ITSM-containing inhibitory receptors PD-1 and BTLA using cell-based, biochemical, biophysical and domain swapping assays. The primary aim being to better understand how these receptors discriminate between binding Shp1 and/or Shp2, and the orientation of Shp1 and Shp2 engagement. These are major unresolved questions in the field that the authors go some way to addressing in a methodical, rigorous, clear and concise manner. Findings are convincing, correlate well with previous findings and internally, and are complemented with excellent schematics, making it easy to comprehend.

Major comments

The authors focus primarily on binding affinities to explain differential binding of Shp1 and Shp2 by PD-1 and BTLA ITIMs and ITSMs, but this is only part of the story. Avidity, compartmentalization, stoichiometry of kinases, and relative abundance of Shp1 and Shp2 are also important aspects of the discriminatory mechanism that are not addressed. Competition assays would go some way to addressing the latter point and should be at least be considered and discussed.

Similarly, authors do not address how distortion of the pY binding pocket of Shp1 and Shp2 nSH2 domains in the auto-inhibited conformation is released, allowing the domain to engage with phopho-ITIM/ITSM. Again, this should be at least discussed. Current binding studies do not address this issue.

Minor comments:

Phosphorylation should be indicated in schematic representations in Figures 3, 6 b, c.

Cellular and physiological significance should be further discussed, as well as broader implications of findings to other ITIM/ITSM-containing receptors in other lineages.

Significance

Findings from this study advance our knowledge of how inhibitory checkpoint regulatory receptors discriminate between Shp1 and Shp2, which has important implications for understanding how the unique biochemical, cellular and physiological functions of these receptors and phosphatases are dictated. Indeed, findings lay the foundation for a universal mechanism, that may apply to all ITIM/ITSM receptors in other cell lineages, and perhaps novel ways of targeting these interactions therapeutically.

Compare to existing published knowledge

Although largely correlative with previous studies, findings from this study start to fill major gaps in our knowledge of these biochemical processes, in a highly rigorous, concise and clear manner. Findings from previous studies were more 'piecemeal', whereas this study consolidates and advances important nuances of these interactions. Moreover, it lays the foundation for further structural, physiological and therapeutic studies.

Audience

The immune receptor signaling community and beyond, including any lineage in which ITIM/ITSM-containing receptors play a major role in regulating cellular responses.

Your expertise

ITIM/ITSM-containing receptors, kinase-phosphatase molecular switches, cellular reactivity to extracellular matrix proteins

Referee Cross-commenting

Generally agree with reviewer's comments. Constructive overall and fair. Although I was thinking additional competition experiments, I do not think necessary. Over the top for this study. Hence, 1 month should suffice to revise accordingly.

Referee #1

Evidence, reproducibility and clarity

This paper explores an interesting problem of SHP1/SHP2 preferences of inhibitory immunoreceptors. The author are quick to point out that many of their individual data points confirm published results at some level, but the power of the paper is in the parallel analysis of both PD1, which is strongly biased towards SHP2 and BTLA, which is biased towards SHP1. This gives them the opportunity to test the predictions of descriptive experiment by making simple mutated receptors with swapped ITIM or ITSM domains.

The work is very well done and generally the authors are quite careful and precise about the language used to describe results, in general.

The results are quite striking in that the find plenty of evidence for transient interaction of SHP1 with PD1 based on the biophysical measurements, but don't detect the interactions in pull down or in "in cell" microcluster recruitment experiments. In describing the pull-downs they discuss the issue of dissociation during washing potentially missing interactions that are taking place. I would prefer that the pull down is fine evidence for binding, but lack of pull down is not evidence for lack of binding. They should double check that this language is consistent. Also, unless something has changed in the microcluster binding experiments, this in situ recruitment of SHP2 to PD1 is only observed or a 2-3 minutes and then can't be detected, the situation for SHP2 becoming the same as it is for SHP1. If the kinetics are different in the cleaner systems that have now developed they should show this in a primary figure as this would be then different when what is reported previously.

The gap in this study is lack of any functional analysis. The Jurkat model could be quite useful as they have a relatively clean system for asking if the transient binding of SHP1 to PD1 has any functional impact, which they have not yet followed through on. Does PD-1 recruited SHP2 have any impact on function after the 5 minutes? Furthermore, the authors need to keep in mind that mice deficient in SHP2 respond to anti-PD1 checkpoint therapies (Rota, G., Niogret, C., Dang, A. T., Barros, C. R., Fonta, N. P., Alfei, F., Morgado, L., Zehn, D., Birchmeier, W., Vivier, E., & Guarda, G. (2018). Shp-2 Is Dispensable for Establishing T Cell Exhaustion and for PD-1 Signaling In Vivo. Cell Rep, 23(1), 39-49. https://doi.org/10.1016/j.celrep.2018.03.026). This is an important issue to discuss in light the the very interesting binding analysis the authors have performed. But I think the functional analysis can be part of a future paper.

I would suggest that the title be modified slightly from "SHP1/SHP2 discrimination" to "differential SHP1/SHP2 interaction" and leave discussion of discrimination until they have the functional data integrated over times that are relevant to T cell transcriptional regulation (1-2 hrs). The functional analysis can be in another paper, but it would be interesting to have a paragraph in the discussion raising the outstanding issues beyond stable binding detected by the pull-down and microcluster recruitment experiments- what are the implications for function. Could the transient interactions in the noise of the steady state and equilibrium measurements be functional?

I would summarise that the work is outstanding as biochemistry and biophysics and it should be published nearly as is. I'm suggesting minor revisions in that the changes are just to text, but I think this is important and somewhat nuanced aspect of the paper that will make it even more helpful to readers.

Significance

The authors generate a detailed descriptive data set about the component interaction of SHP1 and SHP2 SH2 domains with PD1 and BTLA intracellular domains. They then test hypotheses generated from the descriptive data set to better define the nature of the interactions and why PD1 recruits primarily SHP2, while BTLA mainly recruits SHP1. PD1 is a major driver or the cancer immunotherapy revolution and SHP2 is the major candidate for a signalling effector of PD1. This paper can become the reference paper for the specificity and engineering of this interaction, which will make it highly significant in a very active and still expanding field.

Referee Cross-commenting

I still feel that "discrimination" has a functional/activity connotation that is not addressed at all in this paper, but can be addressed. I'm happy to have the suggestion stand and let the authors decide. They need to live with it once its published. Another suggestion- the citations on regulation are mostly old. A good recent paper is Pádua, R. A. P., Sun, Y., Marko, I., Pitsawong, W., Stiller, J. B., Otten, R., & Kern, D. (2018). Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2. Nature Communications, 9(1), 4507. https://doi.org/10.1038/s41467-018-06814-w .

