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Reply to the reviewers

We thank the reviewers and the editor for the insightful and thorough assessment of our manuscript. In this response to review letter, we have listed the original review (black text) and responded to each critique after it.

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

Yang et al. submitted a manuscript describing the detection of pseudogenes ("retrocopies") of APOBEC3 (A3) genes in primates. The evolutionary history and relationship to specific A3s was analyzed and speculated that the maintained A3 retrocopies had a functional role at least early in the evolution and some may have still now. Functional data on some of the expressed retrocopies are presented on L1 and HIV.

The authors claim that "retrocopying expands the functional repertoire of A3 antiviral proteins in primates". While almost of the genetic findings were published recently (Ito et al. 2020), the authors should more clearly describe how their data differ or confirm the data of Ito et. al.

We thank the reviewer for their helpful comments which have guided revisions to our manuscript. We have taken steps to clarify the dramatic differences between our work and the recent publication from Ito, Gifford, and Sato.

Foremost, we respectfully disagree with the reviewer that the genetic findings in our work were contained within the Ito, et al manuscript. Using a computational screen of assembled mammalian genome, the Sato group cataloged the gain and loss of APOBEC3 genes during the evolution of mammals. They found a fascinating correlation between the dynamics of A3s and ERVs that formed the precis of the paper. From their genome-wide search for A3s, Ito et al describe several retrocopies of A3s in two New World Monkey species, one of which retains a full-length open reading frame, leading to the statement that this gene may be functional.

We note that the retrocopies found in the Ito et al paper span only two of the more than 20 species in which we identify A3 retrocopies. Further, as a result of the breadth of our search for A3s, we find additional retrocopies in the same two New World Monkey species that were examined in the Ito et al paper. Finally, our study also examined functional capabilities of these additional A3s. These differences are highlighted by reviewer 3 who writes that relative to Ito et al, our manuscript studies the phenomenon of A3 retrocopies “more deeply both by in silico analyses and cell culture experiments.” Reviewer 3 also summarizes the most important difference in our studies – our work presents a “conceptual advance that the antiviral gene expansion has achieved not only via tandem gene duplication but also via gene retrocopying”.

Lastly, we want to point out that the findings of our manuscript and Ito et al. 2020 were made concurrently. Indeed, throughout the preparation process of this manuscript, we were both aware of each other’s findings and shared preprints with each other. Most of the participating journals in Review Commons have “scoop protection” mechanisms that typically extend 6 months after the publication of the first article (Ito et al was published Jan 2020), and our article was first submitted to Reviewer Commons on February 14, 2020. Therefore, we feel confident that the ‘no scoop’ policy applies to the minimal overlap between our paper and that of Ito et al.

Nevertheless, we have modified the text to more clearly acknowledge the parallel finding of some New World monkey retrogenes in the Ito, et al. paper.

The functional data (Fig. 6) are interesting, but in the current form not complete. The authors have to show protein expression in the transfected cells (A3, L1, HIV) and level of encapsidation into viral particles. In addition, please analyze if the retrocopies express cytidine deaminase active enzymes.

We thank the reviewer for this comment, and we have added a Western blot of the six long-ORF-containing retrocopies as Figure S5. In this blot (from early in the project), we detected protein production in 293T cells for 3/6 retrocopies. In later optimizations of subsets of this blot, we were able to detect expression of the marmoset A3G and the other two marmoset retrocopies (marmoset-2 and marmoset-4). Despite optimization attempts, we were unable to detect protein for one of the retrocopies that restricts HIV-1ΔVif (capuchin-C1). Unfortunately, at this time the included blot is the only one we have in which all 6 constructs are included on a single blot. Optimally, all 6 constructs would be side-by-side in a single blot with optimized conditions, and we are happy to complete this experiment as soon as we are able to return to our lab after the SARS-2 quarantine is lifted. However, we think the added blot shows that some of the retrocopies produce protein and the absence of detectable protein from capuchin-C1 could suggest that this retrocopy is especially potent in its restriction function or an idiosyncratic problem with detecting this protein using Western blot analyses.

We have not previously tested our lentiviral particles for levels of encapsidation of protein from each retrocopy. The value we see in this experiment is in explaining why some of the retrocopies that are expressed in producer cells may not restrict in target cells. While we note that precedent in the literature suggests that A3 proteins which restrict HIV-1ΔVif are invariably encapsidated, we would be happy to carry out this experiment when our lab reopens.

In response to the reviewer’s request to test deaminase activity for each retrocopy, we note that Figure 4 shows the intactness of the deaminase motif in each retrocopy. However, we feel that a description of the mechanisms of restriction of these retrocopies is not a major point of this paper and is beyond the scope of the current investigation.

Reviewer #1 (Significance (Required)):

Minor advance compared to Ito et al. 2020.

We respectfully and rigorously disagree with this assessment. Please refer back to the reviewer’s first comment. We defer, again, to Reviewer 3’s assessment that our work presents a “conceptual advance that the antiviral gene expansion has achieved not only via tandem gene duplication but also via gene retrocopying”. Moreover, we must point out that the Ito et al 2020 paper was entirely computational; indeed, several retrogenes that could computationally be predicted to be ‘dead’ were confirmed by us as having antiviral activity.

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

**Summary:**

Yang et al. study the expansion of APOBEC3 (A3) cytidine deaminases genes in primates. Authors find A3 retrocopies in several lineages in primates using Blast searches. Some are old and some are species specific. Some have disablements and some have intact ORFs. Authors study their mode of evolution, expression and functionality. Authors have performed detailed analyses including functional analyses. Some A3 retrocopies are broadly expressed and some have retained ability to restrain retroelements. I agree with the authors that their data supports that retrocopying has contributed the turnover in the repertoire of host retroelement restriction factors. Authors show that some retrocopies have remained active for long periods of time and they still show that they can restrict retroelements/retrovirus. This work provides an interesting example of immune system diversification. This study of the A3 family of proteins that are part of the vertebrate innate immune system and the data supporting turnover of these kind of immune system genes is strong. The work underscores that this is a way immunity genes evolve and it has parallels in the evolution of the TRIM gene family of immune genes. I just have a few comments. I think the work can gain from analyzing some aspects of the data in more detail and presenting the big picture in a summary table, even if it is just supplementary.

**Major comments:**

A3I is in many species. Does this mean it was preserved (i.e., functional for a while)? For how long have disabling mutations been accumulating? Can we get a sense of that? Even for other retrocopies, do we have a sense of how recent has the pseudogenization been? If it is very recent that means that the gene was active until not long ago.

Our analyses suggest that A3I was born in the common ancestor of simian primates and pseudogenized before the Catarrhini/Platyrrhini split. It is possible that A3I was functional within this extended period (~12-15 million years), but the presence of a shared truncating stop codon amongst all simian A3Is suggests the gene was no longer full-length at the time of diversification of the simians. Instead, the simian LCA likely encoded an A3I with a predicted ORF of 261 codons; if this truncated ORF were functional, it was then further truncated/pseudogenized with additional frame-breaking mutations which follow the phylogeny of primates.

We estimated the timeline of pseudogenization of each retrocopy using the species distribution of each syntenic retrocopy. We also note that we find full-length ORFs in three retrocopies which have been retained for a period of time at least as long as the age of the last common ancestor of the four New World monkeys. These old but intact retrocopies motivated our simulations of ORF retention rates (Figure 5).

In the PAML analyses test could be performed to test if the rate of evolution that are higher or lower than 1 for particular genes are actually significantly higher or lower than 1 for the particular gene comparing the likelihoods of the modes with the given rate with the one with the rate fixed to 1. Is there enough power to do this?

We thank the reviewer for pointing out this omission in our analysis. We did perform these tests and find a significant p-value for two of the nodes p=0.058 and p=0.025 respectively). We have updated the legend for figure S4 to incorporate these p-values

Page 9. It seems to me that the synteny data Figure S2 reveals they are derived from independent retroposition events and not duplications of segments because those would include flanking genes. Is this correct? Authors could comment on that.

Yes, we think that each retrocopy we show in Figure S2 is likely created via an independent retrotransposition event. We have clarified in the text that Figure S2 shows the genes used to establish synteny to support orthology of the retrocopies shared amongst multiple species and that each of these ortholog groups presumably originated via distinct retrotransposition events.

In figure S4, I am not sure why orthologous genes are not grouped together in the phylogeny and why p is smaller than 0.05. How should that figure and the probability be interpreted?

We thank the reviewer for their comments on this figure. First, the reviewer identified an error in the tree in which the branch labels for ‘night monkey-C2’ and ‘night monkey-SS1’ were inadvertently switched. The corrected tree now follows the pattern expected by the reviewer. Second, we employed RELAX to “determine whether selective strength was relaxed or intensified in one of these subsets relative to the other” (Wertheim, et al. MBE 2014). In this case, the p-value corresponds to the finding that the retrocopies (test branches) show intensification of selection relative to the intron-containing A3Gs (reference branches).

We have modified Figure S4 and the associated text to more clearly explain the specific hypothesis test we report.

It would be good to have a summary table that summarizes what genes have support for past or current functionality (preservation for long time or recent pseudogenization, expression, purifying or positive selection, ability to restrict retroelements) and in what lineages.

We agree with this reviewer suggestion. We have added the additional information including the number of frame disrupting mutations as a measure of age, intactness, and ability to restrict retroelements to Table S1. Thanks to this suggestion, Table S1 now serves as the master table to summarize the analyses of each retrocopy.

**Minor comments:**

1. Page 3. Authors say "...the exons and UTRs..." but UTRs are part of exons. Authors could talk about exons only that include protein-coding regions and UTRs.

Changed the text to "exons".

Page 7. I would say disabled instead of "... becoming degraded by mutation."

Fixed according to the reviewer's suggestion.

I would say neutral evolution not neutral selection.

Fixed according to the reviewer's suggestion.

Reviewer #2 (Significance (Required)):

This work provides an interesting example of immune system diversification. Authors study the APOBEC3 family of proteins that is part of the vertebrate innate immune system and the data supporting turnover of these kind of immune system genes. The work underscores that this is a way immunity genes evolve and it has parallels in the evolution of the TRIM gene family of immune genes revealing patterns in the mode of evolution of immunity genes. The audience of this work will be people interested in evolution of immunity, arms races and gene diversification and all evolutionary biologists interested in adaptation. I work in the field of comparative genomics and molecular evolution.

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

**Summary:**

This manuscript by Yang et al. is an well-written, intriguing paper highlighting the evolutionary significance of the gene creation via "retrocopying". The authors investigated the expansion of antiviral A3 genes via retrocopy in Primates, and found that A3G-like retrocopies have been generated repeatedly during primate evolution. A part of A3 retrocopies found in New World monkeys retained full length open reading flames and anti-lentiviral capacities. Interestingly, the spectrum of anti-retroelement activity of A3 retrocopies was different from the original (i.e., intron-containing) A3G gene in these species, suggesting the occurrence of the functional differentiation followed by gene amplification. However, one of the main findings that many A3 retrocopies are present in New World monkey is in-line to a previous report (i.e., Ito et al., 2020, PNAS), and the experimental validations were based on the human (not New World monkey's) retroelements. Nevertheless, this study deeply investigated the possible importance of A3 retrocopies for the host defense system evolution both by in silico analyses and cell culture experiments. This study provides the findings that can potentially expand our knowledge on the evolutionary arms races between retroelements and the hosts.

**Major:**

To strengthen the impact of this work, it would be better to increase the numbers of retroviruses in which the anti-retroviral capacities are investigated. I understand that it is difficult to examine retroviruses or L1s that are colonized naturally with New World monkeys, but I suppose it is not so difficult to investigate a variety of representative retroviruses such as murine leukemia virus (MLV) or the reconstructed human endogenous retrovirus K (HERV-Kcon). This additional experiment would be helpful to highlight that the spectrum of anti-retroviral activity of A3 retrocopies is divergent from the original A3G gene in these species and strengthen the concept to be proposed by this study.

The reviewer raises a fascinating question about whether retrocopies might have different restriction abilities relative to the other A3s in a given species. First, we feel that showing activity against one pathogen is sufficient for our claim that some of the A3 retrocopies have antiviral potential. Second, we discuss in the paper the idea that HIV-1 is not the actual target of these (or any) innate immune genes in New world primates. We argue that any other targets we might test would also be surrogates for the ‘true’ target of these genes.

**Specific:**

1, Since the authors found the expansion of "functional" repertoire of A3 retrocopies specifically in New World monkey, it would be better to rephrase the title as "Retrocopying expands the functional repertoire of APOBEC3 antiviral proteins in New World monkeys".

We thank the reviewer for this comment but point out that a large portion of our manuscript presents our work on primates outside the New World monkeys. The reviewer is correct to note that our finding of restriction activity is limited to New World monkey retrocopies, but we feel that the current title will attract a broader audience and reflects the broader relevance of this work.

2, It might be better to add a figure summarizing which A3 retrocopies in which species retain nearly full length ORFs. For example, how about making a figure like Fig. 2A for all the four representative New World monkey species?

We agree. We have added the length of the longest ORF for each retrocopy to Table S1.

3, Fig. 3

It would be helpful to clarify that which cell of the heatmap corresponds to the intact A3 retrocopies.

We have added labels to indicate the intact A3 retrocopies and adjusted the legend accordingly.

4, Page 4, line 5

It would be better to replace the word "protected" with "escaped" because this retrocopy subset should include the ones that are intact but not functional.

Changed as suggested.

5, Page 4, line 25

It would be better to rephrase "the common ancestor of mammals" as "the common ancestor of placental mammals" because A3 gene is absent in Marsupial.

Changed as suggested.

6, Page 5, line 5

Please rephrase "ongoing" as "recently-occurred".

Changed as suggested.

7, Page 6, line 19

I checked the multiple sequence alignment in File S1 and suspect that the codon (alignment) position of the shared premature stop codon is 261 (not 264).

We thank the reviewer for pointing out this discrepancy. We have revised the text to reflect the correct position of the shared stop.

8, Page 6, line 23

I could not understand the meaning of the sentence "Intriguingly, one lineage-specific mutation...".

Please specify the position of mutation which the authors mentioned (in File S1 or Fig. 1B).

This portion of the text refers to a reversion of a stop codon in the orangutan A3I; specifically, the stop codon shared in all simians acquired a second mutation that created a longer ORF in only this species. We have removed this sentence from the text for the sake of clarity.

9, Page 12, line 8

Please refer Fig. S4 here.

Changed as suggested.

10, Page 12, line 8

Please say "Significant relaxed selection was not detected" rather than "Our analysis detected no relaxation...".

Changed as suggested.

11, Page 12, line 8

Fig. S4 indicates "p=0.015", but the authors regard it as "not significant"?

We thank the reviewer for pointing out this confusing wording. We employ RELAX to “determine whether selective strength was relaxed or intensified in one of these subsets relative to the other” (Wertheim, et al. MBE 2014). In this case, the p-value corresponds to the finding that the retrocopies show intensification of selection.

We have modified Figure S4 to more clearly explain the specific hypothesis test for this p-value. We have also modified the text to clarify this point.

12, Page 12, line 9

Please here refer the data showing the claim "Instead, these A3G retrocopies have evolved more rapidly than...".

Changed as suggested; see previous point.

13, Page 12, line 11

Did the authors perform the statistical test on the dN/dS ratio analysis? If so, please mention the result of the test.

Yes we did. Please refer to Reviewer 2’s ‘Major Point 3’.

14, Page 12, line 15

It would be better to modify the phrase "show evidence of recurrent selection for functional innovation"

Changed as suggested.

Reviewer #3 (Significance (Required)):

This study provides a conceptual advance that the antiviral gene expansion has achieved not only via tandem gene duplication but also via gene retrocopying.

Compare to existing published knowledge.

Although one of the main findings that many A3 retrocopies are present in New World monkey is in-line to a previous report (i.e., Ito et al., 2020, PNAS), this study investigated the above finding more deeply both by in silico analyses and cell culture experiments.

Audience.

Evolutionary biologists and researchers in the field of viruses (particularly retroviruses including HIV-1) and transposable elements would be interested in this work.

Your expertise.

Bioinformatics, genome biology, viruses, and transposable elements

Technological literacy is when you are completely competent with the use of technology and are open to learning new things about newer devices. There are three aspects to technological literacy which are capabilities, knowledge, and critical thinking. I would say capabilities and knowledge are the most prevalent and the ones outmost known. Some people would know all the features within their device or piece of technology and utilize all of it. I know my friend’s mom who always gets the latest IPhones and hasn’t used Google maps at all. The next important thing is knowledge which can be mentioned as an another word is basically knowing what technology to use at which moments. Sometimes your laptop may be better to type and print things. While purchasing a laptop knowing more about the specifications allows you to know what exactly you are buying. After this course ends, I would say I would focus on learning more and just getting the mere exposure of new technology would be a good start.

I think the thing that struck me the most in this class would probably be the amount of readings and the wide array of readings we were exposed to. I am lucky to have chosen this class this semester my final semester at the U. I believe as young millennials we do inherently follow some of the principles that were taught in this class. In the future though I will be more open minded about technology and have learned about how technology literacies can change throughout other peoples lives.

Frankly, I don't think this is accurate as is a misreading of the piece. There is no cure, but we can be sure that if any one is discovered by most any country, it would be widely known and reported on. Anything reported now as a 'cure' is undoubtedly unproven and anecdotal.

Karyn Oliver confirms the idea that a monument can reflect the viewer's beliefs and values. I think she is challenging the idea of collective memory here by saying "monuments are established wth the assumption that we as a nation have collectively decided..." She thinks that not all people have equal power to shape societal narratives, so the monuments are often one sided and not as "collective" as they may seem.

Again, the intro is highly disconnected from the rest of your story. A good introduction should not only introduce us to who the main characters are, it should also demonstrate what the theme of your story is going to be. As it is, the characters Jessica and Stephanie do not appear anywhere again, while the main characters Akilles and Mike appear far later on.

Disgusting

The importance of self-compassion in tempering the brittleness of self-efficacy

The main reason is that most people’s risk tolerance is very low, because self-efficacy (defined as “a person’s conviction or confidence about his or her abilities to mobilize the motivation, cognitive resources or courses of action needed to successfully execute a specific task within a given context”) is remarkably fragile. When it comes to trying and learning new things, people have difficulty transferring success in one arena to even highly related ones. Even small failures lead to learned helplessness so quickly, we learn to protect against that eventuality by not trying new things unless success is guaranteed.

The primary risk of entrepreneurship and other free agent lifestyles is not financial or even social — it is the risk to a person’s very self-concept as someone who does what they set out to do.

What we need if we want to change behavior at this fundamental level is to replace predictive models of behavior change—do this and you’ll get that —with exploratory models.

Stories may actually be a more accurate way of describing how people think about and use mental models of behavior change. Stories, like emergent systems, only move in one direction. They cannot be rolled back and played again. This irreproducibility suggests the importance of another form of psychological capital that is also highly correlated with successful behavior change: self-compassion. They are two sides to the same coin — you need self-efficacy to believe you can do it, but you equally need self-compassion to be ok when you don’t. Self-compassion aids change by removing the veil of shame and pain that keeps you from examining the causes of your mistakes (and often, leads you to indulge in the very same bad habit as a way of forgetting the pain). Self-forgiveness is the first step in fostering an invitational attitude that is open to feedback and learning, from yourself and others.

There is something about the turning of this coin — between efficacy and compassion — that I believe lies at the heart of the experimentation framework I’m envisioning. And the more I think about it, the more I suspect compassion is the far more radical and important side.

Taken from a graduation address delivered at West Point, which is just so good and worth quoting many sections of at length

That’s really the great mystery about bureaucracies. Why is it so often that the best people are stuck in the middle and the people who are running things—the leaders—are the mediocrities? Because excellence isn’t usually what gets you up the greasy pole. What gets you up is a talent for maneuvering. Kissing up to the people above you, kicking down to the people below you. Pleasing your teachers, pleasing your superiors, picking a powerful mentor and riding his coattails until it’s time to stab him in the back. Jumping through hoops. Getting along by going along. Being whatever other people want you to be, so that it finally comes to seem that, like the manager of the Central Station, you have nothing inside you at all. Not taking stupid risks like trying to change how things are done or question why they’re done. Just keeping the routine going.

We have a crisis of leadership in America because our overwhelming power and wealth, earned under earlier generations of leaders, made us complacent, and for too long we have been training leaders who only know how to keep the routine going. Who can answer questions, but don’t know how to ask them. Who can fulfill goals, but don’t know how to set them. Who think about how to get things done, but not whether they’re worth doing in the first place. What we have now are the greatest technocrats the world has ever seen, people who have been trained to be incredibly good at one specific thing, but who have no interest in anything beyond their area of exper­tise. What we don’t have are leaders.

What we don’t have, in other words, are thinkers. People who can think for themselves. People who can formulate a new direction: for the country, for a corporation or a college, for the Army—a new way of doing things, a new way of looking at things. People, in other words, with vision.

That’s the first half of the lecture: the idea that true leadership means being able to think for yourself and act on your convictions. But how do you learn to do that? How do you learn to think? Let’s start with how you don’t learn to think. A study by a team of researchers at Stanford came out a couple of months ago. The investigators wanted to figure out how today’s college students were able to multitask so much more effectively than adults. How do they manage to do it, the researchers asked? The answer, they discovered—and this is by no means what they expected—is that they don’t. The enhanced cognitive abilities the investigators expected to find, the mental faculties that enable people to multitask effectively, were simply not there. In other words, people do not multitask effectively. And here’s the really surprising finding: the more people multitask, the worse they are, not just at other mental abilities, but at multitasking itself.

One thing that made the study different from others is that the researchers didn’t test people’s cognitive functions while they were multitasking. They separated the subject group into high multitaskers and low multitaskers and used a different set of tests to measure the kinds of cognitive abilities involved in multitasking. They found that in every case the high multitaskers scored worse. They were worse at distinguishing between relevant and irrelevant information and ignoring the latter. In other words, they were more distractible. They were worse at what you might call “mental filing”: keeping information in the right conceptual boxes and being able to retrieve it quickly. In other words, their minds were more disorganized. And they were even worse at the very thing that defines multitasking itself: switching between tasks.

Concentrating, focusing. You can just as easily consider this lecture to be about concentration as about solitude. Think about what the word means. It means gathering yourself together into a single point rather than letting yourself be dispersed everywhere into a cloud of electronic and social input. It seems to me that Facebook and Twitter and YouTube—and just so you don’t think this is a generational thing, TV and radio and magazines and even newspapers, too—are all ultimately just an elaborate excuse to run away from yourself. To avoid the difficult and troubling questions that being human throws in your way. Am I doing the right thing with my life? Do I believe the things I was taught as a child? What do the words I live by—words like duty, honor, and country—really mean? Am I happy?

So it’s perfectly natural to have doubts, or questions, or even just difficulties. The question is, what do you do with them? Do you suppress them, do you distract yourself from them, do you pretend they don’t exist? Or do you confront them directly, honestly, courageously? If you decide to do so, you will find that the answers to these dilemmas are not to be found on Twitter or Comedy Central or even in The New York Times. They can only be found within—without distractions, without peer pressure, in solitude.

“Your own reality—for yourself, not for others.” Thinking for yourself means finding yourself, finding your own reality. Here’s the other problem with Facebook and Twitter and even The New York Times. When you expose yourself to those things, especially in the constant way that people do now—older people as well as younger people—you are continuously bombarding yourself with a stream of other people’s thoughts. You are marinating yourself in the conventional wisdom. In other people’s reality: for others, not for yourself. You are creating a cacophony in which it is impossible to hear your own voice, whether it’s yourself you’re thinking about or anything else. That’s what Emerson meant when he said that “he who should inspire and lead his race must be defended from travelling with the souls of other men, from living, breathing, reading, and writing in the daily, time-worn yoke of their opinions.” Notice that he uses the word lead. Leadership means finding a new direction, not simply putting yourself at the front of the herd that’s heading toward the cliff.

