Beyond, OK. Now back again.

Note; First use of OSGrid tag?

Reviewer #2 (Public Review):

The pleiotropic adaptive effects of mutations have been extensively characterized, with numerous examples of both specialist and generalist adaptations. However, it is less clear whether and how these pleiotropic patterns change over the course of evolution for a given evolving species. The authors attempt to answer this question by characterizing the pleiotropic fitness effects of ~150 adapted S. cerevisiae populations at 200-generation intervals over 1000 generations of evolution. The authors provide strong quantitative evidence that patterns of pleiotropy are highly dependent on the evolution environment, and changes substantially over relatively short evolutionary timescales (1000 generations). They further show that even with a fixed genotype and selective environment, replicate populations can substantially vary in their pleiotropic profiles (again, in an environment-dependent manner), and this variation tends to increase over evolutionary time. The authors thus provide substantial evidence that the evolution of "generalist" or "specialist" types is not deterministic but rather strongly dependent on both the specific evolving system and evolutionary stochasticity.

The authors use DNA barcodes to uniquely tag each of their evolving populations, then use bulk fitness assays to measure the fitness of each population at 5 different timepoints across five different environments. These methods are well established, and appear to have been implemented correctly. The resulting data clearly answers the proposed question, and supports the major claims of the paper.

Most previous studies of the pleiotropic effects of adaptation characterize a relatively small number of independently evolved lines, limiting our ability to draw quantitative conclusions (but see Kinser et al 2020 eLife). Furthermore, I am unaware of any large-scale study characterizing how the pleiotropic effects of mutations change over evolutionary time. While the specific experiments and data are not of use to the broader community, the major findings substantially advance our understanding of the evolutionary process and open up new avenues of both theoretical and empirical research.

Also see

• Trophic coherence determines food-web stability | PNAS
• https://hypothes.is/search?q=tag%3A%27trophic+coherence%27

§ 15 (1) S. 1 2. InfSchMV i.V.m. § 6 (1) Nr. 4 2. InfSchMV

more

What did he not have more time to decide?

SciScore for 10.1101/2021.08.03.455003: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.08.04.21261592: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

This part is a bit hard to follow. I would suggest to first explain more generally how does the 'thresholding step' work - as in how does this step reduce overestimation? Then proceed by explaining the ins and outs of the thresholding step. Did you specifically use the same method as in Cirtwill and Hambäch (2021)? This part was unclear to me.

.quiz 确实我当下最严重的问题。如果保存素材，和管理好素材

使用readwise后，加上最近加入了inline tag的技巧。我现在的素材已经到达了block和card的级别了呢。怎么才能有效管呢？

.cmt 没有解决我当前的问题，但是提供了一些有意思的工具和使用场景

.ctk 现在的我也能更明确的定义自己当前碰到的问题了，就是如何管理inline tag级别的素材。啧啧，感觉搞定了以后readwise和flomo的价值对我就能起飞了。

.cmt 记得之前尝试过一阵子bear，吸引我的点就是inline tag，看来当时的灵感是对的。因为现在很依赖inline tag，针对卡片和block级别内容做标注。

碎片的这tag很有意思诶。就和我的randomstuff很像，但是碎片两个字儿更有趣一点

¿A qué hemos llegado?

SciScore for 10.1101/2021.07.30.21261234: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your code and data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

这个解题思路简单，实现起来也不复杂。对于添加相应tag有设计外加权限限制即可。不知道notion和飞书支持不支持。

imp. 什么是高质量内容

搜索关键字，而且应该看到过很多次了。所以，之后的批注要有钩子，不管是基于问题还是基于黑体字的展现形式还是基于inline tag

SciScore for 10.1101/2021.07.30.454436: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.30.454437: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 37. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.25.453673: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.28.454072: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

In Roam use the #inbox tag

Paper Discovery:

• Research Rabbit
• Connected Papers
• Citation Gecko
• Papers With Code

Zotero SciHub - for downloading papers into one's Zotero instance

Academic Networking

• lens.org (also good for discovery)
• OrcID
• Impact Story

Ginko App (trees and cards interface) for writing with interesting import and export

around 2:56: A bit too much Andy Matuschak worship? Pretty sure he didn't invent the so-called Andy Mode. Index cards pre-dated them surely as did Ward Cunningham's Smallest Federated Wiki. There are many other idex-card UIs prior to Matuschak.

Map of Content (MOC) apparently comes from How to Make a Complete Map of Every Thought You Think by Lion Kimbro.

• it's a glorified Table of Contents really

Plugins he's using:

• 3:22:15 add codemirror matchbrackets js
• 3:23:31 advanced tables
• 3:26:09 Better word count
• 3:26:41 calendar
• 3:27:32 copy code block
• 3:28:25 cycle through panes
• 3:29:55 Dataview
• 3:30:33 editor syntax highlight
• 3:30:43 extended mathjax
• 3:31:08 file explorer note count
• 3:32:04 full-screen mode
• 3:32:23 highlgiht public notes
• 3:33:11 kanban
• 3:33:35 kindle highlights
• 3:33:56 metatable
• 3:34:24 mindmap
• 3:35:36 NLP dates
• 3:36:10 pane relief
• 3:36:42 paste URL
• 3:37:21 periodic notes
• 3:37:44 recent files
• 3:37:59 relevant line number
• 3:38:33 show current open note
• 3:38:45 review
• 3:39:43 sliding panes
• 3:40:42 super charged links
• 3:41:11 random note
• 3:41:39 tag wrangler
• 3:42:22 templater
• 3:46:05 zoom

textsniper for OCR and potentially text-to-speech, apple only, so leark for others.

MathPix

Should one flag this URL, or the earliest capture of this URL at archive.org? Does that depend on the purpose, the context by which the annotation is to be used?

Edit: Is there another tag that could be used here?

work flow of roambrain/Maarten van Doornm page, example of catch knowledge

Cf. Leviticus or Ireland. Don't maximize efficiency. Leave slack in the line.

DOI: 10.1016/j.celrep.2019.12.012

Resource: (Sigma-Aldrich Cat# A2220, RRID:AB_10063035)

Curator: @ethanbadger

SciCrunch record: RRID:AB_10063035

What is this?

DOI: 10.1016/j.cell.2019.11.003

Resource: (GenScript Cat# A01738, RRID:AB_2622223)

Curator: @ethanbadger

SciCrunch record: RRID:AB_2622223

What is this?

DOI: 10.1016/j.immuni.2019.10.006

Resource: (Cell Signaling Technology Cat# 3724, RRID:AB_1549585)

Curator: @ethanbadger

SciCrunch record: RRID:AB_1549585

What is this?

I've always had history and philosophy around me from a relatively young age. Some of this stems from a practice of mnemonics since I was eleven and a more targeted study of the history and philosophy of mnemonics over the past decade. Some of this overlaps areas like knowledge acquisition and commonplace books which I've delved into over the past 6 years. I have a personal website that serves to some extent as a digital commonplace book and I've begun studying and collecting examples of others who practice similar patterns (see: https://indieweb.org/commonplace_book and a selection of public posts at https://boffosocko.com/tag/commonplace-books/) in the blogosphere and wiki space. As a result of this I've been watching the digital gardens space and the ideas relating to Zettelkasten for the past several years as well. If you'd like to go down a similar rabbit hole I can recommend some good books.

DOI: 10.1016/j.str.2019.09.006

Resource: (Cell Signaling Technology Cat# 2276, RRID:AB_331783)

Curator: @ethanbadger

SciCrunch record: RRID:AB_331783

What is this?

DOI: 10.1016/j.str.2019.09.006

Resource: (Cell Signaling Technology Cat# 3724, RRID:AB_1549585)

Curator: @ethanbadger

SciCrunch record: RRID:AB_1549585

What is this?

DOI: 10.1371/journal.pone.0222924

Resource: (Sigma-Aldrich Cat# C3956, RRID:AB_439680)

Curator: @Naa003

SciCrunch record: RRID:AB_439680

What is this?

DOI: 10.12688/wellcomeopenres.15403.1

Resource: (Sigma-Aldrich Cat# H9658, RRID:AB_260092)

Curator: @Naa003

SciCrunch record: RRID:AB_260092

What is this?

DOI: 10.1016/j.celrep.2018.07.065

Resource: (Diagenode Cat# C15200190, RRID:AB_2737029)

Curator: @evieth

SciCrunch record: RRID:AB_2737029

Curator comments: Diagenode Cat# C15200190,HA tag monoclonal antibody

What is this?

Task related plugin for Obsidian.

<small><cite class='h-cite via'>ᔥ <span class='p-author h-card'>Eleanor Konik</span> in 2021-07-17: Obsidian Mobile, Community Events & Graph Tips (<time class='dt-published'>07/29/2021 11:06:38</time>)</cite></small>

DOI: 10.7554/eLife.48318

Resource: (MBL International Cat# PM020, RRID:AB_591224)

Curator: @Naa003

SciCrunch record: RRID:AB_591224

What is this?

DOI: 10.7554/eLife.48318

Resource: (MBL International Cat# M180-3, RRID:AB_10951811)

Curator: @Naa003

SciCrunch record: RRID:AB_10951811

What is this?

DOI: 10.1128/mBio.01876-19

Resource: (Qiagen Cat# 34660, RRID:AB_2619735)

Curator: @Naa003

SciCrunch record: RRID:AB_2619735

What is this?

DOI: 10.3390/biom9080368

Resource: (Covance Cat# MMS-101P-1000, RRID:AB_291259)

Curator: @Naa003

SciCrunch record: RRID:AB_291259

What is this?