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

This study reveals the role of WW-PLEKHAs (PLEKHA5, 6 and 7) in the basolateral targeting of copper (Cu) transporter ATP7A. The Authors suggest that the WW-PLEKHAs/PDZD11/ATP7A interaction directs Cu-induced trafficking of ATP7A to the basolateral surface of epithelial cells. Suppression of WW-PLEKHAs impairs basolateral delivery of ATP7A and causes increased intracellular Cu levels. On the contrary, WW-PLEKHAs do not seem to participate in the retrieval of ATP7A back to the Golgi once the Cu levels return to basal values. To support these notions the manuscript provides a substantial set of the data, which were achieved with a wide repertoire of methods. In my view, this manuscript could be of interest to a broad readership, ranging from cells biologists to medical doctors. However, further revision should address the concerns outlined below.

Major points:

1. The Authors claim that at basal Cu conditions ATP7A resides in the TGN regardless of PDZD11 or WW-PLEKHAs depletion (Figs. 3, 4 and Fig. S6, S7). However, colocalization with TGN marker and its quantification are not shown. Thus, the colocalization of ATP7A with TGN marker (Golgin 97 should work in all cell types) has to be shown and its quantification (Pearson coefficient) has to be provided for control and all KO cells.

Response: We thank the Reviewer for this comment. We plan to carry out the IF colocalization of ATP7A with Golgin97 and quantifications for WT and KO clonal lines at basal Cu conditions.

1. Along the same line, ATP7A colocalization with TGN marker and its quantification also has to be conducted for the Cu washout experiments.

Response: We plan to carry out the IF colocalization of ATP7A with Golgin97 and quantifications for WT and KO clonal lines for the Cu washout conditions.

1. The authors say that upon addition of Cu ATP7A labeling was detected along lateral contacts, and near the apical and basal plasma membranes (Fig. 3B, WT). Here again "near apical" localization of ATP7A has to be clarified. This could either represent the ATP7A pool that still remains in the Golgi (which is usually close to apical surface in polarized epithelial cells) or the ATP7A pool delivered to the apical membrane of the cells. However, apical targeting of ATP7A would be odd considering previously published data that shows basolateral localization in polarized epithelial cells. Thus, the authors have to show whether "apical" ATP7A overlaps with TGN marker or with an apical marker (Gp135).

Response. This Reviewer is correct. Effectively we believe that the localization of ATP7A that we observe in cysts is not apical, but sub-apical, as shown for the localization of PLEKHA5, where colocalization with the apical marker gp135 clearly shows a different localization (Fig. 2I).

Therefore, we will carry out co-localization of ATP7A with gp135 in MDCK cells (the monoclonal antibody does not work on mCCD cells) and the labeling of the micrographs in Fig. 3B and 4B will be revised (sub-apical instead of apical). The labeling of PLEKHA5 sub-apical pool will also be revised (sub-apical and not apical) in Fig. 2.

1. PDZD11 or PLEKHA6/7 KOs lead to an ATP7A pattern, which looks like pretty large scattered vesicles that do not overlap with basolateral marker. What are these round ATP7A structures, endosomes? Colocalization assessments with EEA1 (early endosomes), VPS35 (sorting endosome) and LAMP1 (late endosomes) would be needed to clarify this. Alternatively, these vesicles could represent a fragmented Golgi with ATP7A inside. To establish this, labelling with TGN marker at these conditions is required.

Response: We thank the Reviewer for this comment. To clarify the nature (endosomes, Golgi, etc) of the membrane vesicles where ATP7A is localized in KO lines we will carry out double IF colocalization of ATP7A with either Golgin97 or early/sorting (which are mostly overlapped) endosome or the late endosome markers.

1. Biotinylation experiments. The Authors say that KO of either PDZD11, or PLEKHA7, or both PLEKHA6 and PLEKHA7, but not PLEKHA6 alone, decreased ATP7A levels at the basolateral surface of mCCD cells (Fig. 3G), while a small decrease in the basolateral levels of ATP7A is observed in PLEKHA5-KO, but not PLEKHA6-KO MDCK cells (Fig. 4G). Honestly, it is tough to see this. In Fig. 4G all ATP7A bands in the biotinylated fraction look similar. In Fig. 3G, the P11 and P6/7 KO bands of biotinylated ATP7A might be a bit less intense than in WT, while the P6 KO signal looks even more intense that WT. More convincing blots with quantification have to be provided for both figures.

Response: We will carry out additional immunoblots and quantifications of the biotinylation experiments results.

1. Along the same line. Why was apical biotinylation of ATP7A not included? It absolutely should be done to understand whether any KO induces apical mistargeting of ATP7A.

Response: The levels of ATP7A at the apical surface upon basal or elevated copper are negligible and not physiologically relevant, as established by previous biotinylation studies (for example Greenough et al AJP 2004, and Nyasae et al AJP-Gastrointest Liver Physiol 2007). We will carry out IF analysis of WT and KO cells with ATP7A and apical markers (ex. gp135) to clarify if the subapical labeling for ATP7A is or not on the apical membrane. Importantly, LESS, and not more subapical labeling is detected in KO lines (Fig. 3B, Fig. 4B), as we pointed out in the results section. Therefore, the KO lines do not show increased apical (mistargeting of) ATP7A.

1. Copper metabolism. The authors say that KO of either PDZD11 or PLEKHA6/7 results in higher Cu levels. What does this mean in terms of physiology and pathology? In the context of Menkes disease one has to show that this intracellular Cu increase is due to a reduction in Cu release from the cells. So, Cu release from the cells into medium has to be measured by ICP-MS or Cu64. On the other hand, it would be important to understand whether Cu accumulation in KO cells is toxic. To this end viability of KO cells should be tested in Cu dose-response experiments.

Response: The focus of this paper is the molecular mechanisms of ATP7A targeting to the BL plasma membrane, rather than a quantitative analysis of copper transport by and analysis of physiology/pathology of copper homeostasis ATP7A in our WT and KO cell lines. Our measurement of the intracellular copper using the CF4 probe was designed as a physiological readout to confirm that altered localization at the BL plasma membrane correlates with reduced copper extrusion, as it can be hypothesized. This said, to address this point, we plan to carry out an ICP-MS analysis of intracellular copper in selected WT and KO lines, after loading cells with different amounts of copper, and at different times after return to basal copper levels. CF4 and ICP-MS generally track, but they do measure distinct copper pools: CF4 measures exchangeable Cu pools while ICP-MS measures total Cu pools. We will also carry out a crystal violet analysis (see Gudekar et al, Scient. Reports, 2020) of the viability of WT and KO cells in the absence and presence of low or elevated copper levels, as suggested by the Reviewer.

1. How critical is WW-PLEKHAs or PDZD11 deficiency in terms of Cu metabolism? Are there genetic disorders or mouse phenotypes associated with their loss of function? If yes, do these phenotypes include any impairment of Cu metabolism?

Response: To our knowledge, no genetic study has addressed the role of WW-PLEKHAs and PDZD11 in Cu metabolism in vivo. PLEKHA7-KO mice are viable and were not reported to display any phenotype consistent with grossly altered Cu metabolism (Popov et al 2015). Mice KO for either PLEKHA5 or PLEKHA6 or PDZD11 have not been described. However, if WW- PLEKHAs have redundant functions in the trafficking of ATP7A, one would expect that mutation/KO of only one of them may not yield a significant phenotype. Furthermore, we cannot exclude that additional PDZ-containing proteins may participate in the trafficking of ATP7A, compensating a pathological or experimental loss of PDZD11. So, answering this question will require to generate single, double and triple KO mice for WW-PLEKHAs, and carry out a detailed analysis of in vivo Cu metabolism. This is beyond the scope of this paper. The text of the Discussion will be revised to address this comment.