So solitude can mean introspection, it can mean the concentration of focused work, and it can mean sustained reading. All of these help you to know yourself better. But there’s one more thing I’m going to include as a form of solitude, and it will seem counterintuitive: friendship. Of course friendship is the opposite of solitude; it means being with other people. But I’m talking about one kind of friendship in particular, the deep friendship of intimate conversation. Long, uninterrupted talk with one other person. Not Skyping with three people and texting with two others at the same time while you hang out in a friend’s room listening to music and studying. That’s what Emerson meant when he said that “the soul environs itself with friends, that it may enter into a grander self-acquaintance or solitude.”

Introspection means talking to yourself, and one of the best ways of talking to yourself is by talking to another person. One other person you can trust, one other person to whom you can unfold your soul. One other person you feel safe enough with to allow you to acknowledge things—to acknowledge things to yourself—that you otherwise can’t. Doubts you aren’t supposed to have, questions you aren’t supposed to ask. Feelings or opinions that would get you laughed at by the group or reprimanded by the authorities.

This is what we call thinking out loud, discovering what you believe in the course of articulating it. But it takes just as much time and just as much patience as solitude in the strict sense. And our new electronic world has disrupted it just as violently. Instead of having one or two true friends that we can sit and talk to for three hours at a time, we have 968 “friends” that we never actually talk to; instead we just bounce one-line messages off them a hundred times a day. This is not friendship, this is distraction.

When I was reading this I was using the word bubble whenever I needed it. This was one of the moments that I used it to help me understand what this was saying and that it was telling me that Cassius , Casca, and Brutus were going after Caesar and to also kill Antony when they killed Caesar.

i understand now that they think that Julius Caesar will turn on them all, and be like a snake, which as an egg is harmless, but it can be harmful once it gets out of its shell.

The word bubble on the left helped me understand this a whole lot more. When I first read it, it seemed that they really liked Antony and were going to try to get him on board with the plan but if he disagreed they'd kill him. When reading the further in context, they more feared him and were worried that if he were alive he'd know about the plan to kill caesar so if they were to kill caesar they have to kill Mark Antony too.

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Reply to the reviewers

Reviewer #1:

**Summary**

Jang et al., address the important question of spatially localized or compartmentalized metabolic enzymes with a focus on the glycolytic enzyme PFK1. Using a good strategy of inserting a fluorescent tag at the endogenous PFK1 locus with tissue-specific inducible expression in C. elegans, combined with strong quantitative longitudinal imaging and innovative bioengineered microfluidic-hydrogels to control oxygen availability as well as optogenetic approaches, they show PFK1 condensates, which are not stress granules and not seen in normoxia, assemble with hypoxia. PFK1 condensates are dynamic, reversible, localized at the synapse in neurons, and recruit aldolase, another glycolytic enzyme. Although glycolytic proteins were previously shown to compartmentalize near the plasma membrane, and PFK1 was previously shown to assemble into filaments in vitro and be punctate at the plasma membrane in mammalian cells, evidence for cellular localized PFK1 condensates in animals is highly significant. The work includes strong biophysical characterization of PFK1 phase-separated condensates, but no clear indication of the composition of condensates. More significantly, the findings lack functional significance related to PFK1 activity or glycolytic flux with hypoxia vs normoxia. Despite previous work by this group showing that disrupting subcellular localization of glycolytic enzymes impairs neuronal activity in response with hypoxia, the reader is left with questions on the importance of localized and PFK1 condensates and their make-up .

**Major comments:**

Key conclusions are convincing, and most experimental approaches, biophysical characterization including thermodynamic principles, and data analysis are exemplary and well described. However, as indicated above, the work is limited to a descriptive analysis of cellular localization of PFK1 condensates and their biophysical properties without insights on functional significance relative to enzyme activity - or at least glycolytic flux or metabolic reprogramming with hypoxia. At best, only correlations can be drawn from hypoxia-induced localized PFK1 condensates and the authors' previous report (Jang et al., 2016) on hypoxia-regulated neuronal activity. Some insight or at least prediction in the discussion on the differences in spatially localized PFK1 in muscle vs neurons with regard to metabolic or energy distinctions should be included.

We have added additional discussions on the differences of the spatially localized PFK-1.1 in muscles versus neurons, explaining that in both tissues the cellular enrichment appears to be at sites predicted to have high ATP consumption (lines 128-133; 482-484).

Despite the strong biophysical analysis of condensates, several important features are not determined. First is at best a rudimentary analysis of the composition of condensates and also how PFK1 is assembled into these structures. For the former, is the core of the condensate predominantly PFK1 with perhaps aldolase only recruited to the periphery or is aldolase an integral component of the structure. Hence, is it a PFK1 condensate or a glycolytic condensate? For the latter question, is there a particular orientation for PFK1 in condensates, i.e a collection of filaments as previously reported, which might provide insight on assembly? Finally, and less critical but also important is the criterion for spherical, which is not well defined, and at least some idea or speculation on determinants for a spherical morphology - compared with filaments that have been reported for other non-glycolytic metabolic enzymes.

We have now co-expressed PFK-1.1 and ALDO-1 and examined their dynamic formation during hypoxic conditions. We observe PFK-1.1 and ALDO-1 form condensates simultaneously, with gradual enrichment of both molecules. We now include this new data in Figure 7E and Video 8; lines 422-441, 964-989). We also include genetic data demonstrating the ALDO-1 requires pfk-1.1 to form condensates, and that PFK-1.1 requires aldo-1 as well. Therefore, the enzymes are interdependent on each other to form condensates (Figures 7G, 7H, S7B, and S7C).

The spheroid geometry reflects liquid-like properties, which arises from surface tension of molecules loosely held together via multi-valent interactions. Filamentous arrangements reflect crystalline-like structures resulting from more stable interactions between molecules into solid-like states. While we did not perform high resolution studies, like Cryo-EM, to resolve this question, the spheroid geometry of PFK-1.1 condensates, along with its fluid-like properties, suggest the condensates are liquid-like compartment distinct to filamentous structures. We now add this discussion in lines 467-470.

The work is an important advance in our understanding on the self-assembly of metabolic enzymes by showing hypoxia-induced PFK1 condensates in vivo, their spatially-restricted subcellular localization in muscle cells and neurons, and their biophysical properties, the latter being distinct from those of stress granules. Taken together, these findings are more extensive than many previous reports on the assembly of metabolic enzymes into filaments or condensates, but fall short for new insights on functional significance.

We focus this study on the biophysical characterization of the condensates, and how that results in compartmentalized enrichment of glycolytic proteins. Examination of the functional significance of the phase separation to the enzymatic reactions in vivo is not currently possible because we lack probes we can use in vivo to measure the metabolites resulting from the reaction. We have now added discussion acknowledging this and framing its significance in the context of what has been published in the field (lines 484-492). For example, a recent manuscript in ChemRxiv demonstrated, in vitro, that the enzymatic activity of glycolytic proteins, hexokinase and glucose-6 phosphate dehydrogenase, promote these enzymes condensing into liquid droplets. The authors further found that the condensation accelerated the glycolytic reactions (Ura et al., 2020). This raises the question whether glycolytic proteins compartmentalize, and form condensates, in vivo, which we address in this manuscript. We capture this point in (lines 444-464) where we explain that, while it has long been hypothesized that glycolytic proteins like PFK-1 could be compartmentalized, this remained controversial due to lack of dynamic in vivo imaging. In our study, and through a systematic examination of endogenous PFK-1.1 via the use of a hybrid microfluidic-hydrogel device, we conclusively determine that PFK-1.1 indeed displays distinct patterns of subcellular localization in specific tissues in vivo.

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

This paper reports on the condensation of the glycolytic enzyme PFK-1 in response to hypoxic conditions in neurons of C. elegans. The authors employ a microfluidic-hydrogel device to dynamically monitor the relocalisation of PFK-1 from a mostly diffuse state to clusters in response to hypoxia and show that PFK-1 can undergo multiple rounds of PFK-1 clustering and dissolution. The authors work through the key features of a liquid-like compartment (sphericity, fusion, fast internal rearrangements) and give evidence that PFK-1 may have all three. Finally, the authors tag PFK-1 with the light-inducible multimerization domain Cry2 and find that even without light PFK-1 will constitutively form clusters that sequestrate endogenous PFK-1 as well as other glycolytic proteins. The strength of this work is that it is characterizing what appears to likely be phase separation in the context of a whole animal experiencing a stress that it could encounter in the natural world. A limitation of the work is that it is unclear what the functional implications are of condensates of PFK-1 at the molecular or cell scale.

**Major comments:**

-All experiments were performed using fluorescently tagged PFK-1 expressed from endogenous promoter or from the native genetic locus which is important for excluding overexpression artifacts. However, there is still risk that the GFP tag is driving the assembly process. In order to exclude tag-specific effects that may cause aggregation of the tetrameric PFK-1, ideally a control would be done in which PFK-1 is visualized through immunofluorescence experiments of WT cells. Alternatively, a short tag (e.g HA, His) as epitope for is an alternative .

We used fluorescent tags to observe the dynamic relocalization in vivo. While in the study we have not performed immunofluorescence, we established the validity of the labeling method by: 1) using monomeric versions of GFP; 2) using different fluorophores to show the same condensation phenomenon; 3) performing CRISPR for single copy insertions; 4) Demonstrating that different glycolytic proteins form condensates; 5) demonstrating the GFP-tagged versions of the protein are capable of rescuing the loss-of-function alleles and 6) Now adding new data demonstrating the observed localization specifically depend on the presence of other glycolytic proteins. This last result supports that GFP tag is not driving the assembly process of glycolytic condensate and that the glycolytic condensate formation requires the presence of specific molecules in the pathway. I add that we routinely use fluorophore markers to over a dozen distinct proteins that label subcellular compartments, and we have never observed the dynamic relocalization reported here, with the exception of other glycolytic proteins that interact with PFK, suggesting this is a property specific to glycolytic proteins, and, based on the genetic studies, dependent on the glycolytic reaction. We add and discuss these findings in Figures 7G, 7H, S7B, and S7C; lines 422-441, 964-989.

-For the Cry2-section, the complementation of the pfk-1 mutant supports functionality of the synaptic clustering phenotype. Are there other features of function that can be evaluated or could you look at how Cry-2 vs wt worms recover from different durations of stress or frequencies. Could you see if the Cry-2-fusion will rescue function to a partial-loss-of-function allele or a tetramerization deficient allele? A detailed analysis of the effects of constitutive presence of PFK-1-Cry2 clusters would be necessary to bolster claims that this is fully functional construct. Can enzyme activity be somehow monitored?

We did not observe any difference between wild-type worms and CRY2-expressing worms with regards to their development, survival, locomotive behavior or synaptic phenotype. While we can not discard the possibility that this is not a full rescue, with available tools, we can not distinguish the recue with PFK-1-Cry2 from that of just PFK-1.

-The analysis of the sphericity of clusters (4A) is limited due to the diffraction limit of light which limits an analysis of a compartment of this size. While this is a limitation of the live organism, this should be more clearly acknowledged.

We have included in the Methods section our criteria for quantifying condensates and avoiding diffraction limit artifacts. Briefly, “Considering the resolution limit of a spinning disc confocal (approximately 300nm), any structure with a diameter less than 500nm and an area smaller than 0.2 µm2 was excluded from the analyses”. To better clarify this point, we also now add a description of the criteria used in the main text (lines 242-243).

In addition, we observed that PFK-1.1 condensates are not perfect spheres, but constrained spheroids (which can not be explained by diffraction-limited point spread functions). We can explain the observed spheroid shapes based on liquid-like properties of the condensates, and the constrains of the diameter of the neurite. To better highlight this finding, we have now moved Figure S4E into the main figure (Figure 4B’).

-Fusion experiments (4C) do not fully exclude that clusters overlap instead of merging. It would be beneficial to show the foci for several subsequent frames. One would expect that upon fusion, the condensate size would increase, but video 3 suggests the opposite. It would be useful to quantify condensate size before and after fusion for several separate fusion events. -an alternative possible experiment would be the tagging of PFK-1 with a photoconvertible fluorophore (e.g. Dendra2) and subsequent analysis of fusion events

To better show the fusion events in Figure 4C, we now include all xy, yz, and zx plane views of before and after fusion events of Figure 4C (Figure S5B). We also added a quantification of four independent fusion events in which we compare the sum of the areas of the two puncta before fusion and the size of the area of the single punctum after fusion (Figure S5C). These data support that we are observing fusions events.

-4D). It is unclear if foci are indeed undergoing fission or if two clusters next to each other are moving apart.

For Figure 4D, in all the frames we had recorded, a single structure maintains a continuous signal until fission occurs and splits into two structures. To better present this event, we now include an unabridged version of figure of 4D in the supplement that shows all the frames captured (Figure S5D).

-The analysis of side-by-side growth and dissolution kinetics are interesting and a novel view into the non-equilibrium aspects of phase separation in cells.

-Purification of PFK-1 and in vitro reconstitution of condensates would be supportive of liquid-like characteristics although I don't think it is necessary however it would add a lot to the relevance to show enzyme activity is different +/- condensate state but I am not sure if an easy enzymatic assay exists in vitro.

We agree. But the significance of this particular paper, specifically in the context of the in vitro enzymatic work on glycolytic proteins, is to examine the dynamic in vivo localization and the biophysical characteristics of the condensates. To better underscore this in the context of the field, we add discussion of a recent in vitro manuscript demonstrating that liquid droplet formation of glycolytic proteins affect their enzymatic activity (Ura et al., 2020) (lines 444-464; 484-492). While we see the value of future studies reconstituting the glycolytic particles, we believe that is beyond the scope of this particular in vivo study.

**Minor comments:**

-Stress granules in other organisms (yeast paper) have different composition depending on stress type. To make the claim that the PFK-1 compartments are independent of SGs one would ideally test multiple different SG markers.

We selected the stress granule protein TIAR-1 because it is one of the most studied stress granule markers in C. elegans and it is reportedly one of the core proteins and universal components of stress granules irrespective of a stress type (Buchan et al., 2011; Gilks et al., 2004; Huelgas-Morales et al., 2016; Kedersha et al., 1999). Although we did not include images in the manuscript, we had tested a total of three stress granule markers: TIAR-1, TDP-43, and G3BP1 with similar results. We now added that as data not shown (lines 193-194).

-it should be stated in the main text that the microfluidic-hydrogel device was fabricated following previously published protocols

We have added the reference in the main text (line 170) to supplement what we had written in the Methods section: “A reusable microfluidic PDMS device was fabricated to deliver gases through a channel adjacent to immobilized animals, following protocols as previously described (Lagoy and Albrecht, 2015)”.

-Figure 4b: Y-axis should be changed from probability to fraction of occurrence

We have corrected this in both the figure and the figure legends (Figure 4B).

-The discussion should be less speculative concerning any effects seen in PFK1-Cry2 expressing C. elegans

We have modified the discussion as suggested.

-it is perplexing that a protein known to tetramerize with no disordered or RNA-binding domains forms condensates like this. Is there anything known from other systems of additional interacting proteins that may have features that promote liquidity and serve to fluidize these assemblies?

Condensates can form via multivalent interactions, which include, but is not exclusive, to disordered or RNA-binding domains. Because glycolytic proteins have dihedral symmetries that can facilitate multivalent interactions, we believe these structural properties, in combination with regulated conformational changes, promote multivalent interactions leading to their condensation. We had a statement in the discussion (lines 494-519) now add this more clearly in the results (lines 395-398).

Reviewer #2 (Significance (Required)):

Stimulus-induced phase separation has been observed for dozens of metabolic enzymes from various different pathways (reviewed in Prouteau, 2018). Several studies have published the formation of condensates through PFK-1 in diverse organisms (C. elegans, Yeast, human cancer cells) in response to hypoxia or in some cancer lines also without hypoxia (Jin, 2017, Jang, 2016, Kohnhorst 2017, etc.). A yeast study showed that PFK-1 condensates contain various other glycolytic enzymes and that condensate formation enhances glycolytic rates (Jin, 2017).

This study gives the advance of analyzing the dynamics of PFK-1 condensate formation in vivo in the context of a live animal using a microfluidic-hydrogel device and showing that PFK-1 relocalizes to reversible condensates within minutes of hypoxia. If further appropriate experiments (as mentioned above) are performed, this study would strongly suggest that the underlying process of PFK-1 condensate formation is liquid-liquid phase separation. Ideally, if at all feasible, it would be strengthened if there was some insight into the functional consequences of the condensed assemblies formed in hypoxia. These findings may be interesting to researchers working on glycolysis and metabolism in different cells but particularly in neurons.

Field of expertise

-Phase separation, microscopy, in vitro reconstitution

-no experience with C. elegans biology and do not have a practical handle on ease or difficulties of genetic manipulation of C. elegans or metabolic assays for PFK-1

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

**Summary:**

In this manuscript, the authors focus on the subcellular localization of the key glycolytic enzyme PFK-1.1 in C. elegans, initially in whole animals through GFP tagging of the endogenous locus and subsequently in single cells/tissues using a clever genome editing strategy that permitted tissue-specific expression of GFP-tagged PFK-1.1 from its endogenous locus. They observe that PFK-1.1 localization differs from cell-type to cell-type and can be dynamically reorganized in response to exogenous cues. Focusing on hypoxia, they observe that PFK-1.1 forms foci near synapses in neurons under this stress condition. These foci are not stress granules and they are dissolved upon re-oxygenation. These condensates have properties of liquid droplets and can mature (harden) over time. PFK-1.1 fused to the CRY domain can trigger condensate formation under normoxic conditions, which can co-recruit WT PFK-1.1 as well as aldolase.

**Major comments:**

The conclusions are convincing but the impact could be increased if the authors were able to demonstrate the physiological role that the observed phase separation plays in this stress response. Would it be possible to assess glycolytic flux under hypoxia vs normoxia?

It is currently not possible to assess glycolytic flux in vivo in our system, as we lack metabolic sensors (an active area of work we are trying to address, but will take several years to perform correctly). We have added discussion of new in vitro studies examining the consequences of metabolic flux due to glycolytic compartmentation into liquid droplets (Ura et al., 2020), and the significance of those findings in the context of our in vivo studies (lines 444-464; 484-492).

The authors should comment on viability during the hypoxia time course.

C. elegans can survive anoxic condition for a day (Powell-Coffman, 2010). Our hypoxic conditions last minutes, and we can rescue live C. elegans upon completion of the assays. We now include a description of this in the Methods (lines 1216-1218).

The co-clustering of ALDO-1 and PFK-1.1::mCh::CRY2 in Figure 7 should be properly quantified/statistically analyzed

We quantified the fraction of animals that displays ALDO-1 clustering in PFK-1.1::mCh::CRY2 co-expressing animals, as suggested (Figure S7C).

A control of mCh::CRY2 + ALDO-1::EGFP is missing from the experiments shown in Figure 7. Is the presence of mCh::CRY2 sufficient to drive ALDO-1::EGFP clustering?

As a control for the CRY2 tag promoting the formation of glycolytic condensates, we had co-expressed mCh::CRY2 with PFK-1.1::EGFP, which is insufficient to cause the formation of the condensate (Figure 7C). We have now added a new data where we show that in pfk-1.1 deletion mutants, ALDO-1 condensate formation is suppressed, which further demonstrates the dependency between PFK-1.1 and ALDO-1 (Figures 7H and S7C).

Does hypoxia trigger co-clustering of ALDO-1 and PFK-1.1?

To answer this question, we examined the dynamic formation of ALDO-1 and PFK-1.1 condensates by co-expressing the two proteins together and observed that hypoxia triggers their co-clustering. We now include this in Figure 7E and Video 8.

The authors speculate that hypoxia acts via diminished energy (altered ATP AMP ratios). Can this be measured? To support this hypothesis, the authors may wish to test if similar phase separation is triggered by mitochondrial poisons.

We currently lack sensors that can reliably measure, in vivo, the subcellular changes in energy or metabolic flux in C. elegans neurons. However, we previously did test mitochondrial mutants and observed that in those mutants we observe glycolytic condensates (Jang et al., 2016), supporting the idea that defects in energy production promotes the formation of glycolytic condensates.

**Minor comments:** Is 21% O2 not hyperoxic for worms?

While C. elegans are known to prefer lower percentage of oxygen than those in air, in the lab animals are reared in normal air. We therefore used 21% oxygen present in air as our normoxic conditions.

Can the authors speculate more on how do these condensates exhibit "memory" (how they're able to cluster in the same place repeatedly)? Is there any role for the cytoskeleton in mediating nucleation and/or condensation of PFK and glycolytic enzymes?

When we were testing the reversibility of PFK-1.1 condensates, we were not expecting the reappearance of PFK-1.1 condensates in the same place repeatedly. Our current speculation is that, because many glycolytic enzymes, such as PFK-1.1, are allosterically regulated by nucleotides, AMP/ATP ratio may play a role on where glycolytic condensates appear. In other words, the specific synaptic areas, where PFK-1.1 condensate repeatedly reappeared, may have different AMP/ATP ratio that may trigger the condensation of the glycolytic proteins in those locationsupon conformational changes in PFK-1. We can’t exclude, currently, the presence of nucleating factors at synapses that facilitate PFK-1 clustering, but we have not yet identified them. We now include a discussion of this (lines 494-519).

Do the authors think that these clusters are effectively G-bodies from yeast?

G-bodies from yeast also shows glycolytic proteins changing from its diffuse localization to punctate localization in response to hypoxia (Jin et al., 2017). G-bodies, like C. elegans glycolytic condensates, are forms of subcellular glycolytic organization within eukaryotic cells. Yet, G-bodies take 24 hours to form, while we observe the glycolytic clusters in C. elegans within minutes of hypoxic conditions. We will need to understand the composition and function of both to determine if these forms of glycolytic subcellular organization represent the same structure. We note that glycolytic clusters have also been observed in some human cancer cell lines (Kohnhorst et al., 2017). Observation of glycolytic compartments in multiple different species and cell types suggest that, although the regulation, composition and formation kinetics of the glycolytic condensates may differ, compartmentalization of glycolytic enzymes may be a conserved feature. We now add a sentence discussing this (line 535-537).

Reviewer #3 (Significance (Required)):

It is much appreciated that this study tackles the cell biology of signaling and metabolism, which is a fascinating but difficult to study aspect of molecular biology. This work conclusively documents the dynamic reorganization of metabolic enzymes in vivo in response to physiological stimuli. Such reorganization had been proposed previously but was controversial and difficult to study in a controlled way. This work not only confirms previous observations but further demonstrates that the dynamic reorganization is mediated by a liquid-liquid phase separation. What is lacking is a demonstration that this phase separation is physiologically important. Such observations would generate interest from a much broader audience; the present audience presently targeting people specifically interested in non-membrane organelles per se. The reviewer has expertise in cell signalling and its regulation by phase separation.