DOI: 10.1002/ijc.32609

Resource: (Cell Signaling Technology Cat# 3724, RRID:AB_1549585)

Curator: @Naa003

SciCrunch record: RRID:AB_1549585

What is this?

DOI: 10.1111/cge.13610

Resource: (Cell Signaling Technology Cat# 3724, RRID:AB_1549585)

Curator: @Naa003

SciCrunch record: RRID:AB_1549585

What is this?

Reviewer #1 (Public Review):

Yao Rang and collaborators find that heterologous expression of TMEM120A from mouse and human in cells that lack Piezo1 does not result in poke- or stretch-activated currents in whole cells or excised patches, and further detect no mechano-sensitive currents when the purified human protein is reconstituted in giant unilamellar vesicles. Together with high-quality positive controls with Piezo1, Piezo2 and TMEM63a, the results presented here call into question a previous proposal (Beaulieau-Laroche et al., Cell 2020) that TMEM120A functions as the long sought-after mechano-activated channel responsible for detecting painful touch.

Although the evidence supporting a channel function for TMEM120A is not strong, it remains to be ruled out that the discrepancies between the two studies arise from the different methods that were used to deliver the mechanical stimuli, as mentioned by the authors in the Discussion, or from the C-terminal mCherry tag attached to human TMEM120A in this study that was not present in the construct used by Beaulieau-Laroche et al.

Upon determination of the structure of full-length human TMEM120A in nanodiscs using cryo-EM, the authors find that the protein forms a dimer with six transmembrane helices per subunit and a cytosolic N-terminal coiled coil domain. Surprisingly, the authors find a density attributable to coenzyme-A (CoASH) located within a highly conserved cytosolic cavity at the transmembrane domain. The authors provide evidence from mass-spectrometry and isothermal titration calorimetry (ITC) to demonstrate that CoASH binds TMEM120A, and solidify their conclusions by showing that mutation of a residue close to the CoASH density in TMEM120A disrupts binding measured by ITC. The authors show that a potential ion-conduction pathway in their TMEM120A structure would be occluded by CoASH on the cytosolic side and on the extracellular side by a series of not well-conserved residues. Finally, the authors solve a structure in detergents where no density for CoASH is observed, as expected from spectroscopic data showing that detergent-reconstitution results in loss of CoASH binding. In this structure, a conformational change is suggested to occur on the cytosolic cavity entrance where a loop becomes reoriented to occlude the cavity that is otherwise occupied by CoASH. Together, the data presented paints an intriguing alternative for the function of TMEM120A proteins with a role in metabolism or CoA transport.

Although the main conclusions are well supported by the evidence, it is challenging to appraise many of the interesting structural observations pointed out by the authors because the experimental data (i.e. the density) is in most cases not depicted. Some of these observations for which only the model rather than the experimental data is shown include the hinge-like motif at the dimer interface (Fig. 3C), the CoASH binding site (Fig. 4E and Fig. 4 Supplement 1C), the difference in the conformation of the IL5 loop between the apo and CoASH-bound structures (Fig. 5), the extracellular constriction of the possible ion-conduction pathway (Fig. 4 Supplement 1D and Fig. 5 Supplement 2), as well as the comment that the observed density in the structures cannot accommodate other CoA-derivatives, for which data is not shown. The relatively low resolution at which the data were obtained raises concerns regarding many of these detailed observations.

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

Learn more at Review Commons

Reply to the reviewers

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.

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.

I'm annotating the text using hypothes.is - it looks like you cannot annotate an image with this. You have to sign up to annotate text like this and log in.

Reviewer #1 (Public Review):

Energy in the form of ATP is key for any function of the cell. Most organisms have a series of protein complexes in their membrane that transport proton against a gradient using the redox reactions and this is then used by the enzymes called ATP synthases that generate ATP, the energy needed to sustain life. The first enzyme in this electron transport chain is called complex I and uses the oxidation of NADH to the reduction of ubiquinone, which is coupled to proton translocation. The unique shape of this complex had been realized from early electron microscopy studies and they can be divided into a soluble domain that comprises all the co-factors needed for electron transfer and the membrane domain that has modules for proton translocation. Depending on the organism, this complex can be compact with only the core 14 subunits as found in many bacteria or decorated with a number of other subunits as found in eukaryotes. These additional subunits are implicated in the stability and regulation of the complex. With the advances in cryoEM, in the last few years there has been a flurry of structures from different organisms, the composition and arrangement of subunits, potential functional states and description of the mechanism. An important factor to consider is that these complexes are labile and the detergents which are used for purification can have a profound effect on the structure as well as any conclusion drawn from it. In the field of Bioenergetics, one of the key questions that have fascinated scientists for a long time is how the electron transfer is coupled to proton translocation and few different hypotheses has been put forward.

Adding to the growing number of complex I structures, the manuscript by Kolata and Efremov discusses the structure of a complex I from Escherichia coli, a well-studied enzyme and amenable to easy genetic engineering, essential to verify proposed mechanisms. The key observations of the current work and future prospective are discussed below.

The E. coli complex I is known to be labile and previous attempts to crystallize the whole complex had yielded a low-resolution structure of the membrane domain. The authors have overcome this by introducing a tag in the bacterial genome using CRISPR technology minimizing the number of steps needed for enrichment. The manuscript also addresses one of the concerns of the complex I structures i.e., effect of detergent environment by determining the structure of a mesophilic bacterial complex I in nanodisc. Despite the presence of the lipidic environment, substantial number of particles have only the soluble peripheral arm and only half the number of particles exist as full complex indicating the extreme instability of this complex. By extensive computational classification to address heterogeneity, the maps have been improved both for the full complex and the peripheral arm at a very high resolution (2.1 Å), allowing to build almost a complete protein model as well as a number of water molecules, key in understanding the mechanism. Differences in E. coli complex with structures of other enzymes are discussed, in particular the junction of the peripheral arm and the membrane domain and TMH8 in the NuoM subunit of the membrane domain. With all the structural and biochemical details, the authors propose a coupling mechanism that is different from those proposed previously and involves the idea that protons enter the ubiquinone cavity via the periplasmic or intra-membrane side (in organelles) and this is coupled to 3 protons being transported by the membrane domain in the opposite direction. Thus, for each reduction of ubiquinone 4 protons are translocated in two pumping cycles.

While the mechanism is simple and elegant, the absence of a structure with bound ubiquinone (the cavity for ubiquinone is inferred from other structures) and with NADH, different states observed by classification, dissociation of the peripheral arm - all of which asks for some caution and is a caveat. Nevertheless, these structures are important and will be the platform for further studies (addition of NADH, ubiquinone, pH etc.,) and the proposal can be verified by further biochemical and computational studies.

Reviewer #2 (Public Review):

Summary of what the authors were trying to achieve:

Kolata and Efremov set out to achieve a cryoEM structure of functional E. coli respiratory complex I (proton-pumping NADH-ubiquinone oxidoreductase) reconstituted in lipid nano-discs by single particle cryo-electron microscopy (cryoEM).

Strengths and weaknesses of the methods and results:

A strength of the paper is that E. coli respiratory complex I is one of the most studied homologues of the enzyme with many important functional and mutagenesis studies published. For this reason, the complex I field has been anticipating the structure for some time.

A strength of the paper is the production of a E. coli cell line harboring a Twin-Strep tag on the complex I subunit NuoF using the Crispr-Cas9 system. This allows the authors to develop a single step purification of the complex and will be very useful for any future work on E. coli complex I.

A strength of the paper is the multiple methods used to test the integrity of the nanodisc reconstituted complex (i.e., size exclusion chromatography and mass photometry) and the functional assays demonstrating inhibitor sensitive NADH:Q1 oxidoreductase activity.

A strength of the paper is the high-resolution structure of the peripheral (cytoplasmic) arm of the complex. This structure reveals several features unique to complex I and suggests different strategies for stabilization of the peripheral arm subunits have evolved in different lineages.

A weakness of the paper is the disruption of the complex during cryoEM grid preparation resulting in about half of the observed particles missing the membrane arm and likely also contributing to the disorder and biased orientation seen in the intact complexes. This leads to poor density in the membrane arm for all of the intact complex I structures presented and large variations in the local resolution of the membrane arm focused refinement.

A weakness of the paper is the disorder of important functional regions of the complex, namely the NuoH TMH1, whose disorder is unique to these nanodisc E. coli structures, and the NuoA TMH1-TMH2 loop. As the NuoH TMH1 forms part of the entry to the quinone tunnel of the complex, its absence in the structure leads to concerns regarding the function of the nanodisc preparation. Its absence it curious as this suggests flexibility of the helix, as pointed out by the authors, but the authors also state that there is not enough room in the nanodisc to accommodate this helix (given the visible density for the lipid and membrane scaffold protein). These observations suggest denaturation or unfolding in this region of the complex as opposed to simple flexibility. Unfortunately, the NADH:Q1 functional data do not fully address these concerns at Q1 is far more soluble that the native Q8 substrate of the complex. Although the Q1 activity is sensitive to the inhibitor Piericidin A, which clearly demonstrates that the Q1 reduction is occurring in the native quinone binding site as Piericidin A binds specifically at that site, this does not preclude the possibility of Q1 accessing this binding site via a different path. In fact, the structures indicate that given the flexibility in the connection between peripheral and membrane arms of the complex, the quinone binding site is likely open to the cytoplasm. This leads the authors themselves to conclude that the structures presented are likely disrupted/uncoupled states in which the energy converting mechanism of the complex is not likely possible.