1. Discussion. Could PH domains of WW-PLEKHAS be involved in their basolateral localization, thereby generating a targeting patch for ATP7A? Some publications suggest that the basolateral membrane might be enriched in specific PIPs, which in turn generates a favorable environment for some PH domains. Is this the case for PH domains of WW-PLEKHAS?

Response: This is an interesting hypothesis that should be investigated in future studies (lipidomic analysis of KO lines, overexpression studies, etc), but is outside of the scope of the present manuscript.

Minor points:

1. Fig. 6C. CFP-HA is a negative control but still gives a band (although of lower intensity). So how can one be sure that other interactions are specific? This is particularly worrying because the quantification shows a very minor (less than 1.5) increase in the intensity of bands corresponding to specific interactors.

Response: CFP-HA is used as a “negative control” 3_rd _protein, added to bait (GST-PDZD11) and prey (GFP-ATP7A-Cter) (Fig. 6C). The IB shows that in the presence of CFP-HA the bait binds the prey, which is in agreement with the previously reported interaction between PDZD11 and the C-terminal region of ATP7A (Stephenson et al JBC, 2005). The point of the Figure is to show that the interaction between bait and prey is enhanced in the presence of HA-tagged WW- PLEKHAs (again, CFP-HA is the negative control). We agree that the increase is not huge, but it is nevertheless statistically significant, based on several experiments (Fig. 6E).

1. Page 11. The result section title "WW-PLEKHAs promote PDZD11 binding to ATP7A through PDZD11 (Figure 6)" does not sound right and has to be corrected.

Response: The text was revised ("WW-PLEKHAs promote PDZD11 binding to ATP7A”).

Reviewer #1 (Significance):

Delivery of copper transporter ATP7A to the basolateral surface of epithelial cells is of great importance for maintenance of copper metabolism and, hence for human health in general. Impairment of this process in enterocytes causes fatal Menkes disease. However, the mechanisms driving basolateral targeting of ATP7A remained poorly characterized. This study provides a significant advance in our understanding of these mechanisms and opens new avenues for investigation of how WW-PLEKHAs/PDZD11-mediated targeting of ATP7A might be affected in the context of inherited disorders of copper metabolism.

Reviewer #2 (Evidence, reproducibility and clarity):

This manuscript uncovers new PDZD11 interactors that participate in trafficking of the copper transporter ATP7A from the Golgi/TGN to the cell periphery in response to high copper concentrations. These interactors named PLEKHA5, PLEKHA6, and PLEKHA7 interact with the N-terminal Pro-rich domain of PDZD11 through their WW domains. As PDZD11 interacts with the C-terminal region of ATP7A, the authors investigated the hypothesis that WW-PLEKHAs are required for copper-induced relocalization of ATP7A from the TGN to the plasma membrane where it functions in copper efflux. In vitro pull down experiments verified the formation of ATP7A-, PLEKHAs-, and PDZD11-containing complexes. Using using CRISPR/Cas9 technology, the authors have generated PDZD11-, PLEKHA5-, PLEKHA6-, and PLEKHA6/7-KOs cell lines.

Cells lacking one (or more) of these proteins were examined by microscopy with respect to their ability of targeting ATP7A to the cell periphery in response to copper. Abnormal trafficking of ATP7A in these mutant cell lines (PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-KOs) presumably prevented copper efflux since elevated intracellular copper was detected using the fluorescent copper probe CF4.

Although it is difficult to read across the article's figures and supporting figure files (going back- and-forth repeatedly), the manuscript is generally clear and well written, and the results seem well documented accompanied by a tremendous amount of work.

Comments.

1. Two-hybrid screen occurs in the nucleus. How the authors could explain the fact that the use of PDZD11 as a bait exhibited an interaction with PLEKHA5 and PLEKHA6 (as well as PLEKHA7) in this system? Microscopic analysis of PLEKHA5 showed a cytoplasmic submembrane localization with E-cadherin, whereas PLEKHA6 exhibited a localization along the plasma membrane at apical junctions. In the case of PLEKHA7, it is an adherens junction protein. Furthermore, these three proteins are quite big (1116, 1297, and 1121 AAs, respectively) with their WW regions at their N termini, which involved the expression of very long cDNAs fused to the TA domain. As truly membrane-associated proteins, isn't surprising that a two hybrid approach worked?

Response: We carried out several Y2H screens with a number of different baits, and we have always validated the physiological significance of the high score interactions (Pulimeno et al JBC 2011, Guerrera et al, 2016 JBC, and other unpublished data). So, it is an approach that reliably works very well. The Hybrigenics human placenta library that we used contains fragments of proteins, not the full-length proteins. Fig. 1A shows the preys identified with the Y2H using PDZD11 as a bait. The preys that were found comprise only the N-terminal regions of WW- PLEKHAs, not the FL proteins.

1. Fig. 1C, what are the 4 bands seen for the second blot (anti-HA) in lines 1, 2, 9 and 10? The blot was cut in a way that not enough of the membrane can be evaluated. Why using Ponceau for GST-baits and not using anti-GST antibodies? It would be much better having an uniform method (Western blot assays) to show the data.

This latter comment is true for Fig 1D, E, and Fig 6.

Response: The 4 bands seen for the second blot (anti-HA) in lanes 1, 2, 9 and 10 are non- specific cross-reaction of the antibodies with the baits, that are present in high concentration (and present also where there is no CFP-HA, in lanes 1 and 9). The preys can be identified on the basis of their molecular size. For example, no CFP-HA prey is detected, since its size is intermediate between the baits, thus the negative control is validated. We use Ponceau for 2 reasons: 1) Ponceau can detect very well baits on nitrocellulose membranes; 2) to use GST antibodies we would need to cut the membranes. But cutting membranes is not possible when the size of some preys (in this case, the negative control) is in the same range of sizes as the baits. Thus, if we used anti-GST antibodies we would have to strip and re-probe the membranes, which is not optimal in our experience to elicit good signals.

1. Fig 1B, why Caco-2 cells? All the other experiments were conducted with other cell lines such as mCCD and MDCK. Using different cell lines could give different results.

Response: We used Caco2 cells because the Y2H was carried out with a human bait on a human placental library, and Caco2 are human cells. We also tried to use MDCK cells, but the efficiency of the IP was lower.

1. Fig 1D, it is unclear whether the GST-PDZD11 fusion protein (bait) was present or not when used in pull down assays with GFP alone. This is a clear disadvantage of Ponceau, immunoblot would be much better to use.

Response: The labeling by Ponceau is not optimal in one image (Fig. 1D), probably due to a problem of transfer. But a clearer image for the same pulldown with the same bait is shown in the bottom panel of Fig. 1E (where we show 3 PDZD11 baits, FL, N-term and delta-24), and it clearly shows good normalization of baits. We stain baits with Ponceau for normalization.