As we explain for Reviewer 1, we focus this study on the biophysical characterization of the condensates, and how that results in compartmentalized enrichment of glycolytic proteins. Examination of the functional significance of the phase separation to the enzymatic reactions in vivo is not currently possible because we lack probes we can use in vivo to measure the metabolites resulting from the reaction. We have now added discussion acknowledging this and framing its significance in the context of what has been published in the field (lines 484-492). For example, a recent manuscript in ChemRxiv demonstrated, in vitro, that the enzymatic activity of glycolytic proteins, hexokinase and glucose-6 phosphate dehydrogenase, promote these enzymes condensing into liquid droplets. The authors further found that the condensation accelerated the glycolytic reactions (Ura et al., 2020). This raises the question whether glycolytic proteins compartmentalize, and form condensates, in vivo, which we address in this manuscript. We capture this point in (lines 444-464) where we explain that, while it has long been hypothesized that glycolytic proteins like PFK-1 could be compartmentalized, this remained controversial due to lack of dynamic in vivo imaging. In our study, and through a systematic examination of endogenous PFK-1.1 via the use of a hybrid microfluidic-hydrogel device, we conclusively determine that PFK-1.1 indeed displays distinct patterns of subcellular localization in specific tissues in vivo.

**REFEREES CROSS-COMMENTING** Globally it seems that all reviewers feel that impact would be increased if the physiological consequence of PFK-1.1 condensates was examined. Other, specific comments seem fair.

Crazy to think that diseases we have today were caused by mutations in evolution.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Summary:

In this work the authors investigate the dynamics of cell morphogenesis in a convenient in vivo system. They use the terminal cells of the embryonic tracheal system and comprehensively address how cell shape change (elongation in this case) takes place and how membrane is remodeled during the process. By combining different high-resolution techniques, i.e. in vivo imaging of terminal cells expressing different membrane markers and serial-section electron tomography, they describe the organelle organization (ER, Golgi, different types of vesicles) in the terminal cells during their elongation. They identify the presence of membrane structures/vesicles particularly abundant at the tip of the cell ahead of the growing tube. When they block endocytosis they find increased tube membrane and lack of basal membrane growth. In addition, in tomograms, they observe clear membrane defects, like invaginations that could even connect the tube and basal membranes. This correlates with the absence of vesicles at the tip observed in normal conditions.

The analysis of the nature of the vesicles observed indicated the accumulation of late endosomes and MVB, particularly at the tip of the growing terminal cells. Interfering with the formation of these MVB led to defects in the growth of both the tube and the basal membrane.

Altogether the authors propose a model in which the newly formed membrane (and transmembrane proteins) passes through the ER and Golgi and reaches the apical membrane. The incorporated membrane is then rapidly endocytosed and follows a maturation pathway through MVB, from where different cargoes and membrane would be sorted and recycled back to the apical (tube) membrane, or to the basal membrane through a transcytosis mechanism

Major comments:

Are the key conclusions convincing?

Most of the conclusions presented are convincing and supported by the results observed.

However, to my understanding, one of the key conclusions of this work (that membrane is transcytosed from the apical to the basal domain) is not fully convincing. A critical result to support the author's conclusion of apical-basal transcytosis is to find clear evidence of basal accumulation of a transcytosed marker.

1. The authors show accumulation of FGFR-GFP and Myospheroid as evidence. However, I find the results presented not very convincing. The accumulation of FGFR-GFP at the basal membrane in the control is not very clear in the images and movie presented. In addition, in the shibire mutant, some basal accumulation of FGFR-GFP seems to be detected (particularly in the movie). In the figures the authors show an increase of FGFR-GFP intensity when endocytosis is blocked, but this is not explained in the text.

If we understand the referee correctly, there are two parts to her/his concern:

1. Are the proteins we use in fact present basally in normal tracheal cells, i.e. are they good candidates for transcytosed cargo?

2.Do they change their localisation when endocytosis is disrupted? And this point can be divided into two aspects: a. do they change at the basal membrane? b. do they change at the apical membrane (this latter point is not questioned the referee)?

1. The FGFR and beta-integrin are the only known basal markers in tracheal cells. A major reason for being confident of their presence in the basal membrane, even though they are difficult to visualize, is that the biological function of both is at the basal membrane, with the FGFR receiving growth or chemotactic signals from the surrounding tissue, and integrins anchoring the branches on the underlying tissue. However, it is indeed the case that their expression levels are very low, and it is difficult to visualize them, whether by expression of GFP-labelled constructs or by immunofluorescence. We have pushed to the limit a number of methods to improve the detection, but we seem to be constrained by the biology of these molecules.

In addition to the low but detectable signal at the outer boundary cell, some signal is always visible within the cell, which we had in the past always interpreted as an artefact or background, but for which our findings here might provide alternative interpretations.

1. a. We agree with the referee’s assessment that FGFR::GFP is still detectable in the basal membrane after blocking endocytosis. This is, in our view, no contradiction to our model. The most parsimonious interpretation is that this is the FGFR that had already been delivered basally before we interfered with endocytosis, and which remains there after endocytosis is blocked.

b. In addition to this basal pool of FGFR, cells with blocked endocytosis accumulate abnormally high levels of FGFR at the apical membrane, in fact at much higher intensities than at the basal membrane. This is the more dramatic aspect of the phenotype, and our conclusions therefore rely not so much on a possible reduction of basal signal after blocking endocytosis (which would not be possible to demonstrate reliably), but rather on the abnormally enriched presence in the apical membrane.

A technical point: The increase in FGFR::GFP on endocytosis blockage that we show in Figure S4 corresponds to the cytoplasmic + apical pool of the FGFR. We used the Dof signal in the cell to create a mask of the total cell volume to 3D-segment the FGFR signal. Therefore, this analysis does not take into account the FGFR that is present in the basal membrane. We had explained this only in the methods section but will now describe it more explicitly in the Results section.

On the other hand, and very importantly, how does FGFR localization relates to its activity? The authors show that when endocytosis is prevented dpERK (i.e. a reporter of FGFR activation) is not decreased, indicating that FGFR is normally active. Wouldn't this suggest that FGFR is still localized basally to receive Bnl signal?

Indeed, and this is also what we see. This is not in conflict with any of the results or known functions of the receptor. If endocytosis is blocked, FGFR cannot be internalized and removed from the basal membrane, where it is needed to receive the FGF from the surrounding cells.

Our concern, and the reason for doing the experiment, had been that endocytosis might be required for FGF signaling, and that this might account for the failure of the cell to grow. But it turns out that our results show that this is not the case, at least not for the terminal cell in this time frame.

Actually, as the authors indicate, in larval tracheal cells the intracellular accumulation of the FGFR leads to a reduced FGF signal transduction (Chanut-Delalande et al., 2010), suggesting that reduced FGF signaling activity in these cells is due to less FGFR reaching the basal membrane.

That is true, and again, not inconsistent with our own results. In the cited study, trafficking was blocked at a step downstream of endocytosis. In this experimental situation, the internalisation of the FGFR would therefore occur as normal in these cells, but due to the impaired function of the ESCRT complex, intracellular processing, and therefore potential re-delivery to the basal membrane would be impaired. Furthermore, if (as we now propose) FGFR is also delivered via late endosomes in this context, blocking the ESCRT pathway should also impair initial FGFR delivery. In either way, initial delivery or re-delivery of the receptor being blocked, it is reasonable to assume that reduced signal transduction is the result of reduced basal FGFR.

Thus in our study we see no reduction in basal FGFR and no reduction (and even an increase) in signaling, while Chanut et al see reduced basal FGFR and reduced signaling, and the reason for this is that they interfere with a different step of the membrane trafficking pathway.

1. The results with Myospheroid are not very convincing either, as the authors just show a single confocal section of control and shibire mutants. In summary, I consider that this very important point needs to be better documented before concluding that apical membrane material containing basal cargoes is transcytosed to the basal membrane.

We observed this phenotype in several instances. We will quantify it for the resubmission.

1. Another conclusion that, to my opinion, should be better explained and documented, is the coordination of tube and basal membrane growth. Following the movements of the vesicles the authors conclude that there is a net displacement of these vesicles to the tip of the cell. This correlates with the presence of mature endosomes there. So the results postulate that transcytosis occurs at the tip, and therefore the growth of the basal membrane would occur preferentially at the tip. It has been demonstrated, as the authors indicate, that the tube membrane grows all along the length of the tube. How is then coordinated the tube and basal membrane growth? If, as the authors propose in their model, the tube membrane also grows after a process of endocytosis and recycling, wouldn't it be expected to have preferential tip growth? How do the authors reconcile all these observations with previously published results (Gervais and Casanova, 2010)?

The elegant study by Gervais and Casanova (2010) used a very clever method, which was however entirely non- quantitative (and did not make any claims to the contrary, either). The conclusion that material is delivered to the tube throughout its length was based on looking at the displacement relative to the base and the tip of the cell of short and transient secondary branches (seen for example in Fig. 1H in our paper). If the tube would primarily receive material at the tip, these secondary branches would not change their position with respect to the base of the cell. Instead, these branches tend to be displaced towards the tip, which shows that material is also added between the branch and the base of the cell. These branches are seen only in a fraction of wild type terminal cells and quantification is therefore difficult. Thus, the experiment shows convincingly that material is also delivered behind the transient branch and excludes a model by which all growth occurs only at the tip. But it does not discuss what proportion of the total is delivered along the length vs the tip of the branch.

Our model also does not contest the idea of ubiquitous membrane delivery over the length of the tube, either in the initial delivery step, or during redistribution. On the contrary, the presence along the length of the cell of vesicles carrying FYVE::GFP and Rab7, and of smaller MVB-like bodies in the EM sections, suggest that the pathway can also be deployed at a distance from the tip.

1. The serial-section electron tomography analysis is very interesting and identifies different sorts of vesicles. However, it is very unclear what the different vesicles referred in the models correspond to (in the in vivo imaging for instance). For instance, the small granular or the dense-core vesicles correspond to endocytic vesicles at different stages of maturation?

Based on distribution alone, it is almost impossible to determine which of these vesicles correspond to endosomes or to secretory vesicles, even for the extremely experienced EM experts in the team. We would require high resolution CLEM, and a wide range of fluorescent markers to be able to determine which population of vesicles found on EM correspond to each marker. Due to the broad distribution of these vesicles within the cell and their small sizes right now it would be extremely technically challenging to pursue this question (even though we too would love to know)

1. If there is a constant endocytosis from the apical membrane to generate the basal and build the definitive apical membrane, wouldn't it be expected to find many more vesicles around the tube? Wouldn't it be expected to find coated vesicles around or budding from the tube, as the coated vesicles observed budding from the basal membrane in Fig 2D? Or is the endocytosis observed mediated by non-clathrin coated vesicles?

We agree, and we had observed this to be the case, but had not quantified this. We have now analysed the distribution of coated pits and their density in the apical or the basal membrane of the cell. Overall, we found a higher density of endocytic events in the apical membrane than in the basal membrane. As the reviewer noticed, we also found that the majority of endocytic events in the basal membrane occur towards the tip of the cell. We will add these data to Figure S3.

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

The results of Serp accumulation upon MVB interference can lead to confusion (from line 404). The authors seem to suggest that Serp protein is exclusively produced in the FB and transported by transcytosis to reach the tracheal lumen. However, Serp is also produced in the tracheal cells themselves. In fact, serp expression in the FB seems to be detected by late embryogenesis, while expression in tracheal cells is detected much earlier (Dong et al. 2014; Luschnig et al. 2006; Wang et al. 2006). It was also shown that Serp undergoes a recycling mechanism from the lumen to the lumen, through the endosomes-TGN retrograde trafficking, that may also require Shrub (Dong et al, 2014; Dong et al 2013). Thus, it is unclear (and even unlikely) that the Serp found in the vesicles in Shrub-GFP mutants is derived exclusively from the transcytosed component from the FB. I suggest to better explore this issue or to remove this part.

We agree that the notion that the Serp we see is exclusively derived from the fat body is not correct. We observed Serp accumulation around Shrb::GFP sites in embryos at early and late stages, so it is likely that what we see is the result both of apical-to-apical redelivery of Serp (as reported in Dong et al., 2014a), and transcytosis of Serp from the basal membrane to the apical. We will therefore rewrite this. But regardless of whether we are looking at transcytosis, or apical-to-apical cycling, this experiment still reinforces the idea of our work that late endosomes serve as stations that collect material from and re-deliver it towards various compartments in the cell.

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.

As indicated before, more conclusive results for transcytosis should be provided.

I suggest that the authors determine the presence of apical and basal cargoes (FGFR) in the late endosomes found when Shrub activity is impaired. According to his model, both types of markers should accumulate there. A high accumulation of these markers in those endosomes would reinforce the hypothesis proposed.

We agree with the reviewer that this would be a great experiment. Since we only have an FGFR tagged with GFP we cannot do this experiment using the Shrb::GFP line, so instead we would have to use shrb mutants. This in itself is not a problem (see also below – we will be adding some data), but the experiment would require multi-generation crosses which could only be started once the current Covid-19 restrictions are lifted and labs opened again. Instead, we propose to cite the following supporting evidence. Chanut-Delalande et al., (2010) showed that mutants for components of the ESCRT pathway hrs and stam show intracellular accumulation of overexpressed FGFR::GFP in tracheal cells of the air sac primordium, and Dong et al., (2014a) show that shrb mutants accumulate Crb in late endocytic compartments in tracheal cells of the dorsal trunks. We would suggest it is very likely that terminal cells therefore also accumulate Crb and FGFR in late endosomes in the absence of Shrb. An experiment would be nicer, but we fear this is the best we can do at the moment.

While it has been reported that shrub-GFP act as a dominant negative in different contexts (Dong et al, 2014; Sweeney et al 2006) it is unclear why. So it would be desirable to confirm the results with a loss of function condition (either mutant or RNAi line).

We will now add data on shrb mutants where we find a phenotype that is similar as in Shrb::GFP overexpression.

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

The materials and methods section would benefit from more detailed explanations.

We agree.

Are the experiments adequately replicated and statistical analysis adequate?

Many of the experiments presented in this work are technically very challenging, like the in vivo analyses and particularly the serial-section electron tomography. This prevents having high numbers of replicates on occasions.

Minor comments:

1. In the abstract the authors state: "We show that apical endocytosis and late endosome-mediated trafficking determine the membrane allocation to the apical and basal membrane domains". I think that the authors show that "that apical endocytosis and late endosome-mediated trafficking is required for correct membrane growth", but I am not that sure that they show that it determines the membrane allocation

We agree and will change this.

1. References for the PH-GFP localization in cells should be provided. Which is the evidence that it only localizes to plasma membrane?

Apical localization of PH-GFP is preferential rather than exclusive. The construct has nevertheless been widely used as an ‘apical’ marker, based on the fact that this PH domain binds to PIP2, which is enriched in the apical domain of epithelial cells (e.g. Pilot, et al., 2006; Román-Fernández, et al., 2018).

1. It would be more adequate to always use the same terms to facilitate the reading. For instance, in several figures the membranes are referred as basal plasma membrane and tube membrane, but in others outer and inner membrane

We will go through the entire manuscript and use a consistent nomenclature.

1. Figure 3C,D and corresponding text are difficult to understand. The increase of fluorescence of the inner membrane seems to be very high, even higher than the corresponding to the outer membrane. Can the authors explain better this point and also describe better the method applied in the materials and methods section?

Rather than absolute amounts the graphs show fold increase over the amount of membrane at the beginning of the recording. We had used this representation for two reasons. First, the overall signal intensity can vary from one imaging set to the next, so comparing and representing absolute amounts from different datasets is not easily possible. However, we understand now why the representation in our plots was confusing. We will now show in Figure 3D how the total amount of plasma membrane in shibirets cells increases in the same way as in controls. For Figure 3C, we have found a better way of representing what percentage of the total membrane is present in each compartment as the cell grows. We will rewrite this part to make it clearer, and we will describe more thoroughly in the methods section how the analysis was done.

Significance:

This work represents an important advance for the field for several reasons. First of all it represents a technical advance because the authors are able to combine the traditional genetic analysis with two powerful techniques (in vivo imaging and serial-section electron tomography) to analyze single cell behavior at high resolution (temporal and spatial). In addition it represents a conceptual advance as it proposes a mechanism through which membrane growth is coordinated to regulate cell morphogenesis. The mechanism presented (endocytosis and transcytosis) is not new but they find evidence in an in vivo system.

It was previously known that tracheal terminal cells undergo a process of intracellular tube formation and cell elongation at the same time, but the mechanisms coordinating these two cell events were not known. The proposed mechanism may not only be relevant for the morphogenesis of tracheal terminal cells, but could represent a general mechanism of cell morphogenesis. Therefore, the paper should be relevant for research in the morphogenesis area but also in the cell biology field, as it shows how regulated membrane trafficking can control tissue morphogenesis

REFEREES CROSS-COMMENTING:

I agree with reviewer #4 on her/his comments and suggestions about analyzing the involvement of the recycling endosome in the process.

Reviewer #2 (Evidence, reproducibility and clarity):

In their manuscript, Mathew et al present a model in which transcytosis is utilized to deliver endocytosed apical membranes to supply basal membrane growth. The authors examined the developing terminal cell of the fruitfly tracheal system, which is a well established tubulogenesis model, as these cells form subcellular tubes by apical plasma membrane invagination. The authors show that basal membrane growth stops when endocytosis or endosomal transport is blocked, while the apical membrane grows excessively or membrane material accumulates in the cytosol, respectively.

Significance:

The authors used high-end microscopy (including CLEM and electron tomography) to support their model and in my opinion, the quality and the quantity of the presented data are indeed adequate for this. The text is well written, the figures are of superb quality, and several cartoons help to understand the presented data/experiments. Therefore I highly recommend submitting the manuscript to a cell biology journal in its present form.

Reviewer #3 (Evidence, reproducibility and clarity):

This paper investigates the role of membrane trafficking in growth of the polarized tracheal cell that forms a cellular projection containing a subcellular tube. The authors show that apical endocytosis and late endosome-mediated trafficking determine the membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically, and then appears to be continuously endocytosed, together with apical and basal cargo. The sorting and recycling of apical and basolateral membrane appears to occur in a novel organelle carrying markers of late endosomes and multivesicular bodies (MVBs). Inhibiting endocytosis eliminates this compartment.

The work in this paper is generally of high quality, and with one exception, quite comprehensive. The writing and figures are clear.

Major concerns:

1. A central focus of the paper is that balance between apical and basal membrane and the role of transcytosis in moving membrane from the apical compartment to the basolateral compartment. The current view is that transcytosis in mammalian cells usually goes through the recycling endosomes which are marked by rab11, although there is evidence for some trafficking through MVBs as well. In Drosophila, Rab11 positive recycling endosomes are frequently examined as part of endocytic system analyses. However, rab11 is not used as a marker in this paper and indeed there is no mention at all of recycling endosomes, even though recycling is at the core of the work. Since the authors do not examine rab11 or other possible markers of recycling endosomes, it is unclear whether the organelle they identify as carrying markers of late endosome and MVBs is some MVB/recycling endosome hybrid, or whether the organelle is completely distinct from the recycling endosome. Consequently, it is not possible to assess whether the observed trafficking either uses or does not use involve the recycling endosome. This ambiguity make is difficult to relate the observed trafficking to other systems. Minimally the authors should stain for rab-11 in WT and in some of the conditions where trafficking is perturbed and determine if the MVB-like compartment they are observing is rab11 positive, and whether the recycling endosome are affected by the perturbations. Further experiments may be needed to resolve whether any trafficking is going via the recycling endosome or this new MVB-type structure, but without even preliminary data on the relationship between the MVB compartment and the recycling endosome, its hard to say what might be appropriate or exactly how long addressing this will take. But just staining for rab11 in WT and a few mutant conditions to get a handle on what is up with the recycling endosomes in these cells should take less than a month.

We had done a number of experiments on Rab11 but did not include them because we felt they did not add any crucial insights on the mechanism we describe here. However, as the reviewer rightfully points out, Rab11 is a classical marker for recycling and transcytosis and we agree that the reader should know our results. We found that overexpressed Rab11::GFP as well as endogenously tagged Rab11:YFP are both highly enriched around the tube. Unlike Rab5 which is seen in widely spaced discrete vesicles, Rab11 forms a cloud of small puncta. We found very low overlap of Rab11 with CD4::mIFP-positive vesicles at the tip of the cell. This suggests that Rab11 is unlikely to be directly involved in the transcytosis pathway we describe here.

Loss of Rab11 was harder to analyse; Rab11-RNAi did not show any obvious phenotype, which could be due to high maternal contribution or to low knockdown efficiency, none of which we analysed in detail. Expression of a dominant negative Rab11 resulted in very early defects (reported by Le Droguen et al., 2015) which prevented us from analysing the role of Rab11 in tube formation. But because Rab11 does not localize to the compartment at the tip of the cell, we believe that this structure does not rely on Rab11 to transfer material from the apical to the basal membrane of the cell.

We will add the data on Rab11 distribution in Figure S6.

1. In addition to the above, I would recommend more discussion of how the authors' results relate to membrane trafficking and transcytosis in other systems. The recycling endosome should be considered, and it may be appropriate to draw comparisons to membrane trafficking in neurons that goes through MVBs (e.g. reviewed in VON BARTHELD and ALTICK Prog Neurobiol. 2011 Mar; 93(3): 313-340.). Although neurons are not hollow, they have definite morphological resemblance to tracheal terminal branches.

We thank the reviewer for the observation and will expand the discussion on MVB-mediated transcytosis in other systems.

1. line 204-208 "To test whether raised levels of Crb were responsible for the excessive apical membrane, as reported in other contexts (Pellikka et al., 2002; Schottenfeld-Roames et al., 2014; Wodarz et al., 1995), we knocked down Crb (Fig. S4G-H)." According to the legend for Fig s4, the authors express an RNAi construct against crbs. However, there does not appear to be any quantification of the amount knockdown of crb that was achieved. This is a concern for two reasons: 1) RNAi in the embryonic trachea works poorly for most genes (for unknown reasons) 2) This does not appear to be a clonal experiment but rather a pan tracheal driving of Crb RNAi. Loss of crbs would be expected to have very negative effects on tracheal morphogenesis (although this hasn't been rigorously tested to this reviewer's knowledge), but there doesn't appear to be any adverse effects of pan-tracheal crbs RNAi, suggesting that little if any knockdown of crb was actually achieved.

The authors either need to document the reduction of crbs or remove this paragraph. Preferably, they would be able to document the reduction of crb because they are trying to address an important point and if they can show the apical expansion is crb-independent, that would be an nice result.

Loss of Crumbs definitely is detrimental to embryonic epithelia, to different degrees (Tepass and Knust, 1990). We therefore could not use mutants but expressed an RNAi, which does not abolish Crb completely. We have now determined the degree of crb knockdown in our experiments. We stained Crb in embryos that expressed crb-IR in the entire tracheal system (but leaving the epidermal expression intact) and quantified the amount of Crb in the tracheal dorsal trunks, normalized to the signal in the epidermis. We found that in crb-IR embryos, Crb levels were reduced by around 50% compared to control siblings. We will add these results to Fig. S4 in the new version of the manuscript.

Minor concerns:

1. Lines 250. "By this interpretation, unscissioned membrane invaginations protruding from the subcellular tube would occasionally have touched the basal plasma membrane or its protrusions and fused with it, as transcytosing vesicles would have done in the normal situation."

I am not convinced by the argument that the bridging invaginations are fusing analogously to transcytosing vesicles because a protrusion/nascent vesicle coming from the apical surface should have rabs and V-SNAREs that should dock the protrusion/nascent vesicle with an endosomal compartment, not the basolateral surface. A transcytotic vesicle would have the rabs and V-SNAREs for the basolateral membrane. So it would seem that a fusion of the apical surface directly to the basolateral surface would have to be an ectopic event outside of the normal situation.