A weakness of the paper is the building of atomic models into regions of the map which do not contain sufficient detail to warrant atomic models. This is particularly the case for the intact models of complex I as well as the membrane arm focused maps and results in low map-model correlations (0.58-0.71). The models were clearly highly restrained during refinement, resulting in good geometry, as is necessary for low resolution regions. But being able to restrain the geometry is not sufficient for placing atoms into regions where the density is weak or absent. If additional information was used in building/constraining the model, such as the X-ray structure, the regions of the model that are biased towards the X-ray structure model needs to be made clearer. Also, in several places in the membrane arm map residues bulge out of the density (side chain and main chain) leading to possible frame shifts with respect to the match between subsequent residues in the model and the map (see NuoM Ile168 for example).

A weakness of the paper is that several specific claims are made about the positions of side chains but, when investigated, the density for those side chains is poorly resolved. An example of this is NuoH Lys274, which is in a low-resolution region of the map and although is fit as well as possible must be considered low confidence given the local resolution (nearby residues Phe277 and Phe282 have almost no side chain density for example).

A weakness of the paper is that the conformational changes seen between the membrane and peripheral arm of the complex in the different 3D classes are difficult to interpret. It is unclear if they are mechanistically significant or, perhaps more likely given the amount of broken complex observed, due to partial disruption of the complex before it completely breaks apart.

A strength of the paper is the interesting and original mechanistic proposal put forward by the authors. But a weakness is that it is unclear how this proposal stems from the structural data presented. Also, the arguments presented are difficult to follow in their current form and warrant a more detailed discussion with the requisite thermodynamic treatment. This may warrant a more complete discussion in an appendix or unless the authors can more convincingly show how the data presented in the paper suggests their proposed mechanism perhaps a separate review article. Furthermore, the proposed mechanism, as presented would make a simple prediction that in the absence of NuoM and NuoL (or equivalent subunits in other species) complex I would not pump any net protons. Experiments that are relevant to this prediction have been done in E. coli (NuoL deletion) and Y. lipolytica (nb8m deletion that results in loss of both NuoM and NuoL subunits). See https://pubmed.ncbi.nlm.nih.gov/21417432/ and https://pubmed.ncbi.nlm.nih.gov/21886480/. In both cases the complex is still able to pump protons. The behavior of the NuoL deletion in E. coli is reconcilable with their proposed mechanism as NuoM is still present, however, the case of the nb8m deletion in Y. lipolytica is more difficult to reconcile with their proposed mechanism. The authors would need to address these experiments in order to include their proposed mechanism.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

Overall, despite the many strengths of this paper detailed above it is unclear whether the authors achieved their goal of a structure of functional E. coli respiratory complex I reconstituted in lipid nano-discs. It appears that under the current grid preparation conditions that the complex is under excessive stress resulting in partial denaturation and partial-to-complete dissociation. Given the clear biophysical data presented on the intactness of the complex in solution, this disruption likely occurs during grid preparation and further optimization of grid conditions may resolve this issue. With the current maps more work needs to be done to improve the map-to-model correlation and to clearly indicate the regions in the models where this correlation is low.

Likely impact of the work on the field, and the utility of the methods and data to the community:

As the first structure of respiratory complex I from the important model organism E. coli this work will have a big impact on the field. This is due to the history of studies on complex I that have been performed using this organism. The structure of the peripheral arm presented here is itself a major advance for this field and presents several important new insights into the evolution of complex I in different lineages. Due to the problems outlined above the structure of the membrane arm and proposed mechanism are more difficult to evaluate and if the limitation of the models are not made more clear this could lead to misinterpretation by non-experts in the structural biology.

DOI: 10.7554/eLife.22540

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What is this?

DOI: 10.1016/j.celrep.2019.06.068

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DOI: 10.1002/cne.24428

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Curator comments: GFP Tag Polyclonal Antibody Thermo Fisher Scientific Cat# A-11122 also A11122

What is this?

DOI: 10.1002/cne.24428

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Curator: @Naa003

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Curator comments: GFP Tag Polyclonal Antibody Thermo Fisher Scientific Cat# A-11122 also A11122

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DOI: 10.1002/cne.24147

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DOI: 10.1002/cne.24035

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DOI: 10.1002/cne.24035

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DOI: 10.1002/cne.23752

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DOI: 10.1002/cne.23689

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DOI: 10.1093/nar/gky846

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DOI: 10.1002/cne.24428

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DOI: 10.1002/jnr.23760

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DOI: 10.1002/cne.24158

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DOI: 10.1002/cne.24158

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DOI: 10.7554/eLife.21221

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DOI: 10.7554/eLife.21221

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DOI: 10.1002/cne.24234

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What is this?

I had the very same problem with the #reMarkable. I really wanted to like it, but the missng backlight was a showstopper for me.

Now I take my handwritten notes on an iPad Pro 12.9, which is heavy with a less-than-great recharge cycle of one day.

Indeed, Maha! We hope participants in the Summer Open Pedagogy Adventure series might ponder, and explore here in annotation space.

How /what does annotation activity open up? Is that experience going to be the same for all? Yes we can create safer spaces in private group Hypothes.is annotation, but it is more cumbersome and we trade off perhaps the possible serendipity of connection with peers offering different perspectives.

And Why? Reply here and tag your annotation opsa

And this what we are asking participants in the Summer Open Pedagogy Adventure series to explore and question. Is this framing of students as content creation, which is valuable, the full range of what is open pedagogy? Engage with us here and elsewhere we we look at annotation as a form of open pedagogy, of going being the creation and adoption of OERs but building activities around them.

What do you think of Annotations being freely reused as licensed CC0?

What do our adventurers think? Annotate and tag opsa

And that is what we are doing here in the OE Global Open Pedagogy Summer Adventure (2021) in particular, the interactivity strand.

We can use these tools to augment this text but also reply to others, like a greeting space. So if you are here during our live workshop, or maybe later, reply with a greeting to let us know who you are, how you see (or maybe want to ask about) web annotation as an act of open pedagogy.

And explore the many annotations already present here...

And if you remember, add a tag of opsa to your annotations during this activity -- watch what happens.

Let's go adventuring with web annotation

Reviewer #1 (Public Review):

Yao Rang and collaborators find that heterologous expression of TMEM120A from mouse and human in cells that lack Piezo1 does not result in poke- or stretch-activated currents in whole cells or excised patches, and further detect no mechano-sensitive currents when the purified human protein is reconstituted in giant unilamellar vesicles. Together with high-quality positive controls with Piezo1, Piezo2 and TMEM63a, the results presented here call into question a previous proposal (Beaulieau-Laroche et al., Cell 2020) that TMEM120A functions as the long sought-after mechano-activated channel responsible for detecting painful touch.

Although the evidence supporting a channel function for TMEM120A is not strong, it remains to be ruled out that the discrepancies between the two studies arise from the different methods that were used to deliver the mechanical stimuli, as mentioned by the authors in the Discussion, or from the C-terminal mCherry tag attached to human TMEM120A in this study that was not present in the construct used by Beaulieau-Laroche et al.

Upon determination of the structure of full-length human TMEM120A in nanodiscs using cryo-EM, the authors find that the protein forms a dimer with six transmembrane helices per subunit and a cytosolic N-terminal coiled coil domain. Surprisingly, the authors find a density attributable to coenzyme-A (CoASH) located within a highly conserved cytosolic cavity at the transmembrane domain. The authors provide evidence from mass-spectrometry and isothermal titration calorimetry (ITC) to demonstrate that CoASH binds TMEM120A, and solidify their conclusions by showing that mutation of a residue close to the CoASH density in TMEM120A disrupts binding measured by ITC. The authors show that a potential ion-conduction pathway in their TMEM120A structure would be occluded by CoASH on the cytosolic side and on the extracellular side by a series of not well-conserved residues. Finally, the authors solve a structure in detergents where no density for CoASH is observed, as expected from spectroscopic data showing that detergent-reconstitution results in loss of CoASH binding. In this structure, a conformational change is suggested to occur on the cytosolic cavity entrance where a loop becomes reoriented to occlude the cavity that is otherwise occupied by CoASH. Together, the data presented paints an intriguing alternative for the function of TMEM120A proteins with a role in metabolism or CoA transport.

Although the main conclusions are well supported by the evidence, it is challenging to appraise many of the interesting structural observations pointed out by the authors because the experimental data (i.e. the density) is in most cases not depicted. Some of these observations for which only the model rather than the experimental data is shown include the hinge-like motif at the dimer interface (Fig. 3C), the CoASH binding site (Fig. 4E and Fig. 4 Supplement 1C), the difference in the conformation of the IL5 loop between the apo and CoASH-bound structures (Fig. 5), the extracellular constriction of the possible ion-conduction pathway (Fig. 4 Supplement 1D and Fig. 5 Supplement 2), as well as the comment that the observed density in the structures cannot accommodate other CoA-derivatives, for which data is not shown. The relatively low resolution at which the data were obtained raises concerns regarding many of these detailed observations.

Reviewer #2 (Public Review): 

This manuscript describes studies on the structural determinants of activation for the adhesion GPCR (aGPCR) GPR116 both in vitro and in vivo. The authors define key residues for activation on the receptors' N-terminus (the "tethered agonist") and the extracellular loops. Thus, the studies provide novel insights into the structural determinants of GPR116 activation. However, some interpretational issues (detailed below) complicate some of the authors' conclusions. Specific comments are as follows: 

1) Results section, first paragraph, last sentence: The authors write, "These results taken together indicate that the H991A mutant is capable of proper trafficking to the membrane, is able to response to exogenous peptide, but is unable to be cleaved and activated by endogenous ligands in vivo." The last part of this sentence represents an over-interpretation, as the data shown in Figure 1 do NOT show that the non-cleavable receptor is unable to be activated by endogenous ligands in vivo. It is entirely conceivable that a non-cleavable aGPCR could still be activated by endogenous adhesive ligands if those ligands were to change the position of the tethered agonist in manner that alters receptor signaling activity. 