1. In Fig 3A, under basal copper conditions, microscopic image of the PLEKHA6/7-KO seems indicate a distinct pattern of localization for ATP7A in comparison to that of WT. However, this difference does not seem to be highlighted in Fig 3E.

Response: We will re-examine all the micrographs used for the quantification and integrate the data with the results of the colocalization between ATP7A and TGN marker. This should allow us to establish whether there is a dissociation of ATP7A labeling from TGN marker labeling in KO cells, or else a fragmentation of the TGN in the double-KO mCCD cells.

In Figs 3F and 4F, what was the method for quantification?

Response: The methods for quantifications are described in the “Image quantification” section of the Methods. We will add new data about the quantification of co-localization of ATP7A with TGN and endosomal markers.

Along these lines, what is the copper concentration under basal conditions? How much copper was used for elevated copper conditions and what was the time of treatment?

Response: Basal conditions refers to normal cell culture medium (“Cell culture” section of the Methods), and elevated copper is 315 µM of CuCl2 dissolved in culture medium. Cells were treated for 4hr (MDCK) or 5hr (mCCD) when cells were cultured on Transwells, overnight in the case of cysts.

1. Is there any evidence for Atp7A-PDZD11-PLEKHAs association in vivo? Do the authors have assessed these protein-protein interactions using methods such as bimolecular fluorescence in cells?

Response: We have attempted co-IP experiments with endogenous proteins, but they were inconclusive, probably due to the different extraction conditions required to solubilize membrane (ATP7A) and cytoplasmic (WW-PLEKHAs, PDZD11) proteins, and a disassembly of the complex under the conditions required to solubilize ATP7A. We have not tried bimolecular fluorescence, but for the revision we plan to carry out Proximity Ligation Assay (PLA) experiments, which in our hands are very effective in assessing physiological proximity of proteins in cells. Our pulldown experiments however provide evidence that the three proteins form a complex, and that WW- PLEKHAs enhance the interaction between PDZD11 and ATP7A (Fig. 6C-E). This is a mechanism that we have shown occurs also for the complex between PLEKHA7, PDZD11 and Tspan33 (Shah et al, 2018 Cell Rep, Rouaud et al, 2020 JBC).

1. In Fig 5, do the authors have verified the mRNA (or/and protein) steady-state levels of metallothioneins? Probing whether metallothioneins are induced would strongly reinforced their conclusion as to whether an increase intracellular copper levels occurred in PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-Kos cell lines.

Response: We thank the Reviewer for this comment. In the revision we will carry out RT-PCR analysis of the levels of expression of mRNAs for Metallothioneins I and II.

1. In Fig 6E, what was the method for quantitative immunoblot assays? Have you used an Odyssey infrared imaging system (Li-Cor). What was the loading (internal) control under the same analytical method?

Response: The Li-Cor imaging system was used to capture the signals, and intensities were measured in Image Studio Lite program (Li-cor). Signals from the prey (C-terminus of ATP7A) were normalized to signals from the bait (GST-PDZD11) which were used as loading control.

1. In the case of the manuscript section entitled " PLEKHA5, PLEKHA6 and PLEKHA7 show distinct localizations in cells and tissues and define cytoplasmic...", (pages 5 to 7) the reader would benefit having a Table that would summarize all the data. It would be more understandable.

Response: We thank the Reviewer for this suggestion. We will include a Table in the revision.

1. Do PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins exist as multiple isoforms? If that is the case, for each of them, are they exhibiting the same tissue-specific expression profiles as shown in Fig S3? For each protein, if different isoforms exist, perhaps some of them participate in a different way for the targeting of ATP7A?

Response: No PDZD11 isoforms are known, but 15, 5, and 9 different protein-coding transcripts are reported (ensemble.org) for PLEKHA5, PLEKHA6 and PLEKHA7, respectively, the largest ones being the WW-containing transcripts. We focused exclusively on the WW-containing isoforms of PLEKHAs because PDZD11 binds to the WW domains, and the Y2H identified only the WW-containing isoforms of PLEKHA5, PLEKHA6 and PLEKHA7. The observation that the phenotype of PDZD11-KO cells is similar to that of either PLEKHA6-KO, PLEKHA7-KO or double-KO mCCD cells suggests that PLEKHA5, PLEKHA6 and PLEKHA7 WW-containing isoforms act in a complex with PDZD11. This is consistent with the previous observations that highlight a role of the C-terminal region of ATP7A in regulating its traffic, and the binding of the same region to PDZD11. However, we cannot exclude that PLEKHA5/6/7 isoforms that lack the WW domains could participate in the regulation of the targeting of ATP7A, through other, PDZD11-independent mechanisms. The text of the Discussion will be revised to clarify this point.

1. Is it known whether PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins participate in the copper-regulated trafficking of the ATP7B (Wilson) protein? In Fig S3, it is shown that they are expressed in liver, with PLEKHA7 exhibiting a slower migration (protein modification?). Alternatively, are they strictly involved in the regulation of ATP7A (Menkes)? Could the authors discuss about it?

Response: ATP7B lacks the PDZ-binding motif that is responsible for PDZD11 binding, and the C-terminus of ATP7B does not interact with PDZD11 (AIPP1) by beta-galactosidase assays in yeast, unlike ATP7A (Stephenson et al, JBC 2005). For this reason, ATP7B is not expected to be regulated by PDZD11 and WW-PLEKHAs. However, analysis of the localization of ATP7B in our cell lines could be done in future studies. The text of the Discussion will be revised to make this point.

1. The proposed model in Fig 7 is unclear illustrating a nucleus that consumes a lot of space while it is not involved in the proposed mechanism. Cellular proteins that are involved in the proposed mechanism should be bigger and their interactions that lead to formation of protein complexes must be better illustrated as a function of copper availability.

Response: The model of Fig. 7 will be re-drawn to take into account these suggestions.

1. Typo. Line 320: remove "or" and replace it by "and" : ...both PLEKHA6 and PLEKHA7 (Fig. 5A-D).

2. Typo. Line 329: remove (Figure 6) in the title.

Response: The typos were corrected.

Reviewer #2 (Significance ):

This study represents a significant advanced in the copper field.

Reviewer #3 (Evidence, reproducibility and clarity):

Summary

The authors identified some major interesting findings including the key role of WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) in the recruitment of PDZD11 targeting ATP7A to the cell periphery in response to elevated copper. Generating the antibodies against PLEKHAs and PDZD11 and various knock out cell lines and validating their expression in these cell lines and tissues is innovative. Further, the authors showed that copper dependent WW-PLEKHAs and PDZD11 regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

Major comments:

We are in agreement with the manuscript conclusions. Based on the presented studies, the authors propose the in-vivo role of WW-PLEKHAs and PDZD11 in ATP7A trafficking, and how microtubule dynamics and trafficking machinery regulate ATP7A localization. Additionally, investigating the effects of the cell membrane trimolecular complexes ATP7A-PDZD11-WW- PLEKHA on elevated copper would be impactful.