We agree that the fusion events could also be unrelated to the normal physiology of the animal. In other contexts (e.g. the embryonic epidermis, the synaptic bouton) blocking dynamin results in long membrane invaginations as a result of failure in membrane scission. In terminal cells, the apical and basal plasma membranes are very close to each other, and we believe this increases the chance of membrane invaginations meeting and fusion to take place. In addition, the long membrane invaginations we see seem to have been stripped of their clathrin coat suggesting that at least some aspects of ‘vesicle’ maturation proceed even though scission had failed. We also find evidence of small vesicles that resemble the contents of MVBs being deposited within the aberrant membrane invaginations. This suggests that MVBs are able to fuse with these unscissioned tubes and sheets, again indicating that the appropriate molecular markers are present, and the machinery in charge of generating these vesicles is active at the invaginated pits directly. In either case, we will rephrase our interpretations of these data and present it as speculation in the discussion section.

1. Significant figures. This is not a big deal, but the authors are over reporting their significant figures. E.g. "a 7.05-fold increase (+/-2.98 SD)" . With an SD that is 50% the value of the measurement, reporting to hundreds is definitely beyond the accuracy of measurement. Rounding to tenths would be more appropriate.

We agree with the reviewer. We will rephrase this section and use more appropriate metrics. As prompted by Reviewer #1, we modified the analysis that corresponds to this sentence, which also modified the way data is normalized also reducing the spread. This happened because in the new analysis we compare apical and basal signal for each timepoint, which allows better comparison between different cells.

Significance:

As there have not been that many studies on the dynamics of membrane trafficking during morphogenesis, the results should be of broad interest to those studying the endocytic system and the role of membrane trafficking in morphogenesis. However, the paper would be greatly strengthened if the authors considered the recycling endosome in their analysis and write up. As a well-known compartment for trafficking cargo and membrane to both the apical and basolateral surface, it is hard to know how to interpret the observed trafficking without knowing the involvement, or lack thereof, of recycling endosomes in this system.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this work the authors investigate the dynamics of cell morphogenesis in a convenient in vivo system. They use the terminal cells of the embryonic tracheal system and comprehensively address how cell shape change (elongation in this case) takes place and how membrane is remodeled during the process. By combining different high-resolution techniques, i.e. in vivo imaging of terminal cells expressing different membrane markers and serial-section electron tomography, they describe the organelle organization (ER, Golgi, different types of vesicles) in the terminal cells during their elongation. They identify the presence of membrane structures/vesicles particularly abundant at the tip of the cell ahead of the growing tube. When they block endocytosis they find increased tube membrane and lack of basal membrane growth. In addition, in tomograms, they observe clear membrane defects, like invaginations that could even connect the tube and basal membranes. This correlates with the absence of vesicles at the tip observed in normal conditions. The analysis of the nature of the vesicles observed indicated the accumulation of late endosomes and MVB, particularly at the tip of the growing terminal cells. Interfering with the formation of these MVB led to defects in the growth of both the tube and the basal membrane. Altogether the authors propose a model in which the newly formed membrane (and transmembrane proteins) passes through the ER and Golgi and reaches the apical membrane. The incorporated membrane is then rapidly endocytosed and follows a maturation pathway through MVB, from where different cargoes and membrane would be sorted and recycled back to the apical (tube) membrane, or to the basal membrane through a transcytosis mechanism

Major comments:

- Are the key conclusions convincing?

Most of the conclusions presented are convincing and supported by the results observed. However, to my understanding, one of the key conclusions of this work (that membrane is transcytosed from the apical to the basal domain) is not fully convincing. A critical result to support the author's conclusion of apical-basal transcytosis is to find clear evidence of basal accumulation of a transcytosed marker. The authors show accumulation of FGFR-GFP and Myospheroid as evidence. However, I find the results presented not very convincing. The accumulation of FGFR-GFP at the basal membrane in the control is not very clear in the images and movie presented. In addition, in the shibire mutant, some basal accumulation of FGFR-GFP seems to be detected (particularly in the movie). In the figures the authors show an increase of FGFR-GFP intensity when endocytosis is blocked, but this is not explained in the text. On the other hand, and very importantly, how does FGFR localization relates to its activity? The authors show that when endocytosis is prevented dpERK (i.e. a reporter of FGFR activation) is not decreased, indicating that FGFR is normally active. Wouldn't this suggest that FGFR is still localized basally to receive Bnl signal? Actually, as the authors indicate, in larval tracheal cells the intracellular accumulation of the FGFR leads to a reduced FGF signal transduction (Chanut-Delalande et al., 2010), suggesting that reduced FGF signaling activity in these cells is due to less FGFR reaching the basal membrane. The results with Myospheroid are not very convincing either, as the authors just show a single confocal section of control and shibire mutants. In summary, I consider that this very important point needs to be better documented before concluding that apical membrane material containing basal cargoes is transcytosed to the basal membrane.

Another conclusion that, to my opinion, should be better explained and documented, is the coordination of tube and basal membrane growth. Following the movements of the vesicles the authors conclude that there is a net displacement of these vesicles to the tip of the cell. This correlates with the presence of mature endosomes there. So the results postulate that transcytosis occurs at the tip, and therefore the growth of the basal membrane would occur preferentially at the tip. It has been demonstrated, as the authors indicate, that the tube membrane grows all along the length of the tube. How is then coordinated the tube and basal membrane growth? If, as the authors propose in their model, the tube membrane also grows after a process of endocytosis and recycling, wouldn't it be expected to have preferential tip growth? How do the authors reconcile all these observations with previously published results (Gervais and Casanova, 2010)?

The serial-section electron tomography analysis is very interesting and identifies different sorts of vesicles. However, it is very unclear what the different vesicles referred in the models correspond to (in the in vivo imaging for instance). For instance, the small granular or the dense-core vesicles correspond to endocytic vesicles at different stages of maturation?. If there is a constant endocytosis from the apical membrane to generate the basal and build the definitive apical membrane, wouldn't it be expected to find many more vesicles around the tube? Wouldn't it be expected to find coated vesicles around or budding from the tube, as the coated vesicles observed budding from the basal membrane in Fig 2D? Or is the endocytosis observed mediated by non-clathrin coated vesicles?.

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

The results of Serp accumulation upon MVB interference can lead to confusion (from line 404). The authors seem to suggest that Serp protein is exclusively produced in the FB and transported by transcytosis to reach the tracheal lumen. However, Serp is also produced in the tracheal cells themselves. In fact, serp expression in the FB seems to be detected by late embryogenesis, while expression in tracheal cells is detected much earlier (Dong et al. 2014; Luschnig et al. 2006; Wang et al. 2006). It was also shown that Serp undergoes a recycling mechanism from the lumen to the lumen, through the endosomes-TGN retrograde trafficking, that may also require Shrub (Dong et al, 2014; Dong et al 2013). Thus, it is unclear (and even unlikely) that the Serp found in the vesicles in Shrub-GFP mutants is derived exclusively from the transcytosed component from the FB. I suggest to better explore this issue or to remove this part.

- 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.

As indicated before, more conclusive results for transcytosis should be provided.

I suggest that the authors determine the presence of apical and basal cargoes (FGFR) in the late endosomes found when Shrub activity is impaired. According to his model, both types of markers should accumulate there. A high accumulation of these markers in those endosomes would reinforce the hypothesis proposed.

While it has been reported that shrub-GFP act as a dominant negative in different contexts (Dong et al, 2014; Sweeney et al 2006) it is unclear why. So it would be desirable to confirm the results with a loss of function condition (either mutant or RNAi line)

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

The materials and methods section would benefit from more detailed explanations.

- Are the experiments adequately replicated and statistical analysis adequate?

Many of the experiments presented in this work are technically very challenging, like the in vivo analyses and particularly the serial-section electron tomography. This prevents having high numbers of replicates on occasions.

Minor comments:

-In the abstract the authors state: "We show that apical endocytosis and late endosome-mediated trafficking determine the membrane allocation to the apical and basal membrane domains". I think that the authors show that "that apical endocytosis and late endosome-mediated trafficking is required for correct membrane growth", but I am not that sure that they show that it determines the membrane allocation

-References for the PH-GFP localization in cells should be provided. Which is the evidence that it only localizes to plasma membrane?

-It would be more adequate to always use the same terms to facilitate the reading. For instance, in several figures the membranes are referred as basal plasma membrane and tube membrane, but in others outer and inner membrane

-Figure 3C,D and corresponding text are difficult to understand. The increase of fluorescence of the inner membrane seems to be very high, even higher than the corresponding to the outer membrane. Can the authors explain better this point and also describe better the method applied in the materials and methods section?

Significance

This work represents an important advance for the field for several reasons. First of all it represents a technical advance because the authors are able to combine the traditional genetic analysis with two powerful techniques (in vivo imaging and serial-section electron tomography) to analyze single cell behavior at high resolution (temporal and spatial). In addition it represents a conceptual advance as it proposes a mechanism through which membrane growth is coordinated to regulate cell morphogenesis. The mechanism presented (endocytosis and transcytosis) is not new but they find evidence in an in vivo system. It was previously known that tracheal terminal cells undergo a process of intracellular tube formation and cell elongation at the same time, but the mechanisms coordinating these two cell events were not known. The proposed mechanism may not only be relevant for the morphogenesis of tracheal terminal cells, but could represent a general mechanism of cell morphogenesis. Therefore, the paper should be relevant for research in the morphogenesis area but also in the cell biology field, as it shows how regulated membrane trafficking can control tissue morphogenesis

REFEREES CROSS-COMMENTING: I agree with reviewer #4 on her/his comments and suggestions about analyzing the involvement of the recycling endosome in the process.

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Reply to the reviewers

Review comments Rebuttal

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary ** This manuscript describes the X-ray structure determination of two SMAD-DNA complexes confirm that SMAD family proteins bind at least two DNA sequences in a similar fashion, and explores dimer versus monomer formation of the non-DNA bounds forms of the proteins which could influence whether the proteins bind as monomers and dimers. This includes identifying a loop which appears to make a major contribution to this process. There is a lot of experimental work and analysis included. **Major comments: ** The overall conclusions of the manuscript are convincing, but some of the detailed analysis is not clear. The structures look good, the experiments look to be generally well controlled, although some details could be provided in the main text to be clear about what methodology is being used or how analysis was carried out and stepwise conclusions obtained. In particular the analysis of SAXS data is not clear. I'd like to see initial data analysis presented as per the guidelines of Trewhella et al 2017 (PMID: 28876235). There is some mention of data in the SASREF database, but it should be in the supplemental data.

We have prepared a table following this recommendation.

I can't see any evidence for the conclusions about open versus closed monomer state (how good were the fits obtained) - just a graph and a statement. If this can't be better justified please remove the conclusions about these states (they don't really add to the overall conclusions about monomer/dimer which are much less specific), but even the simple analysis supports mostly monomer and small amounts of dimer or higher aggregates. I would also like to see a clear explanation provided about why the MS data supports dimer over other oligomers

We have revised and simplified the SAXS section to clarify the main points. We have re-analyzed the conformations in solution, and the values are presented in new Table S4 and new Figure 3D. We have also included new panels (Figure 3E) and explanations with respect to the IM-MS data (pages 8-9).

State what thermal unfolding experiments are were carried out in the text (and why is the data biphasic?)

The biphasic graphics were interpreted as the presence of dimers and monomers in equilibrium. As suggested by the other reviewer, we have removed these sections as they do not contribute to clarify the main points of our work.

The concept of long versus short loops re domain swapping have been studied in the past but there isn't much reference to this.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373619/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349918/

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039305

https://www.ncbi.nlm.nih.gov/pubmed/22411444

We now mention some related examples in page 8.

**Minor comments: **

The last couple of paragraphs of the introduction are a fairly comprehensive summary of the study overall and the conclusions of the paper. While presaging the key findings and conclusions is fairly common in an introduction this seems to be way too much detail. Unless it is a requirement of the journal, reduce these sections to a couple of sentences and use any other word count to explain your analysis better.

Thank you for this recommendation. We have rephrased and reduced this part of the introduction.

Figures are quite small and hard to see detail at 1X magnification (in both the main and Supplemental figures).

We have removed some panels that were not necessary and increased the size of the figures and labels.

NB.The difference in Tm of SMAD 5 over 8 doesn't seem particularly high as it’s only a couple of degrees (especially when SMAD4 is quite different). The explanation for the Ile>Cys mutation might be about competition of zinc ligation (except that it doesn't seem to cause issues for many zinc finger proteins) but more likely that you've replaced a reasonably bulky hydrophobic sidechain and therefore have lost a bunch of hydrophobic contacts.

We have removed this section entirely.

With respect to the Ile>Cys difference, the residue is located in a loop, and it does not participate in hydrophobic contacts. We still believe that its negative role in the stability of the domains arises from the competition for Zn coordination but we agree with the reviewer that quantifying its specific role is not obvious.

Reviewer #1 (Significance (Required)):

-This paper clarifies concepts about the state of isolated SMAD proteins (thought be largely monomeric in the absence of DNA) and DNA-binding preferences of these proteins. -I don't have specific expertise in the structure/function of SMAD proteins, but the study appears to include sufficient background to place the study in context.

-Audience will mostly be those interested in structure/function of SMAD proteins, with some protein engineers interested in the manipulation of monomeric versus dimer.

-I am a protein chemist and structural biologist with an interest in protein dimerization/oligomerization. I am familiar with most techniques presented, but don't have first-hand experience with IM-MS.

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

**Summary**

The manuscript by Ruiz et al examines how the receptor-activated SMAD (R-SMAD) transcription factors bind DNA, and specifically how the MH1 (DNA-binding domain) of the different classes contributes determining whether they bind as monomers or dimers.

In the context of the full length SMAD proteins, it is thought that hetero-trimers with one SMAD4 plus two R-SMADs are the functional unit. In general, the SMAD1/5/8 R-SMADs respond to BMPs whereas SMAD2/3 respond to TGF beta. However, what is less clear is how the specificity of the gene responses is determined, since all SMADS are able to bind to each of the two sequence classes of response element (GTCT and GC-rich, or 5GC).

Previous structural studies suggest that the major contacts between SMAD MH1 and DNA are very similar, irrespective of the particular SMAD or of whether they bind a TGTC or 5GC element. On DNA, MH1 domains have been observed as dimers, but there has been some concern as to whether this (at least in part) is a crystal artefact, or is perhaps forced by the specific DNA sequences use in these studies. For the BMP R-SMADS this may be less likely, since the amino-terminal helix 1 of one dimer is seen to be dislodged from its own intramolecular interactions allowing it to make contacts with the second MH1 domain in the dimer.

Here the authors test this question of MH1 dimerization and address differences between the BMP responsive and other SMADs. They first show by crystallography that SMAD5 and SMAD8 MH1 domains adopt similar dimeric conformations with the displaced helix 1, and bind to a single 5GC element via one of the MH1 domains. To get at whether these MH1 domains form dimers in solution, they use small angle X-ray scattering, NMR and mass spectrometry, to suggest that the SMAD5 and SMAD8 MH1 domains in solution do not fit with a single conformation, but are better modeled by a mixture of dimer and open monomer. Ion mobility MS also suggested a mix of dimer and open monomer for the BMP SMADs, whereas SMAD3 appeared to be primarily monomer. To test if the MH1 domains themselves encode this potential difference between SMAD5 and SMAD3, they swap loop 1 (6 versus 4 amino acids, between helices 1 and 2) from SMAD3 to SMAD5, and now in solution this chimera appears monomeric, and forms monomers when crystalized with DNA.

Major comments

1. Adding the SMAD3 loop to SMAD5 prevents the open dimer - does the reverse also work? Can you make SMAD3 form SMAD5-like open-dimers by adding the loop 1 sequence from SMAD5?

We have prepared new Smad3 chimeric constructs and we are currently screening crystallization conditions in order to obtain diffracting crystals (if possible). Unfortunately, due to the COVID-19 pandemic, access to our laboratory is highly restricted, while access to synchrotron and mass spectrometry facilities is not available), therefore this work has been postponed until the end of April/May. For this reason, the revised version of the manuscript does not refer to this question. We hope that we will be able to address it in the future.

1. Can the authors include similar schematic models for how the site spacing would be for SMAD2/3-SMAD4 complexes - adding the SMAD2/3/4 model to Figure 5C?

We have incorporated new panels to Figure 5 (Figure 5E,F).

1. The authors comment on the possibility that the dimer conformation dictates the spacing of the sites that will be bound in vivo. In this context, they refer to a previous paper (PMID: 29234012) to suggest differences in site clustering between BMP SMAD and TGF beta SMAD regions of the genome (from ChIP-seq) that fit with the spacing they imply here. However, the major difference shown in this work seems to be between the clustering of GC sites and GTCT sites irrespective of the pathway. Can the authors analyze existing ChIP-seq data to more specifically test the question they raise - ie that SMAD4 bound regions of the genome have different site clustering/spacing depending on whether they are BMP or TGF beta responsive?

Thank you very much for this recommendation. We agree with the reviewer that this information is very valuable and can help support our hypothesis on the different binding preferences of monomers and dimers of MH1 domains. We have performed this analysis and is now included as two new sections. The results are displayed as new Figure 5A,B.

1. I think Figure 2C,D is not really well described in terms of the importance to this work. As it is this data does not really seem to add very much, but perhaps I am missing the importance.

We have entirely removed this section in the new version of the manuscript.

5.Can the authors comment about the compressed GC element or BRE? This seems to be an unfavorable conformation. How might it be bound in vivo, is it an unusual element, or is it relatively widely found? Is it possible that in vitro it binds two MH1 domains, but in vivo might simply act as a normal 5GC, with an additional site nearby?

The BRE domain is less abundant than 5GCs and SBE sites, and in fact, this sequence is not enriched in the ChIP-Seq datasets that we have analyzed. We have included a sentence refering to these findings in page 11.

We have also revised the section comparing the 5GC and BRE-GC site and illustrate this interaction as well as the comparison to our 5GC complex by including two panel in Figure2 that before were displayed as supplementary information. The panels have been edited to clarify the similarities and differences between both complexes. Indeed, the protein-DNA complex made of one BRE motif bound by two MH1 domains as found in the PDB:5X6H crystal structure suffers from several issues, including compactness of the two overlapping Smad-binding motifs that led to distortion of DNA geometry, clashes at protein-protein interface and local cancelation of protein-DNA interactions.

In this new section (page 8) we include the sentence that “we believe that the most probable binding mode in vivo should be that observed in the 5GC and SBE complexes. It seems very unlikely that two MH1 domains would interact with a reduced BRE motif —using half of their protein binding site and causing a high distortion to the DNA structure— if there is the possibility to interact with neighboring sites (Figure 2B,C) using the full protein binding interface and a perfect accommodation to the DNA”.

**Minor comments: ** 1.In Figure 1B is one the two DNAs assumed? In the structure was it two MH1 to one DNA or two of each?

One DNA was hidden for clarity. The crystallographic structure is now shown in full. The crystal structure was solved for a complex made of two MH1 domains bound to a dsDNA molecule that included two Smad-binding sites.

2.Figure 2C and page 9: the stabilization of SMADs in the text and figure do not agree. Maybe just state the exact numbers from the figure in the text.

We have entirely removed this section in the new version of the manuscript.

3.In Figure S1C, can the authors label the retarded complexes on the gels?

Done.

4.Figure 4A - explain the asterisk (presumably the SMAD2 insert).

Yes, it corresponds to the Gly rich region present in loop1. It is indicated now.

5.In Figure 4B, C (and maybe D) can they color helix 1, loop 1, and helix 2 three separate colors, it might really emphasize the effect of the loop if it was more immediately visible.

We have improved these figures but we did not change the colors because the figure was getting even more complicated.

6.The legend to Figure 4 is missing F.

Thank you. This has now been corrected.

Reviewer #2 (Significance (Required)):

The authors conclude that the length of the loop between helices 1 and 2 determines the dimer versus monomer state - a shorter loop as in the BMP SMADS hinders the intramolecular interactions needed for the closed monomeric form, whereas the longer loop in the other SMADS allows the flexibility for these interactions so favors a more closed monomeric form. Showing that the dimers are not forced by crystallography or by binding to fixed DNA elements clearly adds to our understanding of the mechanisms of SMAD function, and it is of interest that the BMP and TGF beta SMADS are different in this respect.

They speculate that this may contribute to the specificity of the responses activated by BMP versus TGF beta signaling based on the requirements for different site spacing depending on whether an open (BMP) or closed (TGF beta) dimer of R-SMADS is present. This idea is likely to be of interest to anyone who studies the responses to the TGF beta superfamily of signaling molecules, and should spur additional experimentation to test it.

As teachers it is important for us to think about the stereotypes we hold of the students we teach. How does it influence teaching and learning?

I think this last point is really excellent. If the ability to repair any infrastructure isn't built holistically into a system the ability for any option other than building over or removing and replacing isn't on the table.

For example when I was a construction inspector for the Township of Lake Como I worked on their artificial aquifer that fed their lakes and houses. They had chosen to build a piping system that had no shut off connectors for outflow and further draining on the north half of the town and on the southern half they had built into those connectors. We were able to reline the southern half of the town's pipes in 5 weeks and we had to remove and replace almost every major water pipe in the town on the north half which took 6 months. A lack of any ability for maintence leads to greater problems.

LOVE THIS! Children want to know 'why' they are here? Their purpose (not the 'purpose' their parents think they should have ;)! When we pay close attention we may find that the innate desires/interests that the children show us are in fact tied to their ultimate purpose. For example, a little boy whose passion for building is supported by providing him wood, tools, etc. so that he can build a table - later we come to find out that this same boy became an adult contractor.

This conclusion proves the point that I made in my alternate annotation. We are obviously able to see so many differences propagated by cultural relevancy phenomenon. In fact, each case study outlines this very thing. There are countless examples down to even when a person becomes an autonomous human entity. So why must we resort to categorizing in such a primitive other-ing way? I think that there could have been many stronger conclusions to this piece and I am disappointed to see the author resort to the insufficient cop out method of binary categorization.

I think it is easy to classify writers if grammar is the only factor taken into consideration. Otherwise, everyone has a different idea of what makes someone a good writer. For some, it may be the ability to make the reader feel something. For others, someone who uses big words and long sentences may be thought of as the best writer in the world. I believe our personal classifications are dependent on our own relationship to writing and what we strive to improve in our own writing.

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Reply to the reviewers

We thank all the Reviewers for taking the time to evaluate our manuscript and providing us with constructive feedback. We are pleased to hear that all Reviewers appreciate the importance and significance of our study, commenting that our conclusions are ‘convincing, are supported by the presented experimental results’ and that our study ‘will yield novel insights into the regulation and function of PALB2 in DNA repair’.

Please refer to our point-by-point response to the specific points raised, in which we highlight a couple of key experiments to be conducted to refine our study in bold. We are grateful for all the reviewers’ remarks and suggestions, which will certainly lead to a substantial improvement in our manuscript.

Point-by-point response:

Reviewer #1 (Evidence, reproducibility and clarity (Required)): Fournier et al. detect acetylation within the chromatin association motif (ChAM) of PALB2 and demonstrate that KAT2 can acetylate these 7 lysine residues within this region. They then generate K to R mutations (7R) or K to Q mutations (7Q) at these sites and perform assays of fluorescence recovery after photobleaching (FRAP) to measure mobility as a measure of chromatin association, RAD51 foci, PALB2 recruitment at sites of laser-induced DNA damage, and sensitivity to olaparib. They find increased mobility of the 7Q mutant of PALB2 but not 7R in the absence of exogenous DNA damage, as well as defects in DNA damage-induced RAD51 foci and resistance to olaparib. On this basis, the authors conclude that acetylation is required for the association of PALB2 with undamaged chromatin and that deacetylation permits mobilization and association with BRCA1 to enable proper DNA repair. While the manuscript is generally well-written, many of the systems are rather elegant, and this study may yield novel insights into the regulation and function of PALB2 in DNA repair, there are some missing experiments to be added and important contradictions that should be resolved in order to fully establish the new model the authors propose.