2) The data shown in Fig. 1B (surface expression of non-cleavable H991A mutant) need to be quantified in some way in order to be interpretable. 

3) Results section, second paragraph, penultimate sentence: The authors write, "These data demonstrate that while the non-cleavable receptor is fully activated in vitro by exogenous peptides corresponding to the tethered agonist sequence, cleavage of the receptor and unmasking of the tethered agonist sequence is critical for GPR116 activation in vivo." However, the non-cleavable GPR116 mutant actually has two key differences from WT: i) lack of full liberation of the tethered agonist sequence, and ii) lack of liberation of a free NTF, which might dissociate from the CTF and have important in vivo physiological actions on its own. Isn't it conceivable that the lack of a freely mobile NTF contributes to the similarity in lung phenotype between the non-cleavable knock-in mutant and the GPR116 knockout? Based on the data shown in Figure 2, how can the authors claim these data demonstrate that unmasking of the tethered agonist is critical for GPR116 activation? The data could equally be interpreted as showing that liberation of a free NTF is critical for the physiological effects of GPR116 in vivo. 

4) Figure 3: If the authors' hypothesis is that the tethered agonist must be liberated in order to allow activation of GPR116, then why do ANY of the Flag-tagged mutant constructs exhibit constitutive signaling activity? Doesn't the N-terminal Flag tag prevent the tethered agonist from being exposed? How can these data be reconciled with the authors' model? 

5) The data shown in Fig. 3D are lacking statistical comparisons, so it is not possible to tell whether any of the differences between the mutants are statistically significant. 

6) The data shown in Fig. 4D (surface expression of the ECL mutants) need to be quantified in some way. 

7) In interpreting the results of the ECL mutations on GPR116 signaling activity, it is unclear why the authors so explicitly propose that these data demonstrate that the tethered agonist must be interacting with ECL2. Isn't it possible that ECL2 mutants with impaired receptor signaling activity simply lock the receptor in an inactive state? In this way, the effects of the ECL2 mutations could be explained without invoking a physical interaction between the putative tethered agonist and ECL2.

SciScore for 10.1101/2021.07.19.21260661: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

This study had several limitations as well as strengths. The collection of breast milk samples were not directly observed and samples were collected with nonstandard sampling time points. Therefore, the breast milk samples collected at the onset of symptoms may not reflect duration of exposure to SARS-CoV-2, which may be attributed for the differences observed in IgA kinetics. We also relied on the maternal report of SARS-CoV-2 test results, symptoms and treatments received, however, all participants completed a semi-structured interview guided by trained study staff who prompted for specifics with the aid of a calendar. Another limitation in our study was lack of reactivity to the S1 protein and its fragments produced in vitro using an E. coli based reaction mixture. This was likely due to the lack of eukaryotic post-translational modifications, including N-linked glycosylation which is abundant in S1. We compensated for this by including purified S protein and RBD in the array and by assaying purified S, RBD and the NTD of S by electro-chemiluminescent immunoassay (ECLIA). The added value of this study comes from the longitudinal assessment of milk antibody levels and the breadth of antigens covered by the protein microarray and ECLIA platforms, coupled with COVID-19 patient cases with unique profiles.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.21.453140: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.20.453054: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.21.453274: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

basics of pull-down assay

"...makes it really difficult to figure "

wilson Huckleberry may have a solution in his pockets already...

Note: First tag in Booklady's space.

How about this for a test?

Reviewer #3 (Public Review):

The structure of the acid-sensing ion channel ASIC1a, a proton-gated cation channel, has been determined in resting, desensitized, and toxin-bound open states. However, the intracellular N- and C-termini are not resolved in any of these structures. Couch et al. sought to outline their structures and any conformational changes associated with ASIC gating using FRET coupled with patch-clamp electrophysiology. The authors inserted fluorescent protein tags at the N-terminus and various positions on the C-terminus of ASIC1 and measured the distance between these tags and the plasma membrane using the membrane-embedded FRET acceptor dipicrylamine (DPA). Using fluorescence lifetime measurements, the authors demonstrated that the N- and C-termini of a given subunit are in close proximity to one another. By observing FRET between fluorescent proteins on N- and C-termini of the same ASIC subunit, the authors demonstrate that there is no substantial rearrangement between the intracellular termini during pH gating. However, they did observe that the N- and proximal C-termini do move relative to plasma membrane during the transition from the resting to the pH-desensitized state.

This paper should be of interest to those working on acid-sensing ion channels and of broader interest to those working on ion channels, receptors, and membrane protein structural biology. The study was well designed and the data are of high quality. The authors took great care to provide a conservative interpretation of their data. I have only minor concerns regarding sources of error, particularly with respect to interpretation of the small effects the authors observe in many of their FRET experiments.

• Figure 2D shows rather small changes in ΔF/F-15 mV between fluorescent protein labels inserted at different positions in the ASIC sequence, particularly for the YFP constructs. As this metric is determined from the top and bottom asymptotes for the Boltzmann fits shown in Figure 2C, it would be useful to have some estimate as to the error associated with the fits at extreme values. Perhaps the authors could provide fits to their data (as in Figure 2C), including confidence intervals, or some similar estimate as to the size of the expected error compared to the effect size in Figure 2D.

• Along those same lines, the authors use an interesting (and potentially generalizable) approach to reducing background from intracellular proteins in their experiments: co-transfecting their channels with empty plasmid DNA. What percentage of the remaining fluorescence signal is the result of intracellular background? How would that affect the data in Figure 2 and 3? Is the ΔF/Fnorm curve for YFP labeled positions in Figure 2-figure supplement 4 so flat because of contaminating background fluorescence?

• In Figure 3D, the FRET efficiency between CFP-cA1-cA1 and N YFP at a 1:15 ratio of the two plasmids is higher than the FRET efficiency between CFP and YFP in the same subunit, even though the authors conclude that fluorescent proteins on the same subunit show considerably more FRET than fluorescent proteins on neighboring subunits. Could this indicate that the N-termini of adjacent subunits are closer together than the N- and C-termini of a single subunit? If, on the other hand, this effect were entirely the result of crowding in the membrane why is FRET efficiency substantially lower when CFP-cA1-cA1 is co-expressed with C4 YFP? Wouldn't this construct produce a similar crowding effect?

• On page 23, the authors state that they detected no pH-dependent changes in FRET between their GFP tag on the N-terminus of ASIC1 and an RFP tag on the channel's C-terminus. However, Figure 4 shows a small, but significant change in fluorescence between pH 8 and pH 7.

• The interpretation of distances between various tagged position on ASIC and the plasma membrane in Figure 2 is based on using two different colored tags with two different distance dependences. However, the interpretation of the data from Figure 5 provided on page 25 is less clear. For example, the reduction in fluorescence from the N-terminal tag is interpreted as the tag moving closer to the plasma membrane. Without similar data from a YFP tag to verify, it seems equally likely that the reduction in fluorescence (at steady state) could result from a movement away from the plasma membrane.

Author Response:

Reviewer #3 (Public Review):

[...] I have only minor concerns regarding sources of error, particularly with respect to interpretation of the small effects the authors observe in many of their FRET experiments.

• Figure 2D shows rather small changes in ΔF/F-15 mV between fluorescent protein labels inserted at different positions in the ASIC sequence, particularly for the YFP constructs. As this metric is determined from the top and bottom asymptotes for the Boltzmann fits shown in Figure 2C, it would be useful to have some estimate as to the error associated with the fits at extreme values. Perhaps the authors could provide fits to their data (as in Figure 2C), including confidence intervals, or some similar estimate as to the size of the expected error compared to the effect size in Figure 2D.

Thank you for this point. We did use Boltzman’s fits to get the asymptotes for each cell and calculate a ΔF/F. However, we could also use a ‘fit free’ approach of simply taking the difference between fluorescence values measured at -180 mV and that at +120 mV, divided by that at -15 mV to normalize for each cell. This approach completely avoids any error associated with fitting the data or imposing any model at all. Using this approach results in slightly different ΔF/F values but the pattern of statistical significance is identical. This new analysis is included in Figure 2 figure supplement 4. It has also been corrected for multiple comparisons.

• Along those same lines, the authors use an interesting (and potentially generalizable) approach to reducing background from intracellular proteins in their experiments: co-transfecting their channels with empty plasmid DNA. What percentage of the remaining fluorescence signal is the result of intracellular background? How would that affect the data in Figure 2 and 3? Is the ΔF/Fnorm curve for YFP labeled positions in Figure 2-figure supplement 4 so flat because of contaminating background fluorescence?

This is a great question. We originally hoped that the CFP and YFP quenching data from different positions could be used to triangulate both a distance from the membrane and a value for background fluorescence assuming that CFP and YFP would yield similar background fluorescences. An analogous approach was used in Zachariassen et al. Proc Natl Acad Sci, 2016 where an equal background was assumed between conformational states within a recording. In the end, the YFP quenching appeared to have a greater background than CFP. We speculate that this may be because the YFP variant we used matures faster than the CFP (mVenus, 17.6 min verses mTurquiose2, 33.5 min; FPbase.org) and hence the YFP matures faster than the ‘new’ channels get to the plasma membrane. However, at present we are uncertain how much of the background fluorescence signal to confidently attribute to this intracellular FP issue.