Additional notes:

1. Figure 4, the authors provide excellent data and images showing localization of PLEKHA, PDZD11 and ATP7A within different cell lines. Nevertheless, showing PLEKHA, PDZD11 and ATP7A localization on membrane of the cell surfaces at elevated copper condition with cell fractionation technique and their interaction through co-immunoprecipitation (co-IP) could validate author's hypothesis. At least the authors should comment on this.

Response: As stated in the response to comment n.6 from Reviewer #2, we attempted co-IP experiments with endogenous proteins, which were inconclusive. Our pulldown experiments provide evidence that the three proteins form a complex, and that WW-PLEKHAs enhance the interaction between PDZD11 and ATP7A (Fig. 6C-E). This is a mechanism that we have shown occurs also for the complex between PLEKHA7, PDZD11 and Tspan33 (Shah et al, 2018 Cell Rep, Rouaud et al, 2020 JBC). We plan for the revision to carry out Proximity Ligation Assay (PLA) experiments, which in our hands are very effective in assessing proximity of proteins in cells, when co-IPs are technically difficult or impossible.

1. Figure 5, Alternatively, intracellular copper levels by ICPMS in the cell lines would strengthen the results. As author's treated the cell lines with very high copper concentration, copper concentration dependent studies would be appreciated to verify how PLEKHA's and PDZD11 response depends on copper concentration

Also, the authors should clearly mention the number of replicates for each experiment and indicate in the figure legends.

Response. We will carry out ICP-MS to evaluate intracellular copper levels as a function of genotype. Depending on the results, we will carry out studies about the dose-dependence of the effects of copper. The number of replicates of the experiment will be mentioned in the Figure legend in the revised text.

Minor comments:

1. Figure1B, co-IP efficiency is lower in Caco-2 cells, therefore endogenous levels of PLEKHA5, PLEKHA6 and PDZD11 in Caco-2 should be checked and shown. Mention the number of replicates for the experiments.

Response. Endogenous levels of proteins are shown in the Input lanes. The low levels of PLEKHA5 in Caco2 cells are consistent with the IB analysis of tissue lysates, showing relatively low levels in intestine (Fig. S3D). The number of replicates of the experiment will be mentioned in the Figure legend in the revised text.

1. Figure 2A, as per result of 2A, E-cadherin labeling is missing. Figure 2M and 2N, author analyzed the co-localization of PLEKHA-5 in presence of nocodazole but not PDZD11. It would be interesting to see the PDZD11 as well after nocodazole treatment.

Response. E-cadherin-labelled panel will be added to Fig. 2A in the revision. We will also show the effect of nocodazole on the localization of PDZD11.

1. The result section title for figure 6 (line 329) is misleading. Also, trimolecular complex PLEKHA's, PDZD11 and ATP7A membrane localization at elevated copper concentration could be shown by immunofluorescence, if possible.

Response. The title of the section was revised, to reflect more accurately the results of Figure 6. It is now “WW-PLEKHAs promote the binding of the C-terminal region of ATP7A to PDZD11”.

Triple IF colocalization of endogenous PLEKHAs, PDZD11 and ATP7A is not possible for 2 reasons: 1) PDZD11 antibodies can only reveal endogenous junctional (clustered) labeling (Guerrera et al JBC2016); the lateral and cytoplasmic labeling is too weak, and can only be appreciated upon overexpression of PDZD11, as shown in Fig. 2B-E (co-expression with selected WW-PLEKHAs highlights how each PLEKHA directs PDZD11 to a different pool). 2) Both antibodies against PDZD11 and ATP7A were raised in rabbits, which makes it technically impossible to do triple labeling. We will address the question of the existence of the ATP7A- containing trimolecular complex by PLA analysis (ATP7A+PDZD11 and ATP7A+WW-PLEKHAs)._

1. General comment: it would be interesting to see the hypothesis and finding in mice model with copper accumulation (for example Atp7b KO mice) as PLEKHA's and PDZD11 are sensitive to copper concentration. Or at least the authors can comment on this future possibility.

Response. We agree with the Reviewer that mouse models could be useful to test the relevance of WW-PLEKHAs and PDZD11 as targets or effectors of copper-sensing mechanisms in vivo.

The text of the Discussion will be modified to envisage these possible future studies

Reviewer #3 (Significance):

In conditions including Menkes disease, occipital horn syndrome (OHS), and ATP7A-related distal motor neuropathy (DMN), characterized by altered intestinal copper metabolism, the new knowledge ATP7A associates with WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 is an important finding for the study of copper homeostasis.

As ATP7A is structurally similar to ATP7B (60% homology), the current study opens the area of the research where WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 could also play role in ATP7B trafficking to address not only Menkes disease but also Wilson disease and other diseases related to altered copper levels.

This is a well written and presented manuscript with excellent mechanistic work utilizing molecular imaging techniques and several confirmatory experiments. I recommend the manuscript to be accepted for publication with minor modifications.

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Referee #3

Evidence, reproducibility and clarity

Summary

The authors identified some major interesting findings including the key role of WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) in the recruitment of PDZD11 targeting ATP7A to the cell periphery in response to elevated copper. Generating the antibodies against PLEKHAs and PDZD11 and various knock out cell lines and validating their expression in these cell lines and tissues is innovative. Further, the authors showed that copper dependent WW-PLEKHAs and PDZD11 regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

Major comments:

We are in agreement with the manuscript conclusions. Based on the presented studies, the authors propose the in-vivo role of WW-PLEKHAs and PDZD11 in ATP7A trafficking, and how microtubule dynamics and trafficking machinery regulate ATP7A localization. Additionally, investigating the effects of the cell membrane trimolecular complexes ATP7A-PDZD11-WW-PLEKHA on elevated copper would be impactful.

Additional notes:

1. Figure 4, the authors provide excellent data and images showing localization of PLEKHA, PDZD11 and ATP7A within different cell lines. Nevertheless, showing PLEKHA, PDZD11 and ATP7A localization on membrane of the cell surfaces at elevated copper condition with cell fractionation technique and their interaction through co-immunoprecipitation (co-IP) could validate author's hypothesis. At least the authors should comment on this.
2. Figure 5, Alternatively, intracellular copper levels by ICPMS in the cell lines would strengthen the results. As author's treated the cell lines with very high copper concentration, copper concentration dependent studies would be appreciated to verify how PLEKHA's and PDZD11 response depends on copper concentration Also, the authors should clearly mention the number of replicates for each experiment and indicate in the figure legends.