**Major comments:** 1.There are some concerns about the interpretation of experiments with the 7R and 7Q mutants of PALB2. For example, in the description of results in Fig. S2C, the authors state "K to R substitutions maintain the charge yet are unable to accept acetylation and hence mimic constitutively non-acetyl lysine". However, in Fig. 4B the association of the 7R mutant with chromatin is similar to WT and in Fig. 7D,E the relative immobility of the 7R mutant is very similar to WT PALB2. Thus, the conclusion that acetylation is required for PALB2 association with damaged undamaged chromatin and for release of PALB2 upon DNA damage does not appear justified. Perhaps the authors need to better consider whether the 7R mutant mimics acetylation because of its charge. Even so, the mutant then maintains the charge normally associated with acetylated PALB2, calling into question whether deacetylation indeed "releases PALB2 from undamaged chromatin".

We agree with the Reviewer’s point that there is no or little difference between WT and the 7R mutant in regard to their enrichment on non-damaged chromatin, as detected by fractionation (Fig 4B), or their mobility, as detected by FRAP (Fig 4D and E). Note that Fig. 7D is our model and Fig. 7E does not exist. As the Reviewer suggests, it is possible that, in contrast to the 7Q mutant, which is defective in both nucleosome and DNA binding (Fig. 2E and F), the 7R mutant may maintain its electrostatic interaction with DNA, while lacking its acetylation-mediated nucleosome interaction, masking the impact of substitutions. This assumption is in line with our model in which ChAM DNA binding assists HR repair, which is supported by the 7R mutant but not by the 7Q mutant. To better dissect the question raised by the Reviewer, we will conduct biochemical analyses of the ChAM 7R mutant, testing its direct interaction with nucleosomes and DNA; the results will be included in the revised manuscript (Experiment 1).

It is also worth noting that full-length PALB2 is enriched at a fraction of H3K36me3-marked exons (which comprise only 1-1.5 % of the whole genome), as shown in our previous genome-wide ChIP-seq analysis (Bleuyard et al., 2017, PNAS). Hence, it is also possible that bulk fractionation or FRAP analyses might not be sensitive enough to highlight the impact of the 7R mutation. Conversely, we foresee that the ChIP-qPCR method, detecting PALB2 association at defined genic regions as shown in Fig S5, will be more appropriate. Thus, in the revised manuscript, we will expand our ChIP-qPCR analyses to further validate our proposed model (Experiment 2).

2.Related to questions of interpreting results utilizing the 7R and 7Q mutants of PALB2, in Fig. 7B,C the 7R mutant but not 7Q supports RAD51 foci and resistance to olaparib similar to WT PALB2. The authors then state in the Discussion that "our work also suggests that caution should be exercised in the use of K to Q substitutions for functional studies of lysine acetylation". Thus, which mutant is giving the correct and reliable results?

We apologise for the miscommunication if this point was unclear. Using biochemical approaches, we established that ChAM acetylation, but not K to Q substitution, facilitates its association with nucleosomes (please compare Fig 2E and Fig 3B). This observation clearly demonstrates that K to Q substitution does not mimic acetylation at these residues, but instead renders PALB2 ChAM functionally null. The PALB2 7Q phenotypes therefore demonstrate the importance of the 7K patch for ChAM function in HR repair, rather than its acetylation status.

Perhaps even more importantly, if results with the 7Q mutant are suspect, the conclusion that deacetylation is required for HR (or DNA repair) is suspect because that is the only case where the authors see a defect in RAD51 foci and resistance to olaparib. Similarly, if the 7R mutant "mimics non-acetyl-lysine" then the fact that it has normal RAD51 foci and resistance to olaparib contradicts the conclusion that deacetylation is required for DNA repair.

Unfortunately, it is currently technically not possible to ‘lock’ the PALB2 7K patch in its acetylated status in vivo (i.e. preventing PALB2 dissociation from active genes). We thus agree with the Reviewer that it is difficult to draw definitive conclusions on the impact of constitutive PALB2 acetylation in HR, although the importance of the 7K-patch for the functionality of PALB2 is evidenced by the 7Q mutant phenotypes. Similarly, strictly speaking, our results using the 7R mutant support the notion that the ‘non-acetylated’ status of the 7K mutant, but not necessarily the dynamics of ‘de-acetylation’ events, can promote HR repair. In the revised manuscript, we will rephrase and clarify these points.

3.There are multiple concerns about Figs. 5 and S5. In Fig. 5A-C, difference in cell cycle progression after synchronization are relatively small and no rationale/interpretation is given for how this may be related to PALB2 function is given. In Fig. 5D,E differences in the levels of gamma-H2AX as a marker of DNA damage between different forms of PALB2 do not become readily apparent until about 6 or more days after addition of doxycycline. As such, it seems that these could be indirect effects and it is unclear how strongly this supports the importance of PALB2 acetylation in the DNA damage response.

We apologise for the miscommunication on these points. We have previously established that steady-state PALB2 chromatin association, jointly mediated by the ChAM and MRG15 interaction, protects a subset of active genes from DNA damage that may otherwise arise from replication-transcription conflicts (Bleuyard et al., PNAS 2017). The results presented in Fig 5 and S5 led us to propose that PALB2 chromatin association is, at least in part, mediated by the ChAM 7K patch, and its acetylation (hindered by 7Q and 7R substitutions, respectively) prevents DNA damage via a similar mechanism, i.e., protecting PALB2-bound genes during replication. This model nicely supports our observations that both 7Q/7R mutants exhibit slow S-phase progression and accumulation of gamma-H2AX over time. These points will be better articulated in the revised manuscript.

In Fig. S5, it is interesting that there are differences in the association of different forms of PALB2 with 3 distinct active loci, but no error bars or measures of statistical significance are given. Further at 2 of the 3 loci, the association of the 7Q mutant is closer to WT than the 7R mutant. Taken together, neither Fig. 5 nor Fig. S5 strongly support the key conclusion that acetylation regulates the association of PALB2 with actively transcribed genes to protect them.

We appreciate this constructive comment. The analysis was conducted once, albeit with three technical replicates, which explains why the results are presented without error bars. Nonetheless, we observe a consistent trend at three different loci, that both 7R and 7Q have chromatin association similar to the empty vector, which is background level (FLAG/IgG ChIP) and does not reflect real binding. The revised manuscript will include the results from three biological replicates with statistical evaluation (Experiment 2).

4.Figs. 6D-G and S6A-D conclude that "DNA damage triggers ChAM deacetylation and induces PALB2 mobilization" based upon FRAP experiments utilizing WT PALB2. But there is no control to demonstrate that this is a specific effect driven by the state of PALB2 acetylation. For example, DNA damage might cause global acetylation changes resulting in relaxed chromatin in which proteins that are not subject to acetylation-deacetylation also show increased mobility.

We thank the Reviewer for this valuable comment. It is true that we cannot formally exclude the possibility that changes in PALB2 mobility are indirect consequence of damage-induced chromatin reorganisation/increased chromatin mobility. However, our analyses clearly demonstrate that ChAM acetylation increases its association with nucleosomes (Fig. 3B), while non-nucleosome binding ChAM-null (7Q or deletion) increases PALB2 mobility (Fig. 2E, Fig. 4E and Fig. S4C). Further, WT PALB2 mobility increases after KAT2 depletion (i.e. reduction of chromatin acetylation of KAT2 targets, hence chromatin compaction) (Fig. 3F), but reduces upon KDAC inhibition (i.e. global increase in acetylation, hence chromatin relaxation) (Fig. 3G). Considering all these observations collectively, the increase in PALB2 mobility detectable upon DNA damage is unlikely to reflect global chromatin relaxation, and that PALB2 acetylation influences its mobility in both challenged and unchallenged cells. This point will be emphasised in the revised manuscript.

5.Fig. 7B shows that the 7Q mutant has diminished RAD51 foci while Fig. S7C,D suggests based upon a different methodology (laser-induced damage) that the 7Q mutant does not affect PALB2 recruitment. Since the issue of recruitment is key to the mechanism proposed, the authors should examine PALB2 foci instead as this may be a more sensitive assay of PALB2 recruitment.

We appreciate the Reviewer’s point. We would like to highlight, however, the well-documented role of BRCA1 in PALB2 recruitment to sites of DNA damage. This supports our notion that the 7Q mutant is recruited to sites of DNA damage, likely mediated via its interaction with BRCA1. As depicted in Fig. 7D, we propose that the 7K patch-mediated PALB2 engagement with damaged DNA, which is disrupted by the K to Q substitutions, is essential for proper RAD51 loading onto DNA, hence RAD51 foci formation and HR repair. This is in line with our observation that PALB2 ChAM deletion, similarly to the 7Q mutant, perturbs damage-induced RAD51 foci formation (Bleuyard et al., EMBO Rep. 2012). We believe that the laser-induced experiments provide high sensitivity and resolution for PALB2 recruitment kinetics, as the data were obtained with real-time live-cell imaging.

6.The authors state in the last sentence of the Results section that "lysine residues within the ChAM 7K-patch are indispensable for PALB2 function in HR" but never test the mutants for HR using reporter assays. The manuscript would be strengthened by performing such assays.

RAD51 foci formation and sensitivity to PARP inhibition are well-accepted readouts for HR repair. Conversely, we have been cautious about existing HR reporter systems, which evaluate gene-conversion or targeting events triggered by a ‘clean’ enzyme-induced DSB, but not an authentic repair of ‘dirty’ DSB induced by IR or olaparib.

7.The model for the role of ChAM acetylation in regulating PALB2 function presented in Fig. 7D is not fully supported by the data presented. Critically, while association with RAD51 and BRCA2 is tested in Fig. S7B, the authors hypothesize that deacetylation is required to release PALB2 to enable association with BRCA1 but this is not tested utilizing the mutants.

We appreciate the Reviewer’s point. It has been demonstrated that PALB2 interaction with BRCA1 is triggered by damage-induced PALB2 phosphorylation (Ahlskog et al., EMBO Reports, 2016), as well as removal of KEAP1-mediated ubiquitylation in S and G2 (Orthwein et al., Nature 2015). Our preliminary analyses further suggest that BRCA1-PALB2 interaction is highly dynamic, and we propose that damage-induced PALB2 modification and its mobilisation jointly facilitate this interaction.

Also, there are some specific points that should be considered in the context of the model. This includes how DNA damage may trigger deacetylation, and whether it is the deacetylated state or the process of deacetylation of ChAM that is critical. Also, if acetylation is important for protecting active genes in the absence of DNA damage, is deacetylation necessary to release PALB2 local or global. This is important, because if it is local there needs to be a specific mechanism for local deacetylation, while if deacetylation is global that could result in transcriptionally active genes becoming unprotected.

We thank this Reviewer for this valuable comment. We agree that, while this study establishes that ChAM is deacetylated upon DNA damage, it remains unclear whether the dynamic ‘de-acetylation’ of PALB2, rather than the ‘non-acetylated status’ of PALB2, is important for HR repair, and whether or not this is a local event. However, we would like to highlight that PALB2-bound genes are mostly periodic, e.g. those required for cell cycle progression (Bleuyard et al., 2017, PNAS). It would therefore be reasonable to speculate that DNA damage triggers the suppression of periodic gene expression as a part of DNA damage checkpoint signalling, possibly in a KDAC-dependent manner, which then allows release of PALB2 without risking DNA damage that could otherwise be caused by replication-transcription conflict. Mobilised PALB2 might then be recruited to sites of DNA damage for HR repair. Further study will be required to fully evaluate this model, for example by identifying the specific KDAC involved in ChAM deacetylation and tracking individual PALB2 molecules, which we consider to be beyond the scope of the present study. In the revised manuscript, we will better describe our model, and further detail the arising questions to be addressed in future studies.

**Minor Comments:** a.Some parts of the Materials and Methods are overly long (such as the subsection on "Protein purification" and "immunofluorescence microscopy") and could be shortened by consolidating experimental details that are largely the same for related processes.

We propose to move these experimental sections to the supplementary information.

b.In the description of Fig. 1D, the statement "7K-patch, which is common to PALB2 orthologs" is misleading since there is not complete conservation of each lysine residue across each ortholog.

We agree with this Reviewer’s comment and will amend the description in the revised manuscript accordingly.

c.Figs. 3E,F and S3B,C perform FRAP in cells with knockdown of KAT2A/B as a surrogate for chromatin association. The authors note that this global reduction in acetylation increases PALB2 diffusion, but there is concern that this experiment is not very informative because the increased mobility may have nothing to do acetylation of PALB2.

Please refer to our answer in response to the Reviewer’s point 4.

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

This manuscript reports the control of PALB2 - chromatin interaction by the acetylation of a particular lysine-rich domain of the protein called ChAM. This acetylation is shown to be mediated by the acetyltransferases KAT2A/B. Following these investigations, the authors made an effort to place their findings in the context of DNA replication and DNA repair. The proposed model is that the acetylation-dependent interaction of PALB2 with chromatin could ensure the protection of the genome during DNA replication and control DNA repair. **Specific remarks** 1 - Based on different experiments, essentially the one shown in Fig. 3B, the authors conclude that the acetylation of the ChAM domain enhances its association with nucleosomes. However, taking into account the experimental setting, this conclusion should be largely tuned down. Indeed, this enhanced acetylation-dependent nucleosome binding was observed when the experiment was carried out in the presence of excess of free naked DNA. Under these conditions, the non-acetylated ChAM fragments became mostly trapped by DNA (clearly shown in Fig. 3C/D), and hence would not be available for nucleosome binding, while the acetylated ChAM fragments would remain available for nucleosome association because of their reduced DNA-binding ability. Consequently, the acetylation of the ChAM domain would only play a role on the availability of PALB2 for chromatin/nucleosome binding and not directly stimulate nucleosome binding. Therefore, the nucleosome-binding capacity of ChAM by itself should not be dependent on ChAM domain acetylation.

If true, this hypothesis could also be relevant in vivo since the poly-K in the ChAM domain could also non-specifically interact with nuclear RNAs and hence its acetylation, by releasing it from nuclear RNAs, would make it available for chromatin-binding. The importance of RNAs in the regulation of PALB2 nucleosome-binding could be tested in the experiments shown in Fig. 2C and 2E by adding RNase to the pull-down medium (WT +/-RNase or addition of increasing exogenous RNAs).

We are grateful for the Reviewer’s detailed comments and find the potential involvement of RNA very intriguing. Indeed, transcriptionally active loci, which are bound by PALB2, are enriched in nascent RNA, and such local RNA may play an important role in promoting the association of acetylated PALB2 with nucleosomes. However, we believe that investigating the role of RNAs in PALB2 nucleosome binding is beyond the scope of this study. As discussed extensively in response to this Reviewer’s point 2 below, we believe the mode of interaction of ChAM with nucleosomes to be highly complex, being jointly mediated by the N-terminal conserved region and the C-terminal lysine cluster. We will discuss these issues more extensively in the revised manuscript.

2 - The real question is as follows. While acetylation makes the protein available for nucleosome binding, which part of the ChAM domain is actually mediating nucleosome binding and whether lysine acetylation could be directly involved in this binding. Another question would be to identify the elements in the nucleosome mediating this interaction, histones (core domain, tails, post-translational modifications, specific histone types), histone-DNA, etc...

We entirely agree with the Reviewer’s question – despite the increasing recognition of the physiological importance of the PALB2 ChAM and our efforts in understanding the mode of association of ChAM with nucleosomes (including the potential involvement of histone tail modifications), this specific question remains enigmatic.

Explicitly, our previous work demonstrated that substitutions of residues within the evolutionarily highly conserved N-terminal part of the ChAM perturb its association with nucleosomes (Bleuyard et al., 2017, PNAS; Bleuyard et al., 2017, Wellcome Open Research). A recent study by the laboratory of Prof Jackson proposed that basic residues across the ChAM are part of a binding interface with an acidic patch of histone H2A in its nucleosomal context (Belotserkovskaya et al., Nat Comm. 2020). Our results presented in this study introduced an additional complexity, showing that the C-terminal 7K basic patch is essential for ChAM-nucleosome interaction. Intriguingly, our study also suggests that the regions flanking ChAM, which are phosphorylated at multiple residues, play roles in regulating ChAM binding to nucleosomes (Fig 2B and C; please refer to our answer to the Reviewer’s minor point 6 too).

We are currently working towards solving the structure of ChAM in complex with a nucleosome, which may help to clarify this very important question. At this point, we think that the question about complete elements for the ChAM interaction with nucleosome is out of the scope of this manuscript, and should be addressed in future work. To make this point clear, we will provide an updated overview of the ChAM elements affecting nucleosome interaction in the revised manuscript.

3 - Taking into account the authors conclusions on the role of ChAM domain acetylation and its impact on PALB2 mobility, in Figure 4D/E, one should expect a difference of t1/2 when wild-type and 7R mutant are assayed by FRAP. At least the measures of t1/2 in the wild-type should have been more heterogeneous compared to the 7R mutant due to the acetylation of the wild-type PALB2 by the endogenous HATs (the impact of endogenous HATs on the wild-type sequence is shown in Fig. 3F). Could the authors comment on this?

We appreciate this Reviewer’s point. As mentioned in our responses to Reviewer 1’s points 1 and 3, we are unable to exclude the possibility that the 7R mutant still maintains its DNA-binding capacity, masking detectable change in its chromatin enrichment and mobility. Also, PALB2 in vivo chromatin association is limited to a small fraction of periodic genes, hence FRAP assay may not be sensitive enough to detect minute but critical differences. We will conduct biochemical assessment of the ChAM 7R mutant and ChIP-qPCR analyses to assess PALB2 binding to specific genes, results of which will be included in the revised manuscript (Experiments 1 and 2).

4 - It would be better to remove the data presented in Fig. 5 since, as currently presented, these investigations remain shallow and do not bring much information on what is happening. The presented data are rather confusing since, in the absence of further investigations, it is not clear which one(s) of the mechanisms involved in the control of DNA replication is controlled by PALB2 and many explanations, including artefacts, remain possible.

The manuscript would gain in interest if the authors would devote the functional studies only to the repair part (Fig.6 and 7).

We feel it is important to show Fig 5, as although the results may appear confusing, they highlight the importance of the acetylation of the 7K patch at the cellular level. Namely, the non-acetylatable 7R mutant fails to support normal cellular growth, likely due to its impaired association with active genes (Fig S5), which might provide in vivo evidence that non-acetylation of the 7K patch promotes PALB2 release from chromatin (please refer to our response to Reviewer 1’s point 3). We are confident that we will be able to clarify this point with our additional ChIP-qPCR analyses (Experiment 2).

**Minor points** 5 - High background of non-enzymatic acetylation of PALB2 fragments makes the identification of KAT2A/B specific acetylation not very convincing. The immunoblot detection of acetylation fragments shown in Figure S1 is much more convincing. Therefore, the authors may consider to present Fig S1 as a main Figure and Fig.1B as a supplementary one.

We will swap or add Fig S1B with or to Fig 1B in the revised manuscript.

6 - It would be interesting if the authors would comment on why the presence of regions flanking the ChAM domain (Fig. 1A, construct #5) significantly reduces chromatin (Fig. 1B) and nucleosome binding (Fig. 1C).

We are grateful for this Reviewer’s comment. Indeed, we noticed that the inclusion of the ChAM C-terminal flanking region perturbs its chromatin association. This region is highly enriched with serine and threonine residues which could be targeted for phosphorylation by cell cycle regulators (CDKs and PLK1) and DNA damage-responsive kinases (ATM and ATR). It is therefore tempting to speculate that, when phosphorylated, this flanking region could mask the basic patch of the ChAM, hence facilitating the release of PALB2 from undamaged chromatin region and its recruitment to sites of DNA damage. In the revised manuscript, we will provide the complete list of PTMs and discuss this point.

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

KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity

The authors describe a series of experiments examining the consequence of acetylation, within a defined motif (Chromatin Association Motif; ChAM), on the cellular roles of the protein PALB2 (Partner and Localizer of BRCA2).

The key conclusions drawn by the authors are generally convincing and are supported by the presented experimental results, which indicate that acetylation of PALB2 by KAT2A/KAT2B modulates its cellular behaviour and response to DNA damage. However please see specific comments below:

**Major Comments**

Expression of full-length PALB2 in the heterologous host E. coli is highly problematic, as the WD40 domain is generally not correctly folded. The authors use the ArticExpress strain to try and solve/alleviate this problem - but it is clear from the materials and methods section that an ATP-wash step has had to be introduced in order to release the recombinant protein from the chaperone system encoded by the ArticExpress system; i.e. indicating poor / mis-folding. Whilst this does not strictly have an effect on the results presented in Figure 1 (detection of in vitro acetylation sites), they have implications for the wider scientific community, as this may lead to the erroneous assumption that is possible to produce functional / folded full-length PALB2 in this way.

We apologise if the manuscript conveyed the message that we are able to produce functionally active, full-length PALB2 in bacteria, which was clearly not our intention. Our aim was to test whether KAT2A was able to acetylate PALB2 in vitro. We agree that the folding and the biochemical properties (e.g. WD40-mediated BRCA2 binding) of the bacterially produced full length PALB2 were not fully assessed. We believe that this does not affect the overall conclusions of this study. In the revised manuscript, we will correct this error to make this point clear.

In vitro modification assays are prone to producing post-translational modifications that are not fully reflective of those observed in vivo, and therefore need to be treated with some caution. This is highlighted by the relatively low modification of K438 in vitro by KAT2A; esp. as this is an acetylation site that has been previously mapped in vivo (by the authors). It would have been useful to include / see the effects on PALB2 function in vivo by modification / alteration of this single site.

We appreciate the Reviewer’s constructive comment. Redundancy of acetylation acceptor residues within a lysine cluster is common, as is also the case for many ubiquitination events, hence we analysed the 7K patch mutant for phenotypic studies. For the same reason, we trust that the outcome of the characterisation of a K438 mutant would not significantly change our conclusions.

Figure 3C and Figure 3D do not fully support or reflect the conclusions drawn by the authors - any peptide containing a cluster of positive charged residues are likely to interact with DNA through charge neutralisation of the phosphodiester backbone, concomitantly any alteration to this region of charge (i.e. via acetylation) will perturb this interaction.

We totally agree with the Reviewer’s view and state, in the main text referring to the results shown in Fig. 3C and D, that “As anticipated, lysine acetylation, which neutralises the positive charge on the lysine side chain, conferred reduced affinity for negatively charged DNA”. In the revised manuscript, we will make this point clearer.

Furthermore, experiments performed with the synthetic acetylated peptides do not agree with those carried out with the GST-ChAM constructs - GST-ChAM interacts with the nicked and linear forms of the pBS plasmid (Figure 2F) but does not interact with the supercoiled form. The WT synthetic ChAM peptide, in contrast, interacts with all three plasmid states at high concentrations. It is suggested that these two figures are removed.

It is true that we cannot exclude the potential difference between GST-ChAM and synthetic ChAM peptide: for example, 26 kDa of GST, which can form a dimer, might mask the full biochemical properties of ChAM in DNA binding. However, we believe that the difference is more likely caused by the concentration of ChAM used. While we used the synthetic ChAM peptides at concentrations of 2.97, 5.94, 29.3 µM for Fig. 3C, we used 5.94 µM of GST-ChAM for Fig. 2F, for which we apologise for the omission of the experimental conditions used. This notion is supported by the side-by-side experiment, which was not shown in the original manuscript. In the revised manuscript, we will make these points clear.

p. 18 : the authors used a PALB2 variant, where the lysines in the 7K patch are mutated to arginine - but don't fully characterise the effects of introducing these particular mutations on the ability of the ChAM fragment to bind to DNA, or indeed to nucleosomes; this is an important control.