• In Figure 3D, the FRET efficiency between CFP-cA1-cA1 and N YFP at a 1:15 ratio of the two plasmids is higher than the FRET efficiency between CFP and YFP in the same subunit, even though the authors conclude that fluorescent proteins on the same subunit show considerably more FRET than fluorescent proteins on neighboring subunits. Could this indicate that the N-termini of adjacent subunits are closer together than the N- and C-termini of a single subunit? If, on the other hand, this effect were entirely the result of crowding in the membrane why is FRET efficiency substantially lower when CFP-cA1-cA1 is co-expressed with C4 YFP? Wouldn't this construct produce a similar crowding effect?

We strongly suspect the N termini of adjacent subunits are closer to each other than N and C of single subunit simply because the N FPs would all be at the same ‘height’ or same depth with respect to the plasma membrane. Thus the measured FRET in this case primarily reflects distances in the x-y plane. This contrasts with the N and C FPs on the same or different subunits where both x-y distances and axial distances come into play.

• On page 23, the authors state that they detected no pH-dependent changes in FRET between their GFP tag on the N-terminus of ASIC1 and an RFP tag on the channel's C-terminus. However, Figure 4 shows a small, but significant change in fluorescence between pH 8 and pH 7.

We have corrected for multiple comparisons within a figure. As a result, this effect is no longer statistically significant (adjusted p value is 0.063).

• The interpretation of distances between various tagged position on ASIC and the plasma membrane in Figure 2 is based on using two different colored tags with two different distance dependences. However, the interpretation of the data from Figure 5 provided on page 25 is less clear. For example, the reduction in fluorescence from the N-terminal tag is interpreted as the tag moving closer to the plasma membrane. Without similar data from a YFP tag to verify, it seems equally likely that the reduction in fluorescence (at steady state) could result from a movement away from the plasma membrane.

This is a very good point. We tried to perform DPA quenching of YFP-containing constructs at pH 6.0, but the acidification resulted in proton-quenching of the YFP fluorescence (Figure 4). We didn’t feel confident in measuring DPA quenching with the concomitant loss of YFP fluorescence due to acidification. Therefore, we relied on the pH 8.0 CFP and YFP data as a starting point (Figure 2). Given the C1 insertion gives the greatest extent of CFP quenching, it is reasonable to place it around the top of the curve. The N position could then be on the left or right side of the hump or peak in the CFP distance curve. The N quenching is comparable to the C2 insertion quenching (Figure 2D, left) yet the N FP is ~ 16 amino acids from the pore-forming membrane helices while the C2 insertions is ~ 40 amino acids away. For reference, the C1 is ~ 24 amino acids. Thus we are reasonably confident the N insertion is on the left side of the hump or peak. A reduction in ΔF/F would indicate movement closer to the plasma membrane. While technically possible that the N position could move further away from the membrane, this would have to be a >25 Å movement. Given there are only 16 amino acids between the CFP and the beginning of TM1 of the channel, we do not think such a dramatic movement outward could occur.

I'm partial to the "Principle of Least Power" in the Axioms of Web Architecture document cited in the bibliography. (The language there better captures the thought and presents it more convincingly, in my opinion.)

Shortcut: https://www.w3.org/DesignIssues/Principles.html#PLP

Reviewer #2 (Public Review):

In the manuscript by Paul W. Maarten and Sidhu A.. et al., the authors surveyed the importance of the DNA binding domain (DBD) and C-terminal domain (CTD) of BRCA2 in response to DNA damaging agents in cells, and the conformations adopted by recombinant constructs. The characterization of these domains are paramount in understanding basic BRCA2 function for novel future exploitation in cancer therapeutics. While the DBD and CTD domain have notable functions in DNA binding, nuclear localization upon DSS1 binding, RPA exchange, and replication fork protection, their role in response to damage and conformational modulation had been unexamined. Studying BRCA2 domain deletion in human cell lines is difficult as human BRCA2 contains a NLS in the C-terminus of the protein. The authors exploit the fact that the murine BRCA2 that has an additional N-terminal nuclear localization sequence to overcome lethality and the study of deletion mutants in human cell lines. Cell survival assays show the DNA binding domain of BRCA2 is most important for cell survival when treated with DNA damaging agents IR, Olaparib, MMC, and Cisplatin. The authors also show this system is functional as they observe the DBD domain is the most important for gene targeting assays that are repaired by homologous recombination. By assessing various DNA damaging agents, the authors highlight the multiple roles of BRCA2 in varying DNA repair processes from DSB repair, BIR, crosslink repair, etc. Interestingly, the C-terminus of BRCA2 does not appear to play a role in to cells when treated with MMC or Cisplatin but plays an important role in mediating self-organization. The authors describe that both the DBD and CTD domains of BRCA2 are important for RAD51 foci formation following IR. Assessing BRCA2 single-particle tracking in live cells, the authors show that the deletion of the DBD and the CTD domain leads to an increased immobile fraction following IR treatment. Using biophysical single molecule analysis, the authors analyzed recombinant BRCA2 DBD , CTD, and double mutants in the presence of ssDNA and interacting protein RAD51. The authors determined these domains are important for BRCA2 self-interactions and BRCA2 conformational rearrangements in the presence of ssDNA supporting in vivo analysis. Biophysical analysis show that the DBD and CTD are important for BRCA2 conformational dynamics that are observed with binding protein RAD51 or DNA substrates.

Strengths:<br> • These studies exploit a murine cellular system to overcome cellular lethality observed in BRCA2 depletion in human cell lines, which allows them to study the mouse BRCA2 protein and associated domain deletions.<br> • The authors also utilize bright photostable fluorophore's called JF646 Halo Tag ligand to study BRCA2, the deletion mutants, and RAD51 using live cell imaging. This is a great technical advancement in observing BRCA2 function in vivo.<br> • The in vivo and in vitro studies both support important roles of the DBD and CTD domain in BRCA2 dynamics.

Weaknesses:<br> • The importance of the in vivo work with these domains and the findings presented is confounded by a lack of biological replicates and clear presentation of statistical analysis within figures in the manuscript.<br> • As both domains are important for response to DNA damaging agents (IR, Olaparib, MMC, and Cisplatin) if a function specification could be made to the deletion mutations this would be most valuable to the field. Assaying molecules with varying substrates (Ex-forked substrates, crosslinked substrates, ssDNA substrates containing DNA lesions) or other protein players (DSS1) may aid in teasing out these roles.<br> • The discussion focuses on DSS1 and the DBD domain, yet the paper lacks any experimental analysis of BRCA2-DSS1. A biophysical analysis with recombinant protein DSS1 may greatly enhance the impact of this work on the field.<br> • It is unclear if the larger BRCA2 assemblies or the deletion mutants in the manuscript form via an oligomerization mechanisms or a phase separated mechanism. Speculation from authors would be valuable.

Reviewer #3 (Public Review):

The biochemical and genetic characterization of BRCA2 has been an ongoing challenge in the DNA repair field as the protein is large, prone to degradation, and expressed at low levels in most cell types. While certain features of BRCA2 have been described previously including its ability to bind and load RAD51 onto resected DNA substrates, much remains to be discovered. In this study, the authors combine genetic studies in mouse ES cells with biochemical analysis to examine the spatial dynamics and molecular architecture of BRCA2. Notably, they utilize an innovative approach coupling endogenous tagging of mouse BRCA2 with a HALO tag to monitor BRCA2 movement within live cells by single particle tracking.

I applaud the authors for achieving a highly technical approach to epitope tagging both endogenous BRCA2 alleles in mouse ES cells and combining this strategy with a HALO tag providing additional utility for a variety of cell biological experiments. By analyzing the endogenous alleles, the authors' system provides physiological levels of protein expression as transcription will be driven by the endogenous promoter thus preserving stoichiometric protein interactions within the cell and avoiding artifacts caused by overexpression.

The authors determine the influence of the DNA binding domain (DBD) and c-terminal binding (CTD) on the dynamic activities of BRCA2. They begin by exposing cells containing 3 different deletion mutants ΔDBD, ΔCTD, and the double mutant ΔDBDΔCTD to four different types of DNA damage (IR, PARPi, MMC, and cisplatin). Notably, ΔDBD displays significant impairment in survival in response to all 4 types of DNA damage. The ΔCTD, in contrast, demonstrates less sensitivity to IR and Olaparib, however, complements as well as WT BRCA2 in response to crosslinking agents MMC and cisplatin. My only criticism in this aspect of the work is that it would have been informative to include a truncated BRCA2 (mimic of a patient pathogenic mutation) or null allele to compare to the survival of the ΔDBD and ΔCTD mutants. I realize that these alleles may be inviable but the authors should clearly state if that was indeed the case.

The authors then go on to demonstrate that the ΔDBD and ΔCTD mutants are recruited to sites of IR damage in a similar manner to WT BRCA2 based on number and intensity of foci. I think it would be informative if the authors provided statistical significance for the graphs depicting the quantitation of foci number and intensity as there do appear to be differences between the mutants and the WT protein. There appears to be a delay in the kinetics of recruitment, especially at the 2 hr timepoint, for the mutants compared to WT BRCA2, which could indicate a defect in the recognition of the DNA damage. Only at the 2 hr timepoint following IR are there less RAD51 foci, and of a lesser intensity, in the three deletion mutants compared to WT BRCA2. Another possibility is the results could be interpreted as a defect in RAD51 loading and/or stabilization of the nucleoprotein filament. While immunofluorescence imaging of DNA repair foci have become common practice to measure protein recruitment to damage, it is impossible to know exactly what is happening in these foci with any granularity.