Minor comments:

1. Figure1B, co-IP efficiency is lower in Caco-2 cells, therefore endogenous levels of PLEKHA5, PLEKHA6 and PDZD11 in Caco-2 should be checked and shown. Mention the number of replicates for the experiments.
2. Figure 2A, as per result of 2A, E-cadherin labeling is missing. Figure 2M and 2N, author analyzed the co-localization of PLEKHA-5 in presence of nocodazole but not PDZD11. It would be interesting to see the PDZD11 as well after nocodazole treatment.
3. The result section title for figure 6 (line 329) is misleading. Also, trimolecular complex PLEKHA's, PDZD11 and ATP7A membrane localization at elevated copper concentration could be shown by immunofluorescence, if possible.
4. General comment: it would be interesting to see the hypothesis and finding in mice model with copper accumulation (for example Atp7b KO mice) as PLEKHA's and PDZD11 are sensitive to copper concentration. Or at least the authors can comment on this future possibility.

Significance

In conditions including Menkes disease, occipital horn syndrome (OHS), and ATP7A-related distal motor neuropathy (DMN), characterized by altered intestinal copper metabolism, the new knowledge ATP7A associates with WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 is an important finding for the study of copper homeostasis.

As ATP7A is structurally similar to ATP7B (60% homology), the current study opens the area of the research where WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) and PDZD11 could also play role in ATP7B trafficking to address not only Menkes disease but also Wilson disease and other diseases related to altered copper levels.

This is a well written and presented manuscript with excellent mechanistic work utilizing molecular imaging techniques and several confirmatory experiments. I recommend the manuscript to be accepted for publication with minor modifications.

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Referee #2

Evidence, reproducibility and clarity

This manuscript uncovers new PDZD11 interactors that participate in trafficking of the copper transporter ATP7A from the Golgi/TGN to the cell periphery in response to high copper concentrations. These interactors named PLEKHA5, PLEKHA6, and PLEKHA7 interact with the N-terminal Pro-rich domain of PDZD11 through their WW domains. As PDZD11 interacts with the C-terminal region of ATP7A, the authors investigated the hypothesis that WW-PLEKHAs are required for copper-induced relocalization of ATP7A from the TGN to the plasma membrane where it functions in copper efflux. In vitro pull down experiments verified the formation of ATP7A-, PLEKHAs-, and PDZD11-containing complexes. Using using CRISPR/Cas9 technology, the authors have generated PDZD11-, PLEKHA5-, PLEKHA6-, and PLEKHA6/7-KOs cell lines. Cells lacking one (or more) of these proteins were examined by microscopy with respect to their ability of targeting ATP7A to the cell periphery in response to copper. Abnormal trafficking of ATP7A in these mutant cell lines (PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-KOs) presumably prevented copper efflux since elevated intracellular copper was detected using the fluorescent copper probe CF4.

Although it is difficult to read across the article's figures and supporting figure files (going back-and-forth repeatedly), the manuscript is generally clear and well written, and the results seem well documented accompanied by a tremendous amount of work.

Comments.

1. Two-hybrid screen occurs in the nucleus. How the authors could explain the fact that the use of PDZD11 as a bait exhibited an interaction with PLEKHA5 and PLEKHA6 (as well as PLEKHA7) in this system? Microscopic analysis of PLEKHA5 showed a cytoplasmic submembrane localization with E-cadherin, whereas PLEKHA6 exhibited a localization along the plasma membrane at apical junctions. In the case of PLEKHA7, it is an adherens junction protein. Furthermore, these three proteins are quite big (1116, 1297, and 1121 AAs, respectively) with their WW regions at their N termini, which involved the expression of very long cDNAs fused to the TA domain. As truly membrane-associated proteins, isn't surprising that a two hybrid approach worked?
2. Fig. 1C, what are the 4 bands seen for the second blot (anti-HA) in lines 1, 2, 9 and 10? The blot was cut in a way that not enough of the membrane can be evaluated. Why using Ponceau for GST-baits and not using anti-GST antibodies? It would be much better having an uniform method (Western blot assays) to show the data. This latter comment is true for Fig 1D, E, and Fig 6.
3. Fig 1B, why Caco-2 cells? All the other experiments were conducted with other cell lines such as mCCD and MDCK. Using different cell lines could give different results.
4. Fig 1D, it is unclear whether the GST-PDZD11 fusion protein (bait) was present or not when used in pull down assays with GFP alone. This is a clear disadvantage of Ponceau, immunoblot would be much better to use.
5. In Fig 3A, under basal copper conditions, microscopic image of the PLEKHA6/7-KO seems indicate a distinct pattern of localization for ATP7A in comparison to that of WT. However, this difference does not seem to be highlighted in Fig 3E.

In Figs 3F and 4F, what was the method for quantification?

Along these lines, what is the copper concentration under basal conditions? How much copper was used for elevated copper conditions and what was the time of treatment?

1. Is there any evidence for Atp7A-PDZD11-PLEKHAs association in vivo? Do the authors have assessed these protein-protein interactions using methods such as bimolecular fluorescence in cells?
2. In Fig 5, do the authors have verified the mRNA (or/and protein) steady-state levels of metallothioneins? Probing whether metallothioneins are induced would strongly reinforced their conclusion as to whether an increase intracellular copper levels occurred in PDZD11-, PLEKHA5-, PLEKHA6-, PLEKHA7-, and PLEKHA6/7-KOs cell lines.
3. In Fig 6E, what was the method for quantitative immunoblot assays? Have you used an Odyssey infrared imaging system (Li-Cor). What was the loading (internal) control under the same analytical method?
4. In the case of the manuscript section entitled " PLEKHA5, PLEKHA6 and PLEKHA7 show distinct localizations in cells and tissues and define cytoplasmic...", (pages 5 to 7) the reader would benefit having a Table that would summarize all the data. It would be more understandable.
5. Do PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins exist as multiple isoforms? If that is the case, for each of them, are they exhibiting the same tissue-specific expression profiles as shown in Fig S3? For each protein, if different isoforms exist, perhaps some of them participate in a different way for the targeting of ATP7A?
6. Is it known whether PDZD11, PLEKHA5, PLEKHA6, and PLEKHA7 proteins participate in the copper-regulated trafficking of the ATP7B (Wilson) protein? In Fig S3, it is shown that they are expressed in liver, with PLEKHA7 exhibiting a slower migration (protein modification?). Alternatively, are they strictly involved in the regulation of ATP7A (Menkes)? Could the authors discuss about it?
7. The proposed model in Fig 7 is unclear illustrating a nucleus that consumes a lot of space while it is not involved in the proposed mechanism. Cellular proteins that are involved in the proposed mechanism should be bigger and their interactions that lead to formation of protein complexes must be better illustrated as a function of copper availability.
8. Typo. Line 320: remove "or" and replace it by "and" : ...both PLEKHA6 and PLEKHA7 (Fig. 5A-D).
9. Typo. Line 329: remove (Figure 6) in the title.

Significance

This study represents a significant advanced in the copper field.

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Referee #1

Evidence, reproducibility and clarity

This study reveals the role of WW-PLEKHAs (PLEKHA5, 6 and 7) in the basolateral targeting of copper (Cu) transporter ATP7A. The Authors suggest that the WW-PLEKHAs/PDZD11/ATP7A interaction directs Cu-induced trafficking of ATP7A to the basolateral surface of epithelial cells. Suppression of WW-PLEKHAs impairs basolateral delivery of ATP7A and causes increased intracellular Cu levels. On the contrary, WW-PLEKHAs do not seem to participate in the retrieval of ATP7A back to the Golgi once the Cu levels return to basal values. To support these notions the manuscript provides a substantial set of the data, which were achieved with a wide repertoire of methods. In my view, this manuscript could be of interest to a broad readership, ranging from cells biologists to medical doctors. However, further revision should address the concerns outlined below.