We appreciate the Reviewer’s comment. Biochemical analyses of the 7R mutant were not conducted, as ChAM produced in bacteria is not expected to be acetylated. Nonetheless, as also linked with the concerns of Reviewers 1 and 2, we recognise the importance of 7R biochemical characterisation for accurate interpretation of in vivo phenotypes. We will assess the DNA and nucleosome binding of the ChAM 7R mutant, which will be included in the revised manuscript (Experiment 1).

Figure 6 : it would be good to show a second supporting example for deacetylation of PALB2 in response to DNA damage - perhaps treatment with MMC?

We appreciate the Reviewer’s comment. Indeed, we have conducted the analysis upon MMC and Olaparib exposure. Curiously, however, no clear change of ChAM acetylation was detectable. Note that, for this experiment, we assessed the acetylation level of a GFP-fusion of ChAM, exogenously expressed in HEK293, along with endogenous gamma-H2AX as a readout of DNA damage signalling. Unlike ionising radiation, which triggered strong induction of gamma-H2AX (Fig. 6), no clear increase of gamma-H2AX was detectable upon MMC/Olaparib exposure. Hence, we propose that the reduction of ChAM acetylation reflects the cellular response to DNA damage. We will make these points clear in the revised manuscript.

**Minor Comments**

p. 16 : 'Our MS analysis of the chromatin-associated GFP-ChAM fragment identified actelyation of all seven lysines within the 7K-patch (Fig. 3A, marked with arrows).

This part of the manuscript is potentially a little confusing, as Fig. 3A references a series of synthetic peptides rather than the GFP-ChAM fragments themselves.

We appreciate the Reviewer’s point. Indeed, Fig. 3A shows 1) MS of the chromatin-associated fraction of GFP-ChAM (the top part with arrows) and 2) a schematic diagram of synthetic peptides that we used for biochemical analyses (the bottom part). In the revised manuscript, we will clarify this point and indicate the MS result and the schematic of synthetic peptides in two separate panels and refer each of them appropriately.

p. 20 : Furthermore, using the FRAP approach, we observed clear differences in diffusion rates of FE-PALB2 following damage by IR, MMC, or olaparib treatment... FE-PALB2 = FL-PALB2?

We apologise for the confusion. In our study, FE-PALB2 refers to Flag-EGFP tagged PALB2 (full-length). This is defined in the text “To this end, a tandem FLAG- and EGFP-tagged full-length wild-type (WT) PALB2 (FE-PALB2)” (p. 17).

Overall, I think you have a great story. There is definitely a beginning middle and end that work cohesively. I am most interested in the organization and it taking over the government. It then eventually did a lot to change society which was cool, and you described the different things well. I know you probably did it on purposely, but congress, state and local governments were left out. I’m guessing in Healthment’s takeover those institutions ended? I also think that you could consider removing your “prologue.” It may not be necessary given that you do mention many of the things in that part throughout the story. For me if I read the story without that part, I think I would focus mostly on the environmental and health affects that Healthment’s formation and governance had on society. You did a pretty good job referencing and including the city as a character as Dr. Bush “asked” for. I think the chapter about Lairen going to get her mom may not be necessary to your overall plot. Lairen and Xavier talking and becoming even closer could probably happen anywhere or anytime in your story. Regardless of some of my “critiques” you have a story that resonates with me and probably many others when it comes to health and that if the government forced it on people, we would be better off. I think though that maybe it would be more than 4% of the US that would say no to it. Great conflicts and resolutions and an interesting outlook and story of a future where a health/environmental group runs the country.

Machines have progressed in such a way though that the previously formed boundary between human and machine is not clear. No longer is the distinction so clear as: humans=consciousness/subjective experience/natural machines=coded devices/bound to man's limitations/artificial Though...I kind of don't think machines are close to consciousness (have the ability to experience "what it is like" to exist) in these progressive technologies, neither when Haraway was writing nor 2020. This may not be all about consciousness but I still think it is at the root of this distinction. It moreso seems like we are just attributing complexity to machines which are human created nonetheless...e.g. I may just not know enough about machines but how are they "self-developing"? Or is she saying they just seem self-developing and that is enough to have social implications?

Beautiful! Silence as a condition of vision. This is brilliant!!!! It made me so happy when I found a way, a phrase that could express why I love the classes Prof. Annabel teaches. I wrote in a piece of paper in order not to forget: "Prof. Annabel makes me believe that the feeling I sometimes have --the feeling of having something to say-- is not false, is not illusory." But now, having read this passage, I may add more to it! I can explain it better. Your classes, the texts you choose for us to engage with, they allow for us to find a path, an always unique path, in which we can celebrate our silence. Not to suffocate with what is not said, but to "bathe [our] imagination" so that when we do break it, it is never to further harm our human relationships. That's so beautiful!

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Reply to the reviewers

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

**General comments**

The manuscript 'Second messenger control of mRNA translation by dynamic ribosome modification' is a very interesting follow up on the research performed by the authors published in 2016. Here, the authors continue their study by determining the impact of the intricate RimABK pathway in Pseudomonas fluorescens on translational reprogramming by controlled modification of ribosomal protein S6 in response to environmental signals. The manuscript is interesting and well written, and the results are sound. However, in my opinion the general conclusion is not supported by experimental data and leaves several potential explanations open. Thus, I suggest to either perform in vitro translation experiments using ribosomes equipped with glutamated S6 to validate translational selectivity, or to soften the language on the working model shown in Figure 12.

The authors would like to thank reviewer 1 for their detailed review of our manuscript. We agree with the reviewer that alternative explanations are possible for the translational changes linked specifically to glutamation, as opposed to rimBK deletion. Our intention when writing the discussion was to clearly distinguish glutamation-specific effects from the large number of indirect translational changes associated with Hfq disruption and other uncharacterised consequences of rimBK deletion. With hindsight, we acknowledge that the discussion and the model in figure 12 should more clearly outline the possible alternative causes for the observed glutamation-specific translational regulation. We have modified the discussion and figure 12 (now figure 10) accordingly.

The reviewer further suggests that we perform in vitro translation experiments using ribosomes equipped with glutamated S6, to prove that glutamation controls translation directly. This is an excellent suggestion that would clarify this important point, and we will certainly attempt it as part of our future analysis of the Rim system. However, at this stage we feel these experiments are beyond the intended scope of this paper, which is to describe the signal inputs and mechanism of the RimABK system and to show evidence for both specific and secondary translational effects of ribosome modification.

**Specific comments**

Figure 1 and S1: The RT-PCR analysis shown here does not allow excluding transcription initiation at alternative promoters downstream of the one determined by 5'-RACE. However, an alternative promoter might contribute to relative ratios between the rimA, rimB, and rimK mRNAs. A Northern blot and/or primer extension analysis could clarify this assumption and would give more detailed insights into the specific regulation.

The reviewer is correct that alternative rim promoters could exist downstream of the amplified 5'-RACE region. To test this hypothesis, we conducted additional RT-PCR experiments to measure expression of rimA (the third gene of the polycistronic rimABK operon) in the same set of conditions as tested for rimK. Relative levels of rimA mRNA do not substantially differ from those seen for rimK, strongly suggesting that the promoter upstream of rimK controls expression of all three rim genes. We have added this dataset to figure S1 and have modified the relevant sections of the text.

Figure 2B: I'm confused by the results shown here! I do only see a reduction of RpsF in the presence of RimA, RimK and cdG. What indicates the modification? Please, explain the interpretation of the result in more detail. Shouldn't the modified RpsF shift due to the addition of glutamate residues?

The uncontrolled activity of RimK acting in the absence of RimB (e.g. the experiment represented in Fig 2B) typically results in a reduction of the unmodified RpsF fraction in the reaction, replaced with RpsF proteins with widely varying numbers of glutamate residues attached to their C-termini. The resulting modified RpsF fraction can appear as a smear of protein density throughout the gel. We have clarified the text surrounding figure 2 to make this more explicit.

Figure 2C: Why does the RpsF modification lead to a supershift? How many glutamate residues are added? Is the smear visible in lane 4 (RpsF+RimK) representing already the slightly modified RpsF protein, which upon addition of RimA results in a supershift? For all SDS-Page analyses shown in the manuscript the validation of the glutamation using the antibodies specific against poly-glutamate would be a great asset to facilitate their interpretation.

Pseudomonas fluorescens RimK appears to have unregulated ligase activity, with many hundreds of glutamates being added to each RpsF protein in the absence of RimB cleavage. In our 2016 paper (Little et al., PLoS Genetics) we use radiolabelled glutamate incorporation and mass spectrometry to show that the supershifted protein smear is composed entirely of RpsF units with C-terminal glutamate tails of varying length. (It is interesting to note that E. coli RimK, which does not have an accompanying RimB protease, can only add 4-15 glutamates to each RpsF protein). We have modified the text slightly to make this clearer.

The reviewer’s suggestion to stain the supershifted RpsF with the poly-E antibody is interesting but would likely only reiterate our published results with radiolabelled glutamate (Little et al. 2016).

Lines 236-238: '...strongly suggesting that the proteomic changes we observe are an active response to modification of ribosomally-associated RpsF proteins.' This is an important suggestion as it allows a flexible and very fast integration of the external signals into a specialized protein synthesis. Thus, it definitely deserves further analysis! Considering that the purified RimA and RimK proteins are available, in vitro modification of RpsF in the context of the purified ribosome would be an important experiment and would greatly increase the quality of the paper. Up to now the selective or specialized translation is pure hypothesis and might also be explained by indirect effects via e.g. increased interaction between the ribosome and HFQ that might mediate interaction with certain mRNAs and thus stimulate their translation.

We agree with the reviewer that direct measurement of translational changes in vitro would tell us a great deal about the mechanism of RimK regulation. This would enable us to confirm whether the glutamation-specific effect is direct, or if it functions through an as-yet uncharacterised indirect mechanism (such as interaction with another translational regulator). As stated above we feel these major experiments are beyond the scope of the current manuscript, although we are keen to do them (as part of a planned structural biology investigation of modified ribosomes). As stated elsewhere in our response, we have extensively revised the discussion text and figure 12 to clarify the limits of our current understanding and highlight the different potential regulatory routes for RpsF glutamation.

Lines 322: '...into a single output: the proportion of all ribosomally-associated RpsF proteins that have C-terminal poly-glutamate tails.' Considering the identification of a group of genes whose translation is altered by rimBK deletion, but not by RpsF glutamation (Class 1, Fig 11B), I would suggest softening this statement. If I interpret the data correctly, they pinpoint to a moonlighting function of the rim-pathway that does not target RpsF!

The genes whose translation is affected by rimBK deletion, but not by RpsF glutamation specifically, include all those genes whose translation is indirectly affected by downstream translational regulators, or through interaction with another affected gene target. As expected, there is substantial overlap between the rimBK and hfq translatomes (Grenga et al. 2017): this analysis can be included in the manuscript as a supplementary table if requested. Importantly, there is very little overlap between the Hfq translatome and those genes that are affected specifically by RpsF glutamation. One possibility is that Hfq interacts with RimK at the ribosome, and the loss of the RimK protein is a major factor in destabilising Hfq function in the ∆**rimK mutant. We have modified figure 12 (now figure 10) and expanded the discussion to include this hypothesis.

While we cannot exclude the possibility that RimK has other cellular targets in SBW25, we think this is unlikely to be a major cause for the results we see here. We have carefully examined the C-terminal peptides of proteins detected in our various proteomic assays and are confident that RpsF is the sole target of RimK in SBW25 under the conditions we tested. We also directly tested RimK interaction with purified Hfq and confirmed that Hfq is not a direct target of RimK modification.

Lines 377-76: '...distinguishing features in the primary or predicted secondary structures of the Rim-mRNAs...' As mentioned already above several indirect options are still open that could confer selectivity to the ribosome.

As stated above, the discussion has been rewritten to more completely reference the possible mechanisms by which RpsF glutamation may lead to translational regulation.

Reviewer #1 (Significance (Required)):

The key concept of the manuscript namely the impact of the intricate RimABK pathway in Pseudomonas fluorescens on translational reprogramming by controlled modification of ribosomal protein S6 in response to environmental signals is novel and will significantly impact the field.

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

**Summary**

The main question addressed by this research is how bacteria adapt to rhizospheric niche through the RimK ATPase glutaminase. This enzyme post-transcriptionally modifies the ribosomal protein RpsF in a process of complex regulation. Regulation is mediated by c-di-GMP that is degraded by the phosphodiesterase RimA and the protease RimB exerts a role opposite of RimK. Novel findings include the finding of RimK acting as a four-state ATPase, depending on the binding of RimA, c-di-GMP or both. Another important finding is the opposite roles of RimK and RimB on the glutamation/deglutamation of RpsF and the tendency to a steady state of four glutamate residues in the RpsF protein. The authors also use proteomics to determine the effect of glutamation, specially at low temperature and under nutrient limitation.

We thank the reviewer for their positive review of the manuscript and address their comments below.

**Major comment**

In my opinion, the results obtained with the Hfq regulation by RimK blur the message. I firmly think that the Ms is very solid with the results obtained in relation with the RimABK/RpsF regulation in P. fluorescens shown as a model in the Figure 12. Moreover, in this final model presented by the authors (fig. 12) they not included the results related with Hfq. These results could be part of another paper.

We agree with the reviewer that the Hfq independent effects of RpsF are an exciting finding and should be a major focus of the paper. That said, we feel that the additional work we have done showing how Hfq is affected by RimK should also be retained in the manuscript in some form. Our data (e.g. figure 8) indicate that Hfq is responsible for a large (indirect) fraction of the ∆rimK phenotype, so understanding how it is affected is important to understand how RimK functions. Based on comments from reviewers 2 and 3 we have reviewed the manuscript text (including data on Hfq) to make the narrative as focussed and clear as possible. We have also redesigned figure 12 (now Fig 10) to consider comments from all three reviewers and have changed the text in the discussion to match this.

**Minor comments**

In figure 4A, what is lane 5?

Lane 5 contains RimB without ADP. The figure legend has been modified accordingly, and we thank the reviewer for highlighting this error.

Line 159 change "suppression of RimK band-shifting" by "suppression of RpsF band shifting"

This has been fixed.

Reviewer #2 (Significance (Required)):

The Ms. is very interesting and deeply describes the relation between environmental conditions, c-di GMP second messenger and the RpsF ribosomal protein posttranscriptional modification in order to respond to low temperatures and changes in nutrient availability. The research developed in this manuscript is original and novel in the field and includes new advances in the signal transduction pathways implicated in the regulation of bacteria adaption to the environment. Besides, the research design and technical methodology is original and includes multidisciplinary approaches of interest to the research community in general.

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

**Summary** Post-transcriptional control of protein abundance is an important yet poorly examined regulatory process by which bacteria respond to signals found in the environments they grow in. The authors' team have previously identified, described and published details around a novel regulatory pathway involving the ribosomal modification protein RimK, regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). In the current manuscript builds considerably on those previous findings and goes several steps further, through various approaches including protein biochemistry, computational modelling, quantitative proteomics and ribosomal profiling, the authors described how the RimABK pathway as a novel translator system that controls bacterial adaption to the rhizosphere in the bacterium Pseudomonas fluorescens. They show that the system achieves this through specific controlled modification of the ribosomal protein RpsF. I read the article with excitement and overall the manuscript describes an extensive data set that will be of considerable interest to many readers in several fields. However, I have made a few points below that the authors need to take on board and address. If these issues are addressed, I believe it will make the presented data much clearer to the reader, tidy up a few ambiguities and make the article a little more accessible to many non-specialist readers.

We thank the reviewer for their thorough and positive assessment of the manuscript. We address their specific points below.

**Major Comments**

1) The major finding described in the manuscript and the one that will be of significant interest to reader is that a novel post-translational ribosomal modification regulatory mechanism involving Rim system controls bacterial adaption. The second messenger cdG only plays a small part in this complicated process. Therefore, I believe the title needs to be revised to capture the scope and key findings of the manuscript.

We are happy to change the title along the lines suggested by the reviewer. We propose: Control of mRNA translation by dynamic ribosome modification as a new title.

2) The authors present a lot of interesting data; however, I found the manuscript a bit of a dense read. I find the key findings are diluted within the text. I would ask that the authors to make it a little more focused. For example, on the regulatory role of RimK and its influence on Hfq and RpsF has been detailed previously so could be placed in supporting information and briefly mention when required. Also, the experiments on the pvdIJ pathway could be removed or placed in supporting information as they are not the main focus of the manuscript. Fig 5 and 6 could be combined as one figure as well.

We have modified the manuscript throughout to make it clearer, more concise, and to focus as much as possible on new findings rather than reiterating what we showed in our last manuscript. In line with the reviewers’ recommendation, we have moved the pvdIJ data into the supplementary material (Fig S3) and merged figures 5 and 6 into one. In addition, to support our data on the importance of RpsF glutamation for ribosomal regulation we used Western blotting to confirm that RpsF4/10glu variants incorporate normally into SBW25 ribosomes in vivo (added as supplemental data Fig S5).

As stated elsewhere, we feel that key data on the relationship between Hfq and RimK should remain in the main manuscript, although we have reviewed the text thoroughly to try to ensure it is as focussed as possible and have moved some results to supplementary material as suggested.

3) The authors propose a four-state kinetic model for RimK ATPase activity with RimA and cdG (described in Fig2 and Table S1). However, later in the manuscript the authors demonstrate that RimB also stimulates RimK ATPase activity, but this seems to have smaller impact than RimA and cdG (Fig 2E, Fig 3A). Why RimB was not included in the ATPase kinetic model of RimK? Does including the RimB data suggest there might be more conformational states for RimK?

Thank you for raising this point. The reviewer is correct in that this data does indeed suggest another level of ATPase activity of RimK. We have added text to the manuscript to reflect this. We have also extended the supplemental Table S1 to include these equations.

4) The authors claim that the suppressive effect of cdG on RimK was depended on the enzyme activity (PDE domain) of RimA. This was tested using an enzymatically inactive RimA variant (RimA-E47A). However, in Fig 3E the amount of RimA-E47A used in the assay seems to be significantly less than wildtype RimA. Additionally, in Fig 2B, the authors show that addition of cdG also stimulates RpsF modification with or without RimA (lane 4-6). I would ask the authors to clarify these points.

It is difficult to directly compare protein variants due to differences in solubility post-purification. Due to difficulties in purifying this (less soluble) form of RimA, co-purifying contaminants have also probably influenced the determination of RimA-E47A concentration to some extent. This restricts us to making largely qualitative statements about protein function, as we do here. Despite its poor solubility and low concentration, RimA-E47A is still able to stimulate RimK. Furthermore, the relatively low concentration of RimA-E47A in our assays would render it at least as susceptible to any effects of cdG addition as WT RimA, meaning we can be confident that cdG has no effect on RimK stimulation by this variant.

Our model incorporates direct stimulation of RimK by cdG alongside its effect on RimA. We show evidence for this in this manuscript and in our 2016 paper.

5) The authors claim that high levels of cdG increase the ratio of RimB protease activity to RimK glutamate ligase activity. However, there is no experiment to provide direct evidence to support this. Please tone down the language used or provide evidence. On the same point Fig 6 was not explained in the main text to support this conclusion. Please include an explanation.

The hypothesis that high cdG levels favour RimB activity over RimK stems from the observation that cdG suppresses RimK activity (by abolishing RimA stimulation) but does not affect RimB. We have data showing that increasing cdG levels suppresses RpsF band shifting in vitro in an assay containing all three Rim proteins (Fig 4). However, we agree the hypothesis that cdG controls the ratio of RimB to RimK activity by controlling the activity of RimK currently lacks explicit, direct evidence and we have modified the text to tone down the language.

An explanation for Figure 6 (now 5b) has been added to the manuscript as requested.

6) In some of the figures/images, for example, Fig2B and Fig 3E, RimA is shown as a major band. However, in other figures/images, for example, Fig 2D, Fig 3D, RimA seems to be two bands. The authors should explain the reason for this.

Based on extensive experimentation, we are confident that the second band present in some of our assays is a cleavage product of RimA. This is an experimental artefact that is linked to concentration and protein stability in vitro. We must stress that the presence of an inactive fraction of RimA in our assays does not affect the conclusions we are able to draw from these experiments. A note has been added to the relevant section of the text.

**Minor comments**

• Line 151, should be RpsF band-shifting instead of RimK.

• Fig 4A there is no legend for lane 5, which made it very difficult to understand the data presented.

Please see above. These two minor errors will be fixed.

• The layout of some figures could be improved.

We have revised the layout of several figures, in line with the reviewer’s suggestion.

• If it is possible to have Fig 11 as a Venn diagram or some intuitive diagram, it will help the readers gain access to the data and understand the results.

We respectfully disagree with the reviewer here. We have tried several different presentation styles for these data, but ultimately considered scatter charts to be the most effective, in line with our previous study of Hfq regulation in Pseudomonas (Grenga et al. Frontiers in Microbiology 2017).

Fig 12 is very neatly laid out. However, I don't feel it captures the dynamic nature of the system. I am just wondering if the authors could break it down so that it describes the changes relating to environmental conditions and/or different cdG levels?

Figure 12 (now Figure 10) has been modified to reflect to comments of all three reviewers.

Reviewer #3 (Significance (Required)):

The manuscript provides detailed evidence to demonstrate a dynamic, post-translational ribosomal modification mechanism which is an important feature of prokaryotic (potentially archaeal and eukaryotic) environmental adaptation. This is an exciting manuscript and one many will wish to read. The data provided will be of interest to scientists working in many fields including microbiology, biochemistry and plant pathology.

I have several areas of expertise including genomics, molecular microbiology, small molecule signalling and regulation, micro-host interaction, adaptation,

Having teachers participate in laboratory classroom residencies has had this impact, but communicating the importance to experts is a challenge, and the tyranny of experts is to deny the importance of practitioners gaining insight or seeing new possibilities.

From what I have been able to gather about the “inner” and “outer” landscapes as presented by Parker Palmer, Laura Rendon and the AACU suggests to me that good teachers are genuinely motivated by not only passion for their field and for the reward reflected in the academic successes of their students, but also the personal growth of their students.  Teaching isn’t something they happen to do but something they must do in order to facilitate students’ social consciousness and empowerment. When we encounter students who may be perceived as unmotivated or lazy, we kick it into gear and get creative with approaches to engaging students where they are. Instead of seeing our students from a deficit perspective, we look for their assets—the qualities of our students that can help them learn, such as their bilingualism, creative interests, hobbies or talents outside of our discipline. What we value in our students isn’t a reflection of what we want them to be, but what they want for themselves. We trust our students’ innate capacity to learn and to acquire confidence in being a college student.

Voices: The voices speak of a key that would either lock/unlock the prison they are in. Whether it is real and they believe it or just a fictitious rumor is either possibility. Yet it seems they want freedom from something. Un/natural time/Desire frustrated (maybe?): If their thoughts are mostly on this key, are they keeping track of the passing time other than day and night? How many days are spent in this prison they believe they are in? Exile: They seemed to be trapped in a prison, in search of a key that may or may not free them. Fragments: The lines go back to the key for three of the lines and prison for two lines. These moments seem to only pause when it is night. Text as plagarism/remixed (maybe?): Coriolanus was mentioned. After looking it up, one result was a tragedy Shakespeare wrote.

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Reply to the reviewers

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

In this study by Wegwitz et al, the authors examine the tumour promoting properties of RNF40 (and the H2B monoubiquitinylation catalysed by it) in Her2 driven breast cancer.