Next, the authors measure BRCA2 movement in the mouse ES cells taking advantage of the HALO tag to track single particles. While technically and visually alluring, it is difficult to extract mechanistic insight from the results. DNA damage induces changes in diffusion leading to BRCA2 molecules with restricted mobility; the authors demonstrated this phenomenon in a prior publication. The deletion mutants appear to have little effect upon BRCA2 mobility.

Finally, the authors utilize scanning force microscopy to analyze binding of the purified human BRCA2 proteins to RAD51 and ssDNA. In the absence of RAD51/ssDNA binding, there is a notable shift in the deletion mutants from oligomeric forms to monomeric compared to full length WT BRCA2. Upon binding to RAD51, there is a dramatic change from multimeric to monomeric forms for the WT BRCA2 (~7% to 74%) with a slight suppression of these changes shown for the deletion mutants. While WT BRCA2 forms extended molecular assemblies upon binding ssDNA, not surprisingly, deletion of the DBD or CTD fail to demonstrate any significant changes in physical architecture. In both situations, the mutant proteins respond to RAD51 and ssDNA in a dampened manner likely due to altered or loss of binding. While the architectural effects of RAD51 and ssDNA binding to BRCA2 are measurable by SFM, it is difficult to reconcile these changes in shape and oligomerization to defects in response to DNA damage and at which specific steps in homologous recombination these physical forms would impact.

Strengths:

1. Generation of mouse ES cells with both endogenous alleles of BRCA2 containing the deletion mutations in addition to a HALO tag is an incredible technical breakthrough and will be a highly valuable reagent for genetic and cell biological studies of mouse BRCA2.<br> 2. The deletion mutants ablating either the DBD or the CTD, or both, is a great genetic approach to understanding the role of these key domains in BRCA2. The response of these mutants (versus WT BRCA2 as a benchmark) to various DNA damage (IR, PARPI, MMC, cisplatin) provides interesting information delineating the roles of these two important domains in BRCA2. For example, the ΔCTD mutant is significantly sensitive to IR and Olaparib, yet complements as well as WT BRCA2 in response to the crosslinking agents MMC and cisplatin.<br> 3. The BRCA2 protein is notoriously difficult to purify and yet the authors succeeded in purifying 4 different forms of the protein for biophysical analysis. While it is difficult to interpret the various forms of BRCA2 by SFM, there are clear differences in the architecture between WT and the three c-terminal mutants. These differences are highlighted upon binding to RAD51 or ssDNA.

Weaknesses:

1. While the separation-of-function result for the CTD deletion in response to crosslinking agents MMC and cisplatin is a novel and compelling result, it would have been informative to compare the survival results and gene targeting assay using a BRCA2 null or mimic of patient mutation (truncating mutation) to see how these 3 mutants stack up against a completely non-functioning BRCA2 allele. Likely, the BRCA2 null alleles are inviable but perhaps a conditional system or truncating allele similar to a patient germline mutation would give a window into response compared to the DBD and CTD deletion mutants.<br> 2. It's not clear in the manuscript what new information we are learning about the mechanisms of BRCA2 in the single particle tracking (SPT) data. The differences in mobility between the mutants and WT BRCA2 seem minimal, but more importantly, it is not immediately clear how these data help us understand the normal cellular functions of BRCA2. No doubt, the technology and innovation to track single particle proteins in the nuclei of cells is impressive, but the authors should clearly explain how we can gain mechanistic insight from the SPT data that is presented in this manuscript.

General Comments:

It is unclear how missing the c-terminal domain (CTD) or the DNA binding domain (DBD) of BRCA2 can be interpreted as having "roles beyond delivering strand exchange protein RAD51" unless a complete biochemical workup of the deletion mutants was performed to detect any alterations in DNA binding, stimulation of RAD51 dependent strand exchange, etc... While interesting and certainly an impressive technical feat, foci imaging and single particle tracking do not provide much information on mechanism (i.e. whether BRCA2 is binding DNA and loading/nucleating RAD51).

The interpretations in the discussion are not overstated, however, I somewhat disagree with the notion that the data, as presented, clarifies the role of BRCA2 beyond its canonical functions of RAD51 loading and nucleation on resected DNA substrates. I would have liked if the authors discussed the idea that it is surprising that mouse ES cells can tolerate complete loss of the DBD, CTD, and loss of both together. Questions that should be addressed in include some of the following: Are proliferation rates compromised compared to WT cells? Are they experiencing replication stress in the absence of any exogenous damage? Further, is there something unique about mouse ES cells that may differentiate BRCA2 behavior that would be expected in somatic human cells?

It is interesting to note that many years ago Ashworth and Taniguchi published back-to-back papers in Nature (2008) describing BRCA2 reversion alleles from in vitro screens of BRCA2 mutant cells selected in cisplatin or PARPi such that some of these reversions resulted in huge deletions of the entire DBD of BRCA2, and yet, they promoted resistance to PARPi. In this context, I would much appreciate if the authors commented on their findings that their constructed DBD deletion is not resistant to PARPi and if they offered some speculation as to why the reversions in those previous studies were.

iffe格式主要是用于在script上引入的方式

SciScore for 10.1101/2021.07.19.452910: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.17.452804: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your code and data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.18.452826: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

We note that our conclusions are based on experiments performed in cell culture and in an artificial animal model and therefore have certain limitations. It will be important to extend this work to a real-world study, which could bridge the gap between genotype, phenotype and epidemiology. In addition, we only considered the effect of ACE2 SNVs. As the serine protease TMPRSS2 can prime the viral spike for direct fusion with the plasma membrane39, 43, SNPs in TMPRSS2 might also impact SARS-CoV-2 cell entry and be useful target of future studies. Moreover, SNPs in the 5’UTR or other non-coding regions of ACE2 were not included in this study and may also be important in regulating ACE2 transcription or translation, leading to varied ACE2 protein levels in vivo that could affect virus entry and the severity of COVID-19. Due to these limitations, we urge caution not to over interpret the results of this study. Of note, SARS-CoV-2 has evolved rapidly in humans, and a variety of genomic changes, including mutations and deletions in the spike protein, have recently been identified44, 45. Given that genetic variants will continue to arise, it is likely that we will see more genomic changes in the spike protein that might affect the spike interaction with human ACE2. Thus, it will be important to continue testing the interactions of these mutated spike proteins with the ACE2 SNVs. In summary, our study suggest that ACE2 polymorphism could impact human susceptibility to SARS-CoV-2 infec...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

An interesting idea Shawn.

SciScore for 10.1101/2021.07.16.21260079: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Statistical and Machine learning analyses: Data analysis was conducted using GraphPad Prism v9, R and Matlab 2021a (Matworks Inc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sample clustering was performed using fuzzy c-means (FCM) clustering method (fcm function running under Matlab).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Matlab</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr></table>

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.07.14.21260544: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

There are a number of limitations to the work presented here. Most importantly, it is not known whether viral isolation is a perfect laboratory correlate for viral infectivity and transmission in humans, which can vary significantly by time, distance, anatomy or mask wearing, and host immune status. We did not specifically convert CT to viral load given the multiple loci and tests investigated and the presence of mixed genomic and subgenomic transcripts for certain qRT-PCR sets. CT values are strongly assay-and instrument-dependent, and so other labs would need to validate the sensitivity of these primers against independent standard curves in order to calibrate assay performance before putting either the WHO-E CT limit or the Mills-sgE assay into use in their own labs. Finally, this work raises several important questions regarding the basic virology of SARS-CoV-2. The variation in viral titers generated from samples harvested at similar levels of CPE is intriguing, especially as we observed relatively little variation in RNA levels seen in these same samples (Supplemental Table S2). Potential variations in the ratio of infectious virus titers to RNA levels has important implications, as current dogma generally assumes a constant relationship between total RNA and levels of infectious virus in clinical samples. Higher levels of viral RNA correlate with poorer clinical outcomes for instance [38], but in general it has been difficult if not impossible to routinely measure infe...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

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Results from scite Reference Check: We found no unreliable references.

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Reply to the reviewers

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

In this manuscript, Mishima et al., designed a reporter system (dubbed PACE, for Parallel Analysis of Codon Effects) to assess the effect of codon usage in regulating mRNA stability in a controlled sequence context. This reporter corresponds to a stretch of 20 repetitions of a given codon (to be tested for its effect on mRNA stability), each repetition being separated by one codon corresponding to each of the 20 canonical amino acids. This stretch is inserted at the 3' end of the coding sequence of a superfolder GFP flanked with fixed 5' and 3' untranslated regions. In vitro transcribed capped and polyadenylated RNAs are then produced from these reporters (each with a specific stretch of repetitions of a given codon), pooled together and injected into zebrafish zygotes to monitor their relative abundance at different time points upon injection.

Using the PACE reporter, the authors were able to obtain a quantitative estimation of the impact of 58 out of the 61 sense codons on modulating mRNA stability. Their results are in agreement with a previous report that estimated the effect of codon usage on mRNA stability using endogenous mRNAs and an ORFeome library (Bazzini et al., 2016). However, contrary to relying on endogenous mRNAs and ORFeome reporters, the advantage of the PACE strategy is that the effect of the codon to be studied can be probed in a defined context, thus avoiding the presence of other motifs or transcript features that could also regulate mRNA stability. Similarly to results from Bazzini et al., 2016, the authors show that blocking translation completely abrogates the effect of codon usage, indicating that translation is required to drive codon-dependent mRNA degradation from their reporters. Also, the extent of codon-dependent mRNA decay is correlated with tRNA abundance and occurs through a process involving mRNA deadenylation as previously described in the zebrafish (Mishima et al., 2016 and Bazzini et al., 2016).