Major points:

1. The Authors claim that at basal Cu conditions ATP7A resides in the TGN regardless of PDZD11 or WW-PLEKHAs depletion (Figs. 3, 4 and Fig. S6, S7). However, colocalization with TGN marker and its quantification are not shown. Thus, the colocalization of ATP7A with TGN marker (Golgin 97 should work in all cell types) has to be shown and its quantification (Pearson coefficient) has to be provided for control and all KO cells.
2. Along the same line, ATP7A colocalization with TGN marker and its quantification also has to be conducted for the Cu washout experiments.
3. The authors say that upon addition of Cu ATP7A labeling was detected along lateral contacts, and near the apical and basal plasma membranes (Fig. 3B, WT). Here again "near apical" localization of ATP7A has to be clarified. This could either represent the ATP7A pool that still remains in the Golgi (which is usually close to apical surface in polarized epithelial cells) or the ATP7A pool delivered to the apical membrane of the cells. However, apical targeting of ATP7A would be odd considering previously published data that shows basolateral localization in polarized epithelial cells. Thus, the authors have to show whether "apical" ATP7A overlaps with TGN marker or with an apical marker (Gp135).
4. PDZD11 or PLEKHA6/7 KOs lead to an ATP7A pattern, which looks like pretty large scattered vesicles that do not overlap with basolateral marker. What are these round ATP7A structures, endosomes? Colocalization assessments with EEA1 (early endosomes), VPS35 (sorting endosome) and LAMP1 (late endosomes) would be needed to clarify this. Alternatively, these vesicles could represent a fragmented Golgi with ATP7A inside. To establish this, labelling with TGN marker at these conditions is required.
5. Biotinylation experiments. The Authors say that KO of either PDZD11, or PLEKHA7, or both PLEKHA6 and PLEKHA7, but not PLEKHA6 alone, decreased ATP7A levels at the basolateral surface of mCCD cells (Fig. 3G), while a small decrease in the basolateral levels of ATP7A is observed in PLEKHA5-KO, but not PLEKHA6-KO MDCK cells (Fig. 4G). Honestly, it is tough to see this. In Fig. 4G all ATP7A bands in the biotinylated fraction look similar. In Fig. 3G, the P11 and P6/7 KO bands of biotinylated ATP7A might be a bit less intense than in WT, while the P6 KO signal looks even more intense that WT. More convincing blots with quantification have to be provided for both figures.
6. Along the same line. Why was apical biotinylation of ATP7A not included? It absolutely should be done to understand whether any KO induces apical mistargeting of ATP7A.
7. Copper metabolism. The authors say that KO of either PDZD11 or PLEKHA6/7 results in higher Cu levels. What does this mean in terms of physiology and pathology? In the context of Menkes disease one has to show that this intracellular Cu increase is due to a reduction in Cu release from the cells. So, Cu release from the cells into medium has to be measured by ICP-MS or Cu64. On the other hand, it would be important to understand whether Cu accumulation in KO cells is toxic. To this end viability of KO cells should be tested in Cu dose-response experiments.
8. How critical is WW-PLEKHAs or PDZD11 deficiency in terms of Cu metabolism? Are there genetic disorders or mouse phenotypes associated with their loss of function? If yes, do these phenotypes include any impairment of Cu metabolism?
9. Discussion. Could PH domains of WW-PLEKHAS be involved in their basolateral localization, thereby generating a targeting patch for ATP7A? Some publications suggest that the basolateral membrane might be enriched in specific PIPs, which in turn generates a favorable environment for some PH domains. Is this the case for PH domains of WW-PLEKHAS?

Minor points:

1. Fig. 6C. CFP-HA is a negative control but still gives a band (although of lower intensity). So how can one be sure that other interactions are specific? This is particularly worrying because the quantification shows a very minor (less than 1.5) increase in the intensity of bands corresponding to specific interactors.
2. Page 11. The result section title "WW-PLEKHAs promote PDZD11 binding to ATP7A through PDZD11 (Figure 6)" does not sound right and has to be corrected.

Significance

Delivery of copper transporter ATP7A to the basolateral surface of epithelial cells is of great importance for maintenance of copper metabolism and, hence for human health in general. Impairment of this process in enterocytes causes fatal Menkes disease. However, the mechanisms driving basolateral targeting of ATP7A remained poorly characterized. This study provides a significant advance in our understanding of these mechanisms and opens new avenues for investigation of how WW-PLEKHAs/PDZD11-mediated targeting of ATP7A might be affected in the context of inherited disorders of copper metabolism.

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Reply to the reviewers

Editor comments:

Thank you for sending your manuscript entitled "In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography" to Review Commons. We have now completed the peer review of the manuscript. Please find the full set of reports below.

We thank the editors of Review Commons and all the reviewers for their insightful comments which helped us to improve our manuscript. We have now modified our manuscript based on the Reviewers’ comments and would like to ask you to consider our revised manuscript for publication.

Reviewer #1:

This manuscript by the Jensen lab surveys a plethora of bacterial outer-membrane projections captured over the years by in situ cryo-tomography under near-native conditions. The authors classify the different visualized structures, highlighting both similarities and differences among them. They further describe molecular complexes that are associated with these projections. The manuscript highlights the abundance of such understudied structures in nature, indicating the need to deepen our exploration into their biological functions and mechanisms of action.

We thank the reviewer for her/his insightful comments that allowed us to improve our manuscript.

The authors should state in the Abstract and Introduction that only diderm bacteria and outer- membrane extensions are included in the study.

Done. We have modified the title, the abstract and the introduction to explicitly highlight this point.

In the Introduction or Discussion the authors should mention the limits of the in situ cryo-tomography, such as the difficulty to observe regions in between neigbouring bacterial cells, and into the thick bacterial cell body.

Done. We have added the following to our revised manuscript:

“Currently, only electron cryo-tomography (cryo-ET) allows visualization of structures in a near-native state inside intact (frozen-hydrated) cells with macromolecular (~5 nm) resolution. However, this capability is limited to thin samples (few hundred nanometers thick, like individual bacterial cells of many species) while thicker samples like the central part of eukaryotic cells, thick bacterial cells, or clusters of bacterial cells are not amenable for direct cryo-ET imaging. Such thick samples can be rendered suitable for cryo-ET experiments by thinning them first using different methods including focused ion beam milling and cryosectioning [30]. Cryo-ET has already been invaluable in revealing the structures of several membrane extensions, including Shewanella oneidensis nanowires [6], Helicobacter pylori tubes [15], Delftia acidovorans nanopods [25], Vibrio vulnificus OMV chains [16], and more recently cell-cell bridges in the archaeon Haloferax volcanii [31].” (Lines 108-118)

Please provide a legend to Table S1 explaining the numbers (organelles?), how many cells were viewed? I think that at least part of it should be included in the main text. Also, there are examples of vesicles emanating from H. pylori. This information is missing from Table S1.