They report, using publicly available data, that increased RNF40 expression is associated with reduced overall and disease-free survival.

Using a mouse model, where they crossed the Erbb2 (mouse Her2) under the control of the MMTV promoter with conditional Rnf40 deletion constructs, the authors found that deletion of Rnf40 simultaneous to Her2 overexpression resulted in a prolonged tumour-free survival, somewhat reduced tumour growth kinetics and tumour incidence.

siRNA silencing of Rnf40 in two Her2 positive breast cancer cell lines resulted in reduced proliferation, clonogenicity and tumour sphere formation and cellular motility.

Transcriptome analysis revealed pathways that could explain the phenotype, like increased apoptosis and actin cytoskeleton regulation. The authors then took further some candidates in the later pathway to investigate the mechanism. They find that Rnf40 loss impacts on actin cytoskeletal dynamics. They also investigate the impact on focal adhesion signalling integrity.

Finally, they investigate the relationship between the transcriptome and H3K4me3 and H2Bub1 landscape in the presence or absence of Rnf40.

The manuscript is convincing regarding the tumour promoting roles of Rnf40, but the key claim that H2B monoubiquitinylation is essential for activation of the Rho/Rock/Limk pathway, where genes are down regulated upon Rnf40 loss resulting in decreased tumourigenicity of cells, is so far not convincing.

"Together, these findings support the hypothesis that the actin regulatory gene network is dependent on direct epigenetic regulation by RNF40 through modulation of H2Bub1 and a trans-histone cross-talk with H3K4me3 levels in HER2-positive BC cells."

Although the correlation is apparent, at this point it's unclear if the phenotype is dependent on the catalytic activity of Rnf40 or it's a non-catalytic effect. Generating a catalytic mutant RNF40 and test it at least in the cell lines studied would be desirable.

We thank the reviewer for this comment and agree that the addition of data with a catalytic mutant RNF40 could strengthen our findings and further clarify mechanisms involved. Thus, in a resubmission we will directly address this point by performing knockdown/rescue experiments with either wildtype or a RING finger mutant RNF40. This will be done by transfecting cells with expression constructs for either wildtype or mutant RNF40 proteins followed by knockdown of endogenous RNF40 using siRNAs targeting the 3’ UTR. Experiments central to our take-home message will be performed (e.g., cell migration, target gene expression, Western blot for H2Bub1, F-actin formation). Together, we hope these experiments will help significantly solidify the message of this paper and further clarify the individual role of RNF40 within the RNF20/40 heterodimer.

**Other comments that need a response:**

1."we investigated RNF40 expression and H2Bub1 levels by immunohistochemical staining of 176 primary BC tumors and 78 brain metastases."

In Fig 1 I can only count 41 primary BC tumours and 73 brain metastases. Numbers don't add up. Also, how is "low" defined as opposed to negative? What is used as controls?

We apologize for this mistake. We corrected the numbers of primary and metastatic HER2-positive specimens used in this study.

2."Moreover, HER2-positive metastatic BC samples showed a particularly high expression of RNF40 compared to primary tumors"

Figure 1 or Fig S1A does not contain data on HER2-positive metastatic BC

We think there might have been a confusion regarding this point. The manuscript does provide information about RNF40 and H2Bub1-staining in primary HER2-positive breast cancer lesions as well as HER2-positive brain cancer metastasis specimens in Fig.1A-C as well in Fig.S1A (representative brain metastases are shown in IHC pictures). This is stated both in the main text as well as in the respective figure legends. However, if for some reason this remains unclear, we would certainly be open to suggestions as to how we can modify the respective sections to improve their clarity.

3."tumors did not display a loss of either RNF40 or H2Bub1 (Fig. 1H) when compared to the adjacent normal mammary epithelium (Fig. S1F)."

I don't understand what I see in Fig S1F, where is the tumour, what is adjacent?

We agree with the reviewer that splitting tumor staining in the main Fig 1 and normal tissues in Fig S1 makes a comparison difficult. We will therefore edit the Fig S1F and provide there an overview of tumor and surrounding normal tissues together with magnifications of the respective areas. This should significantly ease a comparison of both RNF40 and H2Bub1 in tumor and adjacent normal tissues.

4."homozygous loss of Rnf40 (Rnf40fl/fl) resulted in dramatically increased tumor-free survival of MMTV-Erbb2 animals (Fig.1E)." This is overinterpretation of the data, I would not call it dramatic, just significant.

The MMTV-Erbb2 mouse model is a very reliable mouse model for the induction of HER2-positive lesions. In our hands, the tumor incidence in these animals was 100% with a median tumor free survival of 166 days. In comparison, approx. 20% of the Rnf40fl/fl animals (3 out of 14) never developed the disease during the 18 month observation. The animals that still developed lesions had a median tumor free survival of 241 days, which represents a delay of 75 days (45% delay). In light of this, it seems to us that the effect of RNF40 loss on HER2–positive lesions is, indeed, remarkably strong. However, we do not wish to give an impression of over-interpreting or misrepresenting the data. For that reason we modified the wording in the main manuscript according to the reviewer’s suggestion (line 140: “dramatically” was replaced with “pronounced”).

5."loss of Rnf40 led to strongly reduced tumor growth kinetics (Fig.1G)." Is this result significant, I did not see an evaluation of statistical significance in this data.

As suggested by the reviewer, we have performed additional analyses to examine the statistical significance. We have now included the results of these tests in the respective figure.

6."Rnf40fl/fl lesions displayed a heterogeneous pattern of RNF40 expression (Fig.1H), suggesting that the few tumors that did develop in this model were largely caused by an incomplete loss of the Rnf40 allele." If this conclusion is suggested, the authors should check if the "escaper" cells have failed to flox the Rnf40 allele on the genetic/protein level. Otherwise it's not conclusive.

The reviewer brings up an interesting and important point about the heterogeneous loss of RNF40 in “escaper” tumors. Very important to note is that these “escaper” tumors developed significantly later and three animals never developed tumors. Thus, the “escaper” phenotype is rare (at the cellular level) and is likely similar to the selective process that occurs in human tumorigenesis and tumor progression. It is well established through a number of publications that deletion of genes essential for tumorigenesis via Cre-based systems frequently results in a specific selection for the rare instance that the Cre-mediated excision is ineffective. These “escaper” cells can then grow out and proliferate because they do not suffer from deletion of the floxed allele. This effect has also been established when combining MMTV-HER2 and MMTV-Cre. For example, analogous findings were recently published by Costa, et al., in Nature Communications (doi: 10.1038/s41467-019-11510-4) where the MMTV-Cre-mediated deletion of Pak4 resulted in impaired MMTV/HER2 or MMTV-PyMT-driven tumorigenesis, but occasional tumors did appear, which all retained some degree of PAK4 expression. This effect, which we have also seen in our system, was also reported by Miao, et al. in Cancer Research (doi: 10.1158/0008-5472.CAN-11-1015) in 2011. In their work the authors observed that deletion of the Myb gene also impaired MMTV-HER2-driven tumorigenesis and those tumors that developed in Myb flox/flox mice displayed a late onset and invariably retained MYB expression. Similar findings have been reported in a number of other tumor types and with various Cre drivers. Thus, we posit that these findings provide further support for the essential role of RNF40 in HER2-driven tumorigenesis to the extent that rare, RNF40/H2Bub1-expressing “escaper” cells are positively selected for during tumorigenesis and tumor progression.

In order to definitively establish this, we propose performing dual immunofluorescence staining of Rnf40 flox/flox tumors to verify that H2Bub1 is exclusively and universally lost together with RNF40 and that each case of a complete loss of RNF40 also results in a complete loss of detectable H2Bub1 staining. Additionally, we will assess the efficiency of the cre mediated deletion of Rnf40 exons 3 and 4 in Rnf40fl/fl animals by detecting their presence using a conventional PCR approach.

1. Fig S4D - is this clonogenic assay? How many replicates were done, biological technical?

We apologize for the imprecise description of this figure. We edited the manuscript accordingly and included details about the number of replicates.

8."Additionally, treatment with either CYM-5441 (Fig.4J) or …"

Fig 4J is missing! It makes this section rather hard to follow. Fig S4F-G, how many replicates were done, biological technical?

We thank the reviewer for noticing this error. The figures were indeed inappropriately referenced in the text. This error has been corrected.

9."Consistent with our analyses based on changes in H3K4me3 occupancy, genes downregulated upon RNF40 silencing displayed the most prominent decrease in H3K4me3 in the gene body (the 3' end of the peak)"

The impact of these mods changes is hard to judge because they are rather small (I would not use the wording prominent).

As implied by the reviewer, we will replace the word “prominent” with “noticeable”.

Also, are there many other "peak narrowing" genes but they are not downregulated?

The point mentioned here is very interesting. The bioinformatic analyses performed in this study solely focused on the relationship of significantly regulated genes and the H3K4me3 peak narrowing at their TSSs. However, we did not analyze the global regulation of genes showing H3K4me3 peak narrowing near the tSS. As this information might be of high relevance for this study, we propose to investigate this interesting aspect in the revised version of the manuscript.

In fact, our analyses have revealed that a large fraction of genes with H3K4me3 narrowing peaks do not show an appreciable decrease in expression. To better understand the epigenetic features determining the sensitivity of genes to H3K4me3 peak narrowing, we studied the occupancy of several histone marks at differently behaving genes. We discovered that sensitive genes globally present lower occupancy of histone modifications which are known to positively influencing gene transcription. We therefore propose that the epigenetic context (i.e., presence of additional histone modifications) strongly determines whether the loss of H2Bub1, and ensuing narrowing of H3K4me3 near the TSS, results in decreased transcription.

Statistical analysis missing: for example in Fig 2C, Fig 2E, Fig 3G what is n=?, how many technical, biological replicates were analysed?

This information has been added to the revised manuscript.

Fig 4E seems to be a partial duplication of Fig 3D!

The samples in Fig. 3D and 4E originate from independent experiments. In Figure 4D, we indeed provide again PARP and Casp3 signal for siRNF40 samples in order to allow a direct comparison of the effect magnitude between RNF40 depletion and ROCKi treatment.

**Minor:**

Figure referencing: it can be quite confusing to see a different ordering of figures compared to the referencing in the manuscript, for example Fih 1H is referenced in the text before Fig 1F, G. The authors should change the order in the main figures....

We thank the reviewer for pointing this out. We have updated the figure order in the revised manuscript accordingly.

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

In this study, Wegwitz et al propose that the E3 ubiquitin ligase RNF40 is highly expressed in HER2+ breast cancer tumours and correlates with poorer survival, using their own and TCGA data. Contrary to observations suggesting a tumour-suppressive role in other cancers, authors show using RNF40-knockout breast cancer mouse models and in vitro data shat RNF40 promotes tumour growth. RNF40 depletion impairs proliferation, survival and sphere formation by inducing apoptosis. In addition, RNF40 promotes cell migration by upregulating expression of cytoskeletal proteins (ROCK1, VAV3, LIMK2) and their effectors such as phosphorylated cofilin. Authors show elegant partial rescue experiments of the effect of RNF40 depletion on apoptosis and survival.

Given that RNF40 function seems to be context-dependent, findings from this study could have broad significance for other cancers with high RNF40. It also provides some mechanistic data (that should be improved as suggested below) linking this ubiquin ligase to the cytoskeletal machinery and, therefore, control of migration and also proliferation and survival.

Data are well presented and most conclusions are supported by the data. However, there are some gaps at the mechanistic level. Since migration is controlled by RNF40 in vitro, evaluation of metastatic ability in vivo (local invasion for example as suggested below) should be evaluated and would strengthen this part too.

**Major comments**

1. Fig.1A-B, S1A. Specificity of RNF40 antibody should be shown, which could be done quite easily in the tumours from the knockouts. From the datasheets, antibodies recognize human protein only.

We thank the reviewer for this suggestion and apologize for this mistake. The antibody utilized in the IHC studies is actually from Abcam (ab191309) and, in fact, recognizes both species. Table S5 has been corrected accordingly.

It is unclear when the murine tumours were analysed, at endpoint? This should be stated.

We thank the reviewer for this comment. Indeed, all IHC analyses were performed after dissection (endpoint). This information will be added to the manuscript. Kinetic analyses of tumor growth (i.e., Fig. 1G) were performed on the same mouse cohort.

Could authors establish cell lines from the mouse tumours (knockout, partial knockout escapers..)? These could be very useful tools to evaluate key in vitro findings from the study.

The reviewer makes an interesting suggestion. Unfortunately, we were not able to establish cell lines from this model and have since stopped breeding this mouse line (due to the relocation of the principle investigator). However, we did try to generate RNF40-deficient breast cancer cell lines using the CRISPR/Cas9 technology. Interestingly, all attempts failed, supporting the fact that the loss or RNF40 is lethal for the cancer cells. However, to further establish this, for the revision we propose to transfect HCC1954 cells with CRISPR/Cas9 constructs targeting exons 3 and 4, similar to our mouse model. We will then assess the evolution of RNF40-negative cells population over time (i.e., via immunofluorescence staining for H2Bub1). This assay should inform about the expected growth “disadvantage” following RNF40 loss.

Fig.1F-G: since RNF40 controls the cytoskeletal machinery and therefore, migration (Fig. 2G) in the RNF40 knockout tumours, was metastasis (if observed) affected? Or if there was no growth in distant organs detected in the time frame of these experiments, was invasion (and/or pattern of invasion or mode of invasion (morphology of invading cells)) into adjacent tissues affected upon RNF40 depletion? This would add in vivo relevance to the in vitro mechanistic findings, especially since the authors later showed that p-cofilin was also decreased in the RNF40-depleted mouse tumours (Fig.4D).

We agree that the metastasis data from our mouse genetic tumor model would significantly help solidify our findings. Unfortunately, the MMTV-Erbb2 mouse model (overexpressing wildtype Erbb2 gene) only rarely develops distal metastases. In our analyses, we only ever observed two macroscopically visible metastases (one wt/wt and one in an Rnf40flox/flox mouse). However, we feel that the reviewer’s suggestion is a one and will follow this suggestion and attempt to examine possible changes in local invasion of primary tumors into adjacent tissues.

Fig.3: most results using HCC1954 cell line. Key findings should be validated in other cell lines.

We agree with the reviewer about the importance of cross validation of findings using different cell lines. For this purpose, we have now generated data with an additional HER2-positive cell line. These data using the SKBR3 cell line were performed for several of the key experiments. Key findings about phenotypic changes (growth kinetics and colony formation), Ki67 protein levels differences and mRNA regulation of identified regulators of actin cytoskeleton (VAV3, ROCK1, LIMK2 and PFN2) will be included in the revision for both cell lines. Furthermore, as seen in the HCC1954 cell line, an increase of the apoptosis marker cleaved PARP as well as a loss of VAV3 and ROCK1 protein levels was also observed upon RNF40 knockdown in SKBR3 cells. These data will be included in the revised manuscript.

Fig.3A: authors state "both pathways remained intact following RNF40 depletion". However, from those blots, siRNF40 clearly increases pERK and slightly pAKT, which would be unexpected according to previous data in Fig.2. Authors could show quantifications of different blots, or show a more representative blot if increase in pERK was not consistently observed. Was this also seen in SKBR3 cell line?

We thank the reviewer for this comment. Initially, we had anticipated that oncogenic signaling may be decreased in the Rnf40 conditional knockout model. However, much to our surprise, the activity of the downstream signaling actually appears to be increased. In fact, the increase in AKT and ERK1/2 phosphorylation following RNF40 silencing was consistent across different experiments and replicates. While this finding is also consistent with our previous results in an ER-positive system (e.g., see Prenzel, et al., 2011), we do not understand the mechanistic underpinnings of this finding. Importantly though, while consistent, we do not feel that this increase explains the observed phenotype. Nevertheless, to more precisely show the overall change of p-ERK/ERK and p-AKT/AKT, in the revision we will provide a densitometry quantification for both cell lines. We will also modify the sentence to more precisely describe this finding and make the point that since these pathways are not reduced/impaired, they are unlikely to be responsible for the increased apoptosis observed upon RNF40-KD. Western blots assessing p-ERK/ERK and p-AKT/AKT levels in SKBR3 upon RNF40 knock-down will also be added into the supplementary data of the revised manuscript (Fig.S3).

For Fig.3G and Fig.S3A, authors selected genes from this set, how was this done (fold change?). Was expression of the other family members (ROCK2, LIMK1, etc) or of Rho GTPases regulated too?

This information was indeed missing in the manuscript. We have modified the figure legend and the main text accordingly in order to provide the information about the cutoff used in the Enrichr analysis. Regarding the expression of other family members of the actin regulatory network, in the past we performed a more, in depth and focused analysis of our RNA-seq data, broadening our view to other members of the RHO/RAC/CDC42 pathways. While we did identify a few further potentially regulated target genes (e.g. ROS1 or PAK6), these genes were either only weakly expressed or weakly regulated. For this reason, we presumed that these factors could only play a marginal role in the observed phenotype and have focused our attention on the robust part of the signature.

Fig.4B: this may not help, decrease of p-cofilin by Vav3 knockdown is way less dramatic compared to RNF40 depletion or ROCK inh treatment. See comment below regarding other effectors such as Myosin.

Indeed, the consequence of VAV3 loss on p-cofilin levels is less pronounced than the effect observed upon RNF40 knockdown or ROCK1i treatment. Given the fact that RNF40 loss not only affects VAV3 expression, but also has additional direct effects on the expression of other pathway members, this may be expected. We do, however, feel that the VAV3 regulation is likely one component of the effects of RNF40 loss. In addition, it has also been shown that VAV3 is not the only GEF regulating the activity of RHO kinases upstream of ROCK1. Therefore, we would also expect that VAV3 loss only partially reduces ROCK1 activity and therefore only partially phenocopies the effects observed. We will expand the description of these findings in the revised manuscript to reflect these views.

Fig.4C: does ROCK inh reduce RNF40 levels? It may from the immunofluorescence picture.

We thank the reviewer for this comment. In fact, we have examined this possibility. However, no significant changes in RNF40 protein levels were observed upon RKI-1447. If helpful, we can provide Western blot data demonstrating this in the supplemental figures.

Fig.4H-I: the sphingosine 1-phosphate receptor-3 agonist (CYM-5441) partially rescued the effects of RNF40. Since S1P signalling involves Rho GTPase activation -presumably downstream of VAV3 -which is a GEF for Rho, Rac and Cdc42- and upstream of ROCK, LIMK, was activity of these Rho GTPases affected upon RNF40 depletion? This would strengthen the mechanism.

The reviewer points at an interesting aspect of the actin regulation. Indeed we expect that the reduction of VAV3 levels upon RNF40 loss would significantly influence the activity of the downstream client GTPases. However, the measurement of RHO-GTPase activity is tricky and expensive. Furthermore, as mentioned in the previous comment (#7, part 1) VAV3 is only one component of the four major genes encoding critical actin cytoskeleton regulatory proteins regulated upon RNF40 loss, and the only factor upstream of RHO-GTPases. The reduction of downstream ROCK1, LIMK2 and PFN2 levels also influence the activity of this pathway downstream of RHO-GTPase activity. We therefore focused our efforts on assessing F-actin and p-cofilin levels as these may provide more sensitive readouts about the consequence of RNF40 loss on this signaling cascade. However, if the reviewer considers this information as indispensable, we would attempt to investigate changes in Rho-GTPase activity by commercially available Active Rho Detection Kits, although this will significantly delay the resubmission of a revised manuscript.

Related to this, was Myosin II activity (phosphorylated MLC2) affected -since its upstream regulators, especially ROCK are controlled by RNF40?

We thank the reviewer for this insightful suggestion. To address this possibility, we will test this hypothesis for the revised manuscript as suggested by performing Western blot analysis for phosphorylated MLC2.

Fig. S5E:

Authors should consider presenting data of decreased histone methylation of cytoskeleton regulators in main Fig. 5, since this is an important conclusion of this part.

As suggested we will shift the information currently presented in figure S5E to the main figure 5.

Statistics should be revised throughout the manuscript. Comparisons of more than 2 groups should be performed with ANOVA or similar multiple comparison test (instead of t-test).

We thank reviewer for this comment. We replaced statistical tests with the appropriate ANOVA in the respective graphs and updated the legends accordingly.

**Minor comments**

Statement of significance mentions "Anti-HER2-therapy resistance", but this is a misleading since the paper does not deal with therapy resistance. Or are the cell lines used in the study resistant to anti-HER2?

We thank the reviewer for this suggestion. While resistance to anti-HER2 therapy remains one of the major clinical challenges in the treatment of HER2 positive BC lesions, we agree that our data do not strictly address this point. Thus, we have modified the sentence accordingly.

In line with this, authors could add some lines of thought on how RNF40 could be targeted in the clinical/pre-clinical context, which could inform further translational studies.

This is a great suggestion. In the revised manuscript we will include additional text to specifically address this point.

Line 117 "Moreover, HER2-positive metastatic BC samples showed a particularly high expression of RNF40 compared to primary tumors".

Perhaps rephrase, was it that the expression level (intensity) was higher or that the % of positive cells/tumours was higher in the brain mets?

This is a critical point that we will consider in the revised manuscript. We have modified the sentence accordingly to read, “Moreover, the incidence of RNF40-high specimens was higher in HER2-positive brain metastases compared to primary tumors (Fig.1A-B)”.

Fig.1D and S1C. while S1C shows TCGA data, it is unclear which set of patients is Fig.1D (since text says publicly available data, line 118-119), are these their own set of patients (used in Fig.1A-B)? This should be specified in the text, legend.

The origin of the data shown in Fig.1D for relapse free survival of RNF40high and RNF40low patients (KM plotter) is mentioned in the figure legend (kmplot.com) and in the material and method section. However, since this was not apparent, to increase the readability, we have now added a statement about the publically available database of origin for every output graph in the main text as well in the legend and supplementary material.

Line 122. Authors should be careful with this conclusion so far, a correlation between expression and cancer stage/survival does not necessarily mean a tumor suppressive/supportive role.

We thank the review for this comment. We agree with this statement. Therefore, we have carefully rephrased this sentence as following, “In summary, these data demonstrate that RNF40 expression is maintained in HER2-driven primary metastatic BC and that its expression correlates with poor prognosis in these patients.”

Fig. S4E: there are missing labels in graph (control, siRNF40).

The labels have been added.

Panels in some figures are discussed in text randomly and not following same order. For example, Fig.1 (after panel D, then panel H, then back to E, F, G), S3, 4A-C,

We reorganized the order of the different panels of Fig1 to increase the readability. We further screened the main text for similar problems and modified the respective figures accordingly.

Fig.1E: I would suggest changing the line colours, so Rnf40wt-wt line is red and the fl-fl is black, therefore it is similar to panel D (high Rnf40 red, low in black).

We thank the reviewer for this suggestion. Accordingly, we have now indicated low RNF40 expression in red (Figure 1D, 1E and S1B) in the same way that we have indicated RNF40 expression throughout the rest of the study.

Supp Videos: for reviewers and readers, it would help that video has a label while it plays, otherwise after downloading it, video name does not tell whether it is control or RNAi.

As suggested we have renamed the video files of for each condition and added a label informing about the identity of the sample while the video plays.

Reviewer #2 (Significance (Required)):

Given that RNF40 function seems to be context-dependent, findings from this study could have broad significance for other cancers with high RNF40, or even in other pathological contexts -if any- that cursed with high RNF40.

It also provides some mechanistic data (that should be improved as suggested in comments) linking this ubiquitin ligase to the cytoskeletal machinery and, therefore, control of migration and also proliferation and survival. This will also advance the field.