Having validated their PACE protocol, the authors performed ribosome profiling to test whether ribosome occupancy on tested codons is correlated with their capacity to drive mRNA degradation. Their results indicate that, at least for polar amino acids, there is indeed an inverse correlation between ribosome occupancy at tested codons and mRNA stability thus suggesting that slow decoding of codons due to low levels of available cognate tRNA can induce mRNA degradation. The authors further validate this finding by reducing the levels of aminoacylated tRNAAsn (corresponding a polar amino acid) and showing that stability of the reporter RNA carrying a stretch of AAC codons (decoded by tRNAAsnGUU) is reduced. To test whether codon-dependent mRNA degradation in the context of slow ribosome decoding lead to ribosome stalling and collisions, the authors generated a mutant zebrafish strain with impaired expression of ZNF598 (an essential factor of the No-Go decay (NGD) pathway in yeast). They also integrated a known ribosome stalling sequence from hCMV (and a mutant version that does not trigger ribosome stalling) in their sfGFP reporter construct as a positive control for NGD in their assays. Their results indicate that although ZNF598 depletion impairs degradation of the hCMV reporter (as expected), it does not affect codon-dependent mRNA degradation, which appears to occur for most codons through a NGD-independent manner. Finally, through the use of a tandem ORF reporter assay separated by codon tags to be tested, the authors show that destabilizing codons do not stall ribosomes but only lead to their transient slowdown which induces mRNA deadenylation and degradation in a ZNF598-independent manner.

Overall, the manuscript is very well written and pleasant to read. The introduction is well documented and relevant to the study as it allows readers to place the study in the current context of the field while highlighting open questions that have not been addressed yet. The results are clearly presented, the technical approaches are elegant and the conclusions convincing.

Below you will find some major and minor points that, in my opinion, should be addressed by the authors.

**Major point:**

• One interesting aspect of the PACE reporter assay is the possibility to monitor ribosome occupancy in parallel for all codon-tags tested, which the authors did in Figure 3. However, instead of using RNA-seq data to normalize ribosome footprints and obtain ribosome occupancy, the authors used an alternative normalization approach consisting, for each codon-tag, to calculate the number of ribosome footprints with test codons in the A site divided by the number of ribosome footprints with spacer codons in the A site. This approach is elegant and appears to work with codons corresponding to polar amino acids. However, it might have its limitations for other codons.

Indeed, ribosome dwell times (in yeast and mammals) have been shown to respond both to tRNA availability but also to other features such as the nature of the pair of adjacent codons, and the nature of the amino acid within the exit channel (Gobet C et al., 2020 PNAS; Gamble CE et al., 2016 Cell; Pavlov MY et al., 2009 PNAS). However, based on the work of "Buschauer R et al., 2020 Science", only ribosomes lacking an accommodated tRNA at the A site are able to recruit Ccr4-Not to mediate mRNA deadenylation and degradation. Other events that increase ribosome dwell time (and thus occupancy), such as slow peptidyl-transfer, do not lead to Ccr4-Not recruitment and are resolved by eIF5A. It is therefore possible that depending on the nature of the codon that is being tested, ribosome occupancy at test and spacer codons can be biased by the nature of codon-pairs and "dilute" the effects of tRNA availability.

If the authors performed RNA-seq together with the ribosome profiling experiment, it might be interesting to use the RNA-seq data to calculate ribosome occupancy on "tested" and "spacer" codons to check whether using this normalization, they do find a negative correlation between ribosome occupancy and PACE stability. A different approach would be to perform ribosome run-off experiments using harringtonine and estimate the elongation speed across the codon tag. However, I am aware that this experiment could be tedious an expensive.

• Figure 6: Insertion of the Lys x8 AAA stretch in the tandem ORF reporter leads to a decrease in HA-DsRedEx expression compared to that of Myc-EGFP. However, results from "Juszkiewicz and Hedge, 2017" using a similar reporter in mammalian cells indicate that stretches of Lys AAA below 20 repetitions only elicit poor RQC (less than 10% of true ribosome stalling for 12 repetitions of the AAA codon). Instead, most of the loss in RFP signal results from a change in the reading frame of ribosomes due to the "slippery" translation of the poly(A) stretch. I therefore think that it could be important to perform the experiment in ZNF598 KO embryos to validate that the observed reduction in HA-dsRedEx does indeed result from stalling and RQC and not from a change in the reading frame of ribosomes.

On a similar note, how do the authors explain the decrease in signal of the Flag-EGFP and HA-DsRedEx observed when using the Flag-EGFP with non-optimal codons? I understand that RQC occurring through NGD leads to ribosome disassembly at the stalling site and possibly mRNA cleavage (thus explaining the decrease in HA-DsRedEx signal compared to Myc-EGFP). However, I would assume that codon-mediated mRNA decay (even for ORF longer than 200 of non-optimal codons) should trigger mRNA deadenylation, followed by decapping and co-translational 5'to3' mRNA degradation, following the last translating ribosome. I would therefore expect not to see any change in the HA-DsRedEx/Myc-EGFP ratio even for the non-optimal Flag-EGFP reporter. Could the 200 non-optimal codons trigger some background RQC through NGD? Or could there be some ribosome drop-off? It might be interesting to test the optimal and non-optimal Flag-EGFP reporters in the ZNF598 KO background to check whether the observed decrease in the relative amount of HA-DsRedEx results from stalling-dependent RQC.

**Minor comments:**

• The color-coded CSC results from "Bazzini et al., 2016" presented at the bottom of panel B in figure 2 are misleading because many codons (such as PheUUU, AsnAAU, TyrUAC...) are lacking information. I have the impression that the authors used the combined data from the rCSCI (obtained from the reporter RNAs) and CSC (obtained from endogenous transcripts) corresponding to Figure 1F from Bazzini et al., 2016. This data set excluded all codons that were not concordant between the endogenous and reporter CSCs (which are those that are lacking a color code in Figure 2B from this study). However, in the scatter-plot of PACE Vs CSC (from Supplemental Figure 1D of this study), the authors used the complete set of CSC values from Bazzini et al .,2016. Could the authors please use the complete set of CSC values from Bazzini et al., 2016 to color code codons in their Figure 2B?

• Figure 4B. The charged tRNA measurements seem to have been done in a single biological replicate (there aren't any error bars in the chart). I understand that the procedure is tedious and requires a large amount of total RNA to begin with, but it would be preferable to perform it in three biological replicates.

• Supplementary Figure 2B. I do not understand what the figure represents. The legend is quite cryptic and states that the panel corresponds to the information content of each reading frame. More information should be given so that readers can understand how to interpret de figure and extract periodicity information.

Reviewer #1 (Significance (Required)):

Since the seminal work from Jeff Coller's laboratory in 2015 (Presnyak et al., 2015 Cell) showing a global and major role for codon optimality in determining mRNA half-lives in yeast, the role of codon usage in modulating translation and stability of mRNAs has been widely studied in different organisms (including zebrafish and mammals). As stated by the authors in the introduction, most studies have relied on correlation analyses between codon usage and mRNA half-lives from endogenous transcripts or from ORF libraries with fixed 5'UTR and 3'UTRs. This approaches could suffer from the presence of transcript features that can participate in other mRNA degradation pathways, which could limit their use when performing further mechanistic studies.

The work by Mishima and collaborators presents an original reporter assay that allows to evaluate the role of codon usage on regulating mRNA stability in a defined context, thus avoiding the impact of confounding factors that could bias the measurement of mRNA stability. Results obtained using this reporter are in good agreement with previous reports from Zebrafish (Bazzini et al 2016., and Mishima et al., 2016). From this validated reporter approach, the authors further show that codon-dependent mRNA degradation is directly related to tRNA availability and (at least partially) to ribosome occupancy (two factors already suggested as being important for codon-mediated decay in zebrafish, although they were based on correlation analyses). Furthermore, the authors show that codon-mediated mRNA decay occurs during productive mRNA translation and that it is functionally distinct from RQC induced by ribosome stalling. As a consequence, codon-mediated mRNA degradation is independent from the RQC factor ZNF598 (which they also validate for the first time as an important RQC factor in zebrafish). This information is new within metazoans since only in yeast it has been clearly shown that codon-mediated mRNA decay is distinct from RQC induced by ribosome stalling and collisions.

Taken together, the reported findings will be of interest to the community working on mRNA metabolism and translation. It could also interest, more broadly, scientists working on translational selection and genome evolution.

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

In this manuscript, Mishima et al aim to determine if the RNA-mediated decay determined by codon optimality is part of the ribosome quality control pathway, triggered by slowed codon decoding and ribosome stalling or it is an independent pathway.

To this end, the authors capitalize on their previous work to design a very elegant high-throughput reporter system that can analyze individually codon usage, ribosome occupancy and tRNA abundance. This reporter system, called PACE, is rigorously validated throughout the manuscript, because blocking translation with a morpholino blocking the AUG codon demonstrated that the effects no RNA stability are translation dependent.

When most of the available codons are tested using the PACE system, the authors recapitulate codon optimality profiles similar to the ones previously uncovered using transcriptome-wide approaches.

Thanks to the design of the reporter, which alternates repeats of a test codon with random codons, the authors can calculate how quickly a ribosome decodes the test codon on average. With this approach, the authors uncover a negative correlation between RNA stability and ribosome density on codons for polar amino acids and suggest that codon optimality is related to a slower decoding of the codons.

With the PACE reporter validated, the authors can interrogate the system to gain mechanistic insights of codon optimality. First, they test if RNA decay and deadenylation mediated by codon optimality is determined, in part, by the levels of aminoacylated tRNAs available. The authors use a very elegant approach, as they overexpress a bacterial enzyme (AnsB) in zebrafish that degrades asparagine, effectively reducing the levels of tRNA-Asn. The authors demonstrate that AnsB turns a previously optimal Asn codon, AAC, into a non-optimal one. This effect is translated into RNA destabilization and deadenylation, but this effect in not extended to other codons encoding amino acids not affected by Asn. These results provide a direct experimental validation of the previously published observation of tRNA levels and codon optimality.