Done. We added a column to the table indicating the number of cells available for each species. We also added the information about the vesicles in H. pylori to the table. This table is now incorporated into the main text of the manuscript as Table 1.

Please provide an ordered list including all the strains (and IDs of the specific isolates) used in this study and their genotypes.

Done. We added Table S1 to the revised manuscript that contains this information. This table also includes relevant references to all the published papers where these strains were previously used.

The authors describe in detail the H. pylori tubes that seem to be flagellum-core independent. However, the authors found previously (ref 15) that during infection, these structures are dependent on CagA T4SS, and they visualized T4SS sub-complexes in proximity to the point of tube emanation. This should be described and discussed in the text. Also, please indicate if the "host-independent" tubes are similarly dependent on T4SS.

Done. We added the following to the revised manuscript:

“The scaffolded uniform tubes of H. pylori that we observed were formed in samples not incubated with eukaryotic cells, indicating that they can also form in their absence. However, the tubes we found had closed ends and no clear lateral ports, while some of the previously-reported tubes (formed in the presence of eukaryotic host cells) had open ends and prominent ports [15]. It is possible that such features are formed only when H. pylori are in the vicinity of host cells. Moreover, while it was previously hypothesized that the formation of membrane tubes in H. pylori (when they are in the vicinity of eukaryotic cells) is dependent on the cag T4SS [15], we could not identify any clear correlation between the emanation of membrane tubes and cag T4SS particles in our samples where H. pylori was not incubated with host cells. We also show that the tubes of H. pylori are CORE-independent, indicating that they are different from the CORE-dependent nanotubes described in other species.” (Lines 303-313)

Is there any difference in the frequency or length of the tubes in the mutants presented in Figure 4? The flgS mutant in the image exhibits a very short filament; is that typical?

We did not see any significant statistical difference in the number or lengths of the tubes in these different mutants. We added Table S2 to the revised manuscript which details the number of cells we visualized for each mutant and the number of the tubes seen there. In all these mutants the lengths of the tubes ranged between few tens to hundreds of nanometers. In addition, we added Fig. S2 to show more examples of these tubes in each of these mutants.

Minor points:

-Please check full bacterial names that are sometimes missing (e.g., lines 110-112).

Done.

-There is no reference to panel 2G. Please check the references to all panels.

Done. Please see lines 154 and 183 in the main text.

-Lines 181-184: There is no figure related to the formation of teardrop-like extensions from C. pinensis. Please review the text accordingly.

Done. Corrected.

-Line 235, not clear to what "as these" refers to.

Done. We modified the text as the following:

“As these MEs/MVs from S. oneidensis were purified” (Lines 246-247)

-Line 241, not clear what "a secretin-like complex" is, and no reference is provided.

Done. We modified the text as the following:

“In the third category, we observed a secretin-like complex in many tubes and vesicles of F. johnsoniae. Secretins are proteins that form a pore in the outer membrane and are associated with many secretion systems like type IV pili and type II secretion systems (T2SS) [39–41]” (Lines 252-254)

Reviewer #1 (Significance)

As described in this manuscript, even in model bacteria these structures are generated (e.g., Caulobacter forms the hardly studied nanopod extensions). The manuscript also provides visual categories of these structures, defining "extension types" that are likely to be used by the scientific community for years to come, similar to the initial pili classification during the 1960s-70s. It is a "descriptive study," in the positive sense of the term, as it significantly contributes to the field of bacteriology.

We thank the reviewer for her/his kind words and enthusiasm about our work. It is an honor to have our work compared to the seminal pili classification work done in the 1960s-70s by pioneers in the field of bacteriology.

Reviewer #2:

The manuscript "In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography" by Kaplan et al., identifies and catalogues membrane extensions (MEs) and membrane vesicles (MVs) from 13 different species using cryo-electron tomography. Furthermore, they identify and discuss several protein complexes observed in these membrane projections.

The manuscript is beautifully written, interesting, and genuinely got this reviewer excited about the biology. I applaud the authors on their manuscript and have only minor comments and a few thoughts that the authors may wish to think on and discuss.

We thank the reviewer for her/his kind words and insightful comments that allowed us to improve our manuscript.

Some schematics throughout the introduction would be useful to readers new to the field/ outside the field who are not used to these different membrane structure features.

We thank the Reviewer for this suggestion. First, we made an extra figure with schematics showing the cell body and membrane tubes but that was rather redundant with Figure 8. For this reason, we added explicit labels to figure 1 highlighting the cell body and the tubes in these examples to help the reader following that figure and the subsequent ones. However, if the Reviewer has an explicit suggestion/view about the schematics then we would be very happy to do that.

The size of scale bars should be indicated on the figure panels themselves rather than in the figure legend to assist the reader.

Done.

In reference to lines 193-196 - what was the extracellular environment like in these micrographs? Were other cells present? Could it be the extracellular environment/surrounding cells that stimulate pearling? Have the authors considered this? Please discuss if relevant/insightful.

This is a good point. The cells were usually plunge-frozen in their standard growth media (except in H. pylori where the cells were resuspended in PBS and subsequently plunge-frozen). Yes, there are other cells present in the sample, however, usually, only one cell is present in the field of view of the tomogram as areas with multiple cells have thick ice and therefore not amenable for cryo-ET imaging. We added the following to the revised manuscript:

“As usually only one (or part of a) cell is present in the cryo-tomogram, we can’t exclude that differences in the extracellular environments, like the presence of a cluster of cells in the vicinity of the individual cells with pearling tubes, might play a role in this observation” (Lines 198-201).

"Randomly-located complexes" in this reviewers opinion should actually be described "seemingly randomly-located complexes" given there may be an organization present that is beyond the resolution limit of this study.

The is a good point. Indeed, we can’t exclude that these complexes have a preferred localization in specific lipid patches that we can’t detect in our cryo-tomograms. We added the following statement to the revised manuscript:

“These complexes, which were also found in the OM of intact cells, did not exhibit a preferred localization or regular arrangement within the tube at least within the fields of view provided by our cryo- tomograms (Fig. 5a & b).” (lines 227-230).

In reference to lines 287-292 - is it possible this has to do with lipid composition? Have the authors considered this? Please discuss if relevant/insightful.

Done. We added the following to the revised manuscript:

“In addition, differences in the lipid compositions among the various species investigated here might also play a role in the formation of these different forms of projections” (Lines 299-301).

Reviewer #2 (Significance ):

These results advance the field by shedding new light on bacterial membrane extension morphologies. The authors use a cryo-ET to catalogues membrane extensions and membrane vesicles which has not been done before.

This paper is likely to be of interest to structural biologists, biophysicist, membrane protein biologists, virologists and microbiologists.

This reviewer is a single-particle cryo-EM structural biologist with interest in membrane proteins._

We thank the reviewer for her/his enthusiasm about our work described here.