Finally, the authors interrogate the relationship between the codon optimality pathways and the ribosome quality control pathways, that takes care of stalled ribosomes. The authors generate a zebrafish mutant of Znf598, a vertebrate homolog of the yeast protein in charge of resolving stalled ribosomes. Using a maternal-and-zygotic mutant, the authors demonstrate that in these mutant's codon optimality proceeds as usual but ribosome stalling is not resolved, providing evidence for first time that Znf598 is involved in ribosome quality control in vertebrates.

Altogether, this manuscript presents work that builds on the previous findings of the authors and other labs but it is a qualitative leap forward rather than a marginal increment, because the body of work in the current manuscript i) establishes a reporter to dissect the mechanisms of codon optimality, ii) demonstrates that ribosome slow-down but not stalling is part of the trigger of RNA decay mediated by codon optimality, iii) demonstrates that this pathway is independent of ribosome quality control pathway and finally iv) demonstrates that vertebrate Znf598 is involved in the RNA decay mediated by ribosome stalling.

Due to these novel findings, and the rigor of the experimental design, this manuscript should be accepted for publication. The authors should first address the following comments:

**Major comment:**

1. The authors very elegantly demonstrate the impact of AnsB on the stability of the RNA reporter, and it is precisely the simplicity of the reporter that allows the authors to draw clear conclusions. Nevertheless, it would be interesting to determine if the reporter results in embryos injected with AnsB also translate to endogenous genes rich in AAC codons. The authors could perform a polyA-selected RNA-Seq in embryos treated with AnsB to determine if the transcripts rich in AAC codons are destabilized compared to wild-type, thus validating the reporter results in endogenous genes. **Minor comments:**

In figure 5J the authors plot the normalized codon tag levels of the PACE reporter run in the MZznf598 mutant. The authors color code the labels in the x-axis following the PACE results in wild-type (figure 2B). The authors should also plot the wild-type values to have a direct visual comparison of the results trend in both genotypes. The authors focus in the title on the role of Znf598 or the lack thereof in RNA decay induced by codon optimality. However, for the non-aficionados in codon-optimality, ZnF598 is an unknown protein and adds little information to the title. The authors should provide a more informative title, directly pinpointing that codon-optimality is independent of the ribosome quality control pathway.

Reviewer #2 (Significance (Required)):

This manuscript presents work that builds on the previous findings made by the authors and other laboratories but it is a qualitative leap forward rather than a marginal increment, because the body of work in the current manuscript i) establishes a reporter to dissect the mechanisms of codon optimality, ii) demonstrates that ribosome slow-down but not stalling is part of the trigger of RNA decay mediated by codon optimality, iii) demonstrates that this pathway is independent of ribosome quality control pathway and finally iv) demonstrates that vertebrate Znf598 is involved in the RNA decay mediated by ribosome stalling.

In addition to the conceptual findings, the authors establish a new high-throughput reporter system to evaluate the influence of codon optimality in RNA decay.

The work its done in zebrafish embryos, an in vivo model system where codon optimality has been extensively tested by the authors and others, following the stability of reporter and endogenous genes.

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

Mishima et al. address a very timely topic of how the codon composition of the ORF and the associated translation elongation speed affect mRNA stability. Several studies have already shown a strong correlation between codon optimality and mRNA stability - meaning the more "optimal" the codons, the faster supposedly the elongation speed and the more stable the mRNA. Most of these studies were done by analyzing global expression data, with limited follow up, therefore being also impacted by other co-translational mRNA decay pathways and in addition these studies could also not test directly the effect of each single codon on mRNA stability. The authors took a systematic reporter-based assay approach, called PACE, which allowed them to test systematically the effect of codon composition on mRNA decay. By integrating also ribosome profiling data, the authors could nicely show that the speed of translation (measured by ribosome density) correlates with their determined mRNA stability effect of each codon and also the corresponding tRNA levels. However, interestingly this seems to be the case only for codons encoding polar amino acids, but not the ones that encode charged or non-polar amino acids. It will be very interesting to find out why that is? Finally, the authors address if some of the effects they see might be due to ribosome collisions and associated no go decay (NGD). For this they generated a Znf598 mutant by CRISPR-Cas9. Znf598 is the proposed homolog of Hel2, the protein in yeast that is essential for NGD. The authors go on to show that NGD is defective in this mutant, but that codon mediated decay, which is elongation dependent, is not to a large part not dependent on Znf598.

**All minor comments:**

1. It is intriguing why only polar AAs show a tRNA amount specific effect in the ribosome footprint data. Some hypothesis/discussion about this could be expanded further in the discussion or results.
2. On the same token some additional analysis might be helpful. For example, in Figure 2E, the authors group codons in weak, neutral and strong based on PACE measurements and then look at the tRNA expression range for each of the three groups. Could the authors do this also separately for the codons of polar, non-polar and charged amino acids? What do you see - still the same pattern as for all the codons or do again only polar amino acids show the trend?
3. Can the authors elaborate on the development of their PACE system? Why is it designed the way it is? What parameters did they test? For example, why the 20 amino acids tail, did you you test shorter sequences of the amino acid, spacer repeats, etc?
4. The next few questions are a bit more of a technical nature regarding the reporter construct used for PACE.
5. Did all AA pairs (Codon of interest + spacer codon) behave the same in the footprint assay? Does the data have enough information and resolution for this?
6. Was the order of the spacer codons always the same in all the constructs? Could the specific order, if it is consistent, have any unseen consequences (ie. interaction with the exit tunnel)? Did the authors test other orders?
7. Are the spacer codons optimized?
8. Are the codons affected in the NGD mutant the ones that are most different in the Bazzini data?
9. The authors inject directly mRNA into the embryos, therefore avoiding that the reporter mRNA is ever in the nucleus. However, there could be nuclear events (e.g. loading of particular proteins) that might affect the fate of an mRNA in the cytosol, among these the translation efficiency and also stability. Maybe some comment in the discussion as to the effect of missing nuclear factors would be welcome. This is not a criticism; it would just be nice to hear the authors' thoughts on that.
10. Page 6; final paragraph: "Finally, we compared the speed of the ribosome translating mRNA destabilizing codons to that of an aberrantly stalled ribosome." Not sure the authors did that actually. They tested the effect of ribosome slowing down on protein production and mRNA levels and compared that to stalling ribosomes, but did not compare the "speed" directly and I am not even sure what they mean by that in this context. Probably good to rephrase.

Page 7, upper half: ".....by taking the positional effect of codon-mediated decay into account (Mishima and Tomari, 2016)."

This is my limited knowledge of the literature, but I think you should mention what this positional effect is and not just cite a paper.

Very minor, but on page 8 when PACE is introduced, the authors show the different destabilizing effects of the three Ile codons. While that is ok, in the section before, when the authors tested their construct by qRT-PCR, they focused on the two Leu codons. I would also mention them here and do a direct comparison of the qRT-PCR results with the pooled PACE result for these two codons. Based on the figure the two codons seem to behave qualitatively like expected, but I am not sure how good the quantitative behavior matches. The AnsB experiment - the authors only mention data about one of the two Asn codons (AAC), but what about the second Asn codon (AAU) - do you also see an effect on that codon upon overexpression of AnsB as well? AAU is already a quite destabilizing codon and you might not see a further increase in destabilization, but it would be great to know if there was or not. Page 13, second paragraph: More out of interest, but it is quite intriguing that GCG turned into a destabilizing codon (opposite of what one would expect if NGD would play a bit a role). Any speculation why? Page 14, end of page and related to Figure 6C: AAU seems much more destabilizing than AAC. Therefore, I would have expected that the inserted sequence with the AAU codons would lead actually to downregulation of the mRNA and therefore the EGFP and DsRFP total protein signal relative to the construct with the AAC inserted in between, even if the ratio of EGFP/DsRed seems unchanged. However, based on the western blot in 6C the total protein levels seem very similar. Isn't that surprising? Although, AAU obviously allows translation to proceed it should still induce a stronger mRNA decay than AAC and therefore result in less total mRNA (and protein level as a consequence). Did the authors quantify the exact levels of the reporter proteins and mRNA and compared them between the two constructs? Page 15, last sentence: Somehow for me the word "transient" is a bit hard to grasp in this context. What do you mean by that - do you really mean "impermanent" or "lasting only for a short amount of time"? Don't you simply mean "weaker", "less strong"? Page 17, second sentence: I think the authors want to reference here Figure 2E and not Figure 2D.

Reviewer #3 (Significance (Required)):

All in all, I have to say that it was a real pleasure to read this manuscript. The authors were extremely thorough with their experiments and did nearly never overstate any of their conclusions. It is a very insightful story, which in my opinion will contribute greatly to the field of gene expression and posttranscriptional gene expression regulation in particular. The PACE assay, although a bit artificial, gave very clean results, which agree with the previous literature and could be very useful for future studies. Generating the Znf598 mutant and showing that the codon-dependent decay is independent from NGD is a great addition to this paper. Although it is a bit of a pity that we do not see more of a characterization of the Znf598 mutant in this paper, I do agree with the authors that this might take away a bit of the focus of this manuscript and that the mutant deserves actually its own story. I only have very minor comments/questions for the authors that they should be able to address easily. Finally, I can only repeat myself by saying: congrats on this great paper and I fully support publication.