Area of expertise

Actin-myosin cytoskeleton, Rho GTPases-ROCK, cancer, metastasis, cell signalling

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

**Summary:**

Wegwitz and colleagues present extensive and detailed data focussed on the role of the E3 ubiquitin ligase and ring finger protein RNF40 in HER-2 associated breast cancer. It is clear that the role of RNF40 and its major substrate histone H2B (monoubiquitination of histone H2B at lysine 120; H2Bub1) as part of a complex with RNF20, is not a simple one in the context of malignancy. This group, and others, have previously reported on the intriguing role of RNF40 that can in certain circumstances function to suppress tumorigenesis, and in other circumstances function to support tumorigenesis. While H2Bub1 has been shown to be lost in many different malignancies, these investigators show that in HER-2 associated breast cancer, this is not the case. In fact, the results presented in this study show that RNF40-mediated H2Bub1 is important for the expression of genes involved in the actin cytoskeleton and the downstream FAK signalling cascade. Supporting this, mining of a public database showed that RNF40 mRNA was high in HER-2 associated breast cancers and was correlated with a worse prognosis (overall and RFS, relapse free survival). The investigators also used a mouse model (MMTV-Erbb2) generating a tri-transgenic (MMTV-Erbb2; MMTV-Cre; Rnf40flox) that allowed breast tissue specific overexpression of HER2 at the same time as KO of Rnf40, so mimicking the human disease. In fact, mouse tumours recapitulated the human results, including in disease free survival (lower with higher Rnf40), and with less tumours seen when there was less Rnf40 (the Rnf40 floxed tumours appeared heterogenous in staining patterns for Rnf40 and H2Bub1, supporting the concept of "escaper" cells, positive for both Rnf40 and H2Bub1 that would be positively selected during tumorigenesis).

The authors also took a cell biology approach to studying HER2 positive breast cancer and RNF40 using two HER2 positive cell lines (HCC1954 and SKBR3). RNF40 was down-regulated using siRNA and numerous functional studies showed that targeting RNF40 suppressed behaviours consistent with tumorigenesis (proliferation, migration, clonogenic survival, spheroid formation, growth kinetics). Furthermore, down-regulation of RNF40 in the HCC1954 cell line followed by GSEA identified gene signatures associated with apopotosis and the actin cytoskeleton regulatory pathway (e.g. ROCK1, VAV3, LIMK2, PFN2). They further showed that phospo - cofilin (that occurs downstream of ROCK1) was reduced in RNF40 down-regulated cells, also implicated in regulation of the actin cytoskeleton. Phalloidin staining for F-actin showed disruption of the cytoskeleton in RNF40 down-regulated cells. Additionally, the ROCK1 inhibitor, RKI-1447, showed similar effects to depletion of RNF40.

The authors then sought to determine whether the RNF40 associated gene expression in HER2 positive cells was in fact happening through H2Bub1 and the active histone mark H3K4me3 it has been reported to cross-talk with. RNF40 regulated genes (up or down-regulated) showed lower levels of H2Bub1 occupancy compared to non-regulated genes. H3K4me3 was lost in most genes influenced by RNF40 down-regulation, including genes associated with the actin regulatory pathway. The overall conclusion is that RNF40 is a major epigenetic regulator of the actin regulatory gene network in HER 2 positive breast cancer and could be a therapeutic target.

**Major comments: major issues affecting the conclusions.**

(1) What is happening at the gene level for both H2Bub1 and H3K4me3 in the context of RNF20 down-regulation is complex and would benefit from inclusion of a schematic, or a series of schematics describing different scenarios, as the text is quite difficult to follow.

This is a very constructive proposition. We will attempt to follow this suggestion in order to simplify the message of the respective section by providing to schematic illustrations depicting the cascade of events occurring upon RNF40 loss in the cancer cells.

It is not entirely clear that the changes seen in H3K4me3 are a direct result of cross-talk with H2Bub1 (some literature reports that there is no cross-talk between these histone marks for instance). It is also not entirely clear how the other histone marks investigated support the main discoveries of the paper. The authors need to consider this in the way that they present the data and their interpretation of it.

The reviewer addresses an important point about the mechanistic aspect of the RNF40-dependent epigenetic regulation. We and others have shown that RNF40-mediated H2B monoubiquitination is a central step for activation of the COMPASS complex and the TSS-proximal broadening of H3K4me3 (PMID:31733991, 19410543, 22505722, 28209164). However, the situation certainly is not as straight forward as it is in yeast, where the vast majority of H3K4 trimethylation is H2Bub1-dependent. To what degree global H3K4me3 levels are dependent upon the H2B ubiquitin ligases RNF20 and RNF40 appears to vary, depending upon the investigated system (probably the variation in the literature referred to by the reviewer). However, in our work, we reproducibly see widespread H3K4me3 peak narrowing specifically on RNF40-dependent genes, in a context-dependent manner (i.e., genes displaying these effects are different according to the system investigated). To support and consolidate the central function of the H2Bub1-H4K4me3 crosstalk in our system, we propose to perform rescue experiments: siRNAs targeting the 3’UTR of RNF40 will be co-transfected with an expression construct encoding for either a wild type or a ΔRING (catalytic inactive) form of RNF40 lacking the endogenous 3’UTR. The ability of ectopically expressed wild-type, but not catalytic inactive RNF40, to rescue the expression of the identified actin cytoskeleton genes and downstream signaling should provide a solid argument to support the hypothesis of our study. We will also include additional discussion about the potential different H3K4 methyltransferases that may potentially be involved.

(2) RNF40 is known to work in a complex with RNF20 to monoubiquitinate histone H2B at lysine 120 (H2Bub1). In experiments where RNF40 has been down-regulated, did the authors also note down-regulation of RNF20 (as has been previously reported).

This is an interesting question from the reviewer. We indeed observed a consistent reduction of RNF20 protein levels upon RNF40 knockdown (and vice versa) in different cell systems, including the HER2-positive cell lines HCC1954 and SKBR3.

Is the data presented likely to be the result of abrogation of the complex rather than RNF40 specifically?

Although particularly difficult to answer, the use of a catalytic mutant in key experiments should at least partially shed light on this aspect (as proposed in the answer to Reviewer #3’s question 1). In that case, the complex integrity can be maintained while specifically abrogating RNF40 ubiquitin ligase activity.

While I am not asking for experiments to be repeated with down-regulation of RNF20, some consideration of this needs to be included in the Discussion. Is RNF20 also highly expressed in HER2 positive breast cancer (TCGA, KM Plotter data).

We absolutely agree with the reviewer’s point of view. As an obligate binding partner of RNF40, RNF20 indisputably plays an important function in the phenotype caused through RNF40 loss. We will therefore carefully further discuss this aspect in the revised manuscript. Preliminary analyses based on the TCGA dataset point at a high expression of RNF20 in HER2-positive lesions. Furthermore, survival analysis of HER2+ BC patients based on the same dataset showed that patients with high RNF20 expression harbor an unfavorable prognosis, similar to what we have seen with RNF40. We may therefore implement these expression and survival data in the revised manuscript.

**Minor comments: important issues that can confidently be addressed.**

(3) It would appear that immunohistochemistry for RNF40 and H2Bub1 on human samples is only reported as "low" or "high". This is perhaps not dealing with the full spectrum of IHC scores, such as completely absent, although the methods do note a "null" value (no detectable staining). Were there no "null" results? Please define the criteria for "low" or "high".

Indeed, specimens lacking H2Bub1 or RNF40 staining were attributed the “null” scoring. However, while we have observed null staining in other BC subtypes (e.g., see Bedi, et al., 2015), none of the HER2 positive BC samples were found to be negative for either RNF40 or for H2Bub1. However, for the revision, we will provide representative examples of null-stained tumor specimens (from other BC subtypes) for RNF40 and H2Bub1 from the same tissue microarray.

(4) I think there might be some confusion in labelling of Fig 1A and B as the legend states that all breast cancers are on the left and the HER-2 positive on the right, for each of primary tumours and brain mets, but I think one is under the other? Labelling should be checked in this figure.

We apologize for this mistake. This has been corrected in the figure legend accordingly.

(5) What this IHC data doesn't show is whether RNF40 and H2Bub1 levels are always correlated in individual tumours (i.e. RNF40:H2Bub1, high:high OR low:low OR null:null). Can the authors please include and comment on this data.

The reviewer has made a very interesting point here. We will comment on this point in the revision.

(6) Please include overall survival data (KM Plotter) as a panel in figure 1, alongside RFS for RNF40 expression levels (currently in Supplementary).

We added the OS as well the RFS data from the same database next to each other in the main figure.

(7) Spheroid formation looks to only be shown in a single cell line (HCC1954). Was the other cell line not suitable for spheroid studies? Some comment should be made and care taken not to "overclaim" as text notes two cell lines.

SKBR3 are unfortunately not suitable for tumor sphere formation assay. We may provide instead a soft agar assay with SKBR3 cells upon. If needed, we may replace the SKBR3 cell line with BT474 for this specific experiment

(8) It would have been interesting to see results of a GSEA in the mouse mammary tumours as a complement to human. Is there a reason why this wasn't undertaken?

Rnf40fl/fl tumors present a large fraction of “escaper” cancer cells retaining RNF40 expression. For this reason, bulk sequencing of such tumors would likely only provide a “diluted” molecular signature consequent to RNF40 loss. For this reason this experiment has not been done.

(9) Conclusions are made about RNF40 in HER2 positive cells only in the context of H2Bub1 and H3K4me3. Without having conducted similar experiments in HER2 negative breast cancer cell lines / models, it is difficult to draw the conclusion that this is HER2 positive specific. Can the authors either soften some of their conclusions along this line, or consider repeating some of their data in HER2 negative models.

The scope of the study has deliberately been set on HER2-positive malignancies, because former studies already extensively studied the impact of H2Bub1 loss in TNBC and Luminal BC (PMID 28157208, 18832071). We will therefore modify the manuscript text accordingly and soften the appropriate sections as suggested by the reviewer.

(10) RNF40 likely has substrates other than histone H2B. There is a report describing interactions with RNF40 (STARING) and syntaxin for e.g., (Chin et al., 2002 J Biol Chem 277:35071-9). Can the authors please comment on other potential substrates of RNF40 in light of their data that focuses only on its epigenetic role as a regulator of the actin cytoskeleton.

Our study was mainly focused only on the gene expression program driven by RNF40 in HER2+ BC. We therefore do not know nor have we focused on other novel non-histone substrates. We will, however, allude to this possibility in a revised manuscript.

Reviewer #3 (Significance (Required)):

Nature and Significance of the Advance:

Clinically, this work provides a significant advance in that it is zeroing in on HER2 positive breast cancer and generating fundamental data that could underpin development of a new therapy for this malignancy. Conceptually, it is expanding knowledge of how the E3 ubiquitin ligase RNF40 is functioning as an epigenetic modifier of a specific type of malignancy by being important for the actin cytoskeleton.

Work in Context of Existing Literature:

As acknowledged by the authors, this work builds on a previous publication of theirs (Xie et. al., 2017 "RNF40 regulates gene expression in an epigenetic context-dependent manner." Genome Biol). They have other recent papers on RNF40 (Schneider et al., 2019 "The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells", Clin Epigenetics; Kosinsky et al., 2019 "Loss of RNF40 decreases NF-kappaB activity in colorectal cancer cells and reduces colitis burden in mice", J Crohns Colitis). H2Bub1 is one of the least well studied histone modifications and as such, this study of one of its key histone writers, RNF40, is significant in elucidating the significance of this histone mark.

Audience:

This paper will suit a discovery-based science audience interested in epigenomic regulation of malignancy. Further, it will suit those looking for new drug development strategies for malignancy.

My Field of Expertise:

Basic scientist with expertise in epigenetic/epigenomic regulation in malignancy; cell and molecular biology. I felt capable of reviewing all aspects of this paper.

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Reply to the reviewers

Rebuttal_ Preprint RC-2020-00156

We thank the editor for handling our manuscript and both reviewers for their constructive critiques. We provide below a detailed list of results already available and experiments we propose to perform to address the reviewers’ comments and improve the quality of our manuscript.

Reviewer #1

In this manuscript, Obacz et al. investigated the role of IRE1 signaling in regulating the recruitment of myeloid cells in glioblastoma multiforme (GBM) microenvironment. They show that inhibition of IRE1 signaling decreased polynuclear neutrophil (PN) infiltration to GBM tumors in an animal model; conversely, IRE1 activation correlated with higher expression of myeloid cells-attracting chemokines in GBM. They also show that IRE1-XBP1s pathway promotes proinflammatory chemokines in GBM tumor cells through upregulation of UBE2D3, which leads to degradation of the NFκB inhibitor IκB and activation of NFκB downstream signaling. Their finding of a novel IRE1/XBP1s/UBE2D3/NFκB axis is important for understanding the basis of pro-tumoral inflammation in GBM, potentially in other 'immune hot' cancers. The manuscript is well written and the conclusion is well supported by the experiments. However, there are a few critical points that need to be addressed to strengthen their study**.

We thank this reviewer for his/her positive comments on our work and for the suggestions made to improve its relevance

Review#1 point 1: In this study, the authors used the GBM primary cell line RADH87 with stable overexpression of wild-type (WT) IRE1 or a truncated IRE1 variant. The expression of wild-type IRE1 was confirmed by Western analysis (Figure S1D). However, the expression of truncated IRE1 variant was not shown.

Response 1.1. The expression on truncated IRE1 variant (designated as Q780* - 80 KDa) is shown in Fig.S1D, following the expression on wild-type (WT) IRE1 (110 KDa). This point will be indicated in the revised version of the supplemental figure legends.

In addition, without tunicamycin treatment, there was no visible difference in XBP1s expression between the cells expressing WT or the mutant IRE1.

Response 1.2. Under basal condition, XBP1 splicing is indeed limited and therefore, there is no detectable difference in XBP1s expression level between IRE1 WT and Q780*. In contrast, under tunicamycin treatment (acute stress), reduced XBP1 mRNA splicing is observed (Fig.S1D) thus confirming the functionality of the Q780* truncated form. Of note, RNAseq was performed on these cell lines and basal splicing was quantified showing that even though it is an event that occurs at low frequency, it is decreased in cells expressing the Q780* mutant (this information will be added in the revised manuscript, data are available and analyses ongoing).

In the Boyden chamber assay (Figure 1C, D), conditioned medium from these cells were used; it was not described whether the cells were treated (e.g. with tunicamycin) to activate the IRE1 pathway. ** Response 1.3. Cells were not treated with Tunicamycin to excluded the impact of other UPR arms in the induction of cytokines expression/myeloid cells attraction. As a consequence, it is the basal secretome (found in conditioned media) that was used in those experiments to evaluate cell migration. We have now strong evidences that blunting IRE1 signaling (either genetically or pharmacologically) has a strong impact on GBM cells proteome and in particular on their secretome even if under basal conditions (manuscript in preparation). This information together with the fact that basal XBP1 mRNA splicing is reduced in IRE1 signaling deficient (Q780* expressing) cells, indicate that in GBM cells, constitutive IRE1 activity contributes to modulate the composition of their secretome towards chemoattraction of myeloid cells. This point will be further detailed in the results and discussion sections of the revised manuscript.

Review#1 point 2: The evidence that the mRNA expression of UBE2D3 positively correlates with IRE1/XBP1s pathway is weak. First, In Figure 3D, the correlation between the mRNA expression of UBE2D3 and XBP1 does not seem strong. In addition, as XBP1 mRNA level does not reflect IRE1 activation (as opposed to that of XBP1s), the level of XBP1s instead of total XBP1 should be assessed. Furthermore, such correlation should be validated in additional GBM cohorts/datasets.

Response 2. We agree that the correlation between UBE2D3 and XBP1 mRNA levels in TCGA GBM cohort might not be strong. However data presented in Fig3D were significant. Values indicated in green were Pearson’s correlation values (r). This point will be included in the revised figure legends. Moreover, in the revised version of the manuscript we propose to directly correlate the levels of XBP1s mRNA with the expression levels of SYVN1, UBE2D3 and UBE2J1 mRNAs. These data are available from the RNAseq data obtained from the TCGA cohort and already used previously by us (Lhomond et al. Embo Mol Med 2018). In addition, following this observation we have carried out a number of experimental validations using both established and primary GBM cell lines with genetic modifications of XBP1/XBP1s expression as well as ER stress-dependent induction of XBP1s and we clearly demonstrated that XBP1s mRNA levels correlate with UBE2D3 mRNA expression levels (Fig.3G-H, Fig.S2D-E). In addition, in Fig3E using our IRE1 activity signature we have shown a strong correlation between UBE2D3 and XBP1s, which is even more robust than simply correlating the mRNA levels. Data are already available and analyses are ongoing.

Review#1 point 3: The results in Figure 3 indicated that XBP1s acts as a transcriptional regulator of UBE2D3 expression. However, it is not clear whether this effect in GBM cells is direct or indirect. Further experiments such as chromatin immunoprecipitation and reporter assays are required to clarify this point.

Response 3. We agree with this reviewer’s point. Although we have scrutinized the publicly available ChIPseq databases and found UB2D3 among potential XBP1-regulated genes, we did not validate this observation in our model. To address this point we propose to perform ChIP experiments in cells overexpressing a tagged form of XBP1s and validate the presence of UBE2D3 promoter fragments in our experimental system. Moreover, these experiments will also be carried out with endogenous XBP1s (in-house XBP1s antibodies Pluquet et al. Cancer Res. 2013) in our primary GBM lines under basal and ER stress conditions. At last, to further document this, luciferase reporter assays using the UBE2D3 promoter (whose length would be defined based on ChIP experiments and the presence of XBP1s binding sites) upstream the luciferase ORF could be performed. Both ChIP and reporter assays have to be performed.

Review#1 point 4: In addition to UBE2D3, the two other ubiquitin-protein ligases, SYVN1 and UBE2J1, may also be implicated in the degradation of IκB. Did the authors assess their potential role on IκB degradation in their model system?

Response 4. We thank this reviewer for this suggestion. We have previously tested the impact of SYVN1 on IkB degradation with results showing a lot of variation. Indeed even though the trend of our results indicated that SYVN1 silencing appeared to lead to a slight increase in IkB expression, they never reached statistical significance. Variability in the results might be due to the efficacy of SYVN1 silencing and as such we propose to repeat further these experiments with SYVN1 siRNA smart pools to improve silencing efficacy. Moreover, SYVN1 has been shown to also contribute to the ubiquitylation and degradation of IRE1 (Gao et al. Embo Rep 2008; Sun et al. Nat Cell Biol 2015) and has its expression regulated by IRE1 activity (Dibdiakova et al. Neurol Res 2019), it might represent as well a very interesting target to study. Regarding UBE2J1, the situation is less documented. However, it was shown that this E2 works together with SYVN1 in conserved manner to contribute to ERAD (Chen et al. Nat Plants 2016). As such it might also be interesting to test whether the silencing of UBE2J1 impacts on IkB expression. To sum up, we propose to test experimentally whether the silencing of UBE2J1 or SYVN1 or both together impacts on IkB expression (we need to perform the experiments).

Review#1 point 5: The authors only used ectopic expression of relevant proteins to test their hypothesis in U87 and RADH87 cells. It is necessary to validate these findings using siRNAs/inhibitors for IRE1 and UBE2D3 in a GBM cell line that expresses high levels of endogenous IRE1 and UBE2D3.

Response 5. We propose to test the effect of SYVN1 and UBE2J1 silencing on IkB expression in U87 and RADH87 cells in the revised version of the manuscript (see above). In addition to address this reviewer’s comment, we propose to use U87 and RADH87 cells overexpressing IRE1 (Lhomond et al. 2018) and treat them with MKC886, or with siUBE2D3 or with both and evaluate whether in those conditions the NFkB pathway is affected. These experiments should be carried out relatively easily provided that all the recombinant cell lines, drugs and siRNA are already available.

Review#1 point 6: In Figure 3I: The protein expression of UBE2D3 should be shown.

Response 6: We had included control experiments with UBE2B3 expression in FigS3B in the initial version of the manuscript. We will include UBE2D3 expression for Fig3I in the revised version of the manuscript (these data are already available).

Review#1 point 7: In the right panel of Figure 3I: What do the labels #1, 2, 5 mean? Clear descriptions should be provided in the figure legend.

Response 7. Those labels correspond to different RADH87 cell lines stably overexpressing UBE2D3 protein. The validation of UBE2D3 expression using Western blotting will be included in FigS3B of the revised version of the manuscript (data are already available).

Review#1 point 8: In Figure S1D: The expression levels of the truncated IRE1 variant should be shown.

Response 8. The expression on truncated IRE1 variant (designated as Q780*) is shown in Fig.S1D, following the expression on wild-type (WT) IRE1. This point will be indicated in the revised version of the supplemental figure legends.

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Reviewer #2

In the current study, the authors generate evidence supporting a novel pathway downstream of IRE1α/XBP1s in GBM cells involving the activation of an E2-ubiquitin ligase, UBE2D3. In order to do this, they use a combination of patient derived and established cell lines engineered to overexpress IRE1 mutants, XBP1s or UBE2D3. They claim that UBE2D3 is upregulated downstream of XBP1s in GBM cells, and functions to activate NF-kB through the degradation of IkB, thus promoting CXCL2/IL-6/IL-8 production and the subsequent recruitment of monocytes and polymorphonuclear (PN) cells to the tumor microenvironment. However, the article has major shortcomings that need to be addressed before considering its publication

We thank this reviewer for his/her constructive comments on our work.

Review#2 point 1: Fig. 1: Classification of immune cells infiltrating GBM. The characterization of immune infiltrate in GBM is too simplistic. Monocytes, monocyte-derived macrophages and microglia are treated as equivalents along the text (IBA1+), making the story hard to follow. At least in mice, these populations can be easily distinguished based on CD45/CD11b/Ly6C expression (see for example Zhihong Chen et al., Cancer Research, 2017). Can the authors further analyze which of those population are actually affected under IRE1 deficiency and/or UBE2D3 overexpression? On the other hand, it is rather questionable that all CD11b negative cells are exclusively T cells, as suggested in Fig 1B. Can the authors provide evidence and/or references to support their gating strategies?

Response 1: We thank the reviewer for this comment. Our objective was to test the impact of IRE1 modulation on the infiltration of myeloid cells in the tumor, and we did not plan to describe this effect on the complete and detailed infiltrating myeloid populations in GBM which could represent a full study on its own. However, to address this reviewer’s critique we propose to complete the characterization of the myeloid population in our mouse model using IHC by adding Ly6C staining for macrophages and granulocytes. We did not select flow cytometry approach to explore this point as suggested by the reviewer (Cheng, Cancer Res, 2017), but instead IHC was preferred as we thought that the localization of the infiltrated immune cells was important to evaluate (periphery vs. core of the tumor). The information about the localization of immune cells is already available and will be added to the revised manuscript. Concerning the second point raised by this reviewer, the strategy to characterize the immune population in human GBM specimen was to combine CD45 and CD11b markers as previously described by Hussain et al. Neuro-Oncol 2006 and Parney et al. J Neurosurg 2009. Moreover, the analysis of additional markers allowed us to confirm that CD45+ CD11b+ cells were mainly monocytic cells (that also co-expressed CD14, CD168, CD64 and HLA-DR); CD45 low CD11b high cells were granulocytes (CD66B, CD15 and CD16); and CD45 high CD11b low cells were mainly CD3+ T cells. These data are already available and will be added to the revised manuscript.

Review#2 point 2: Fig. 1: RADH IRE1 Q780\ model - Can the authors further validate the IRE1 deficiency of their model cell line RADH87 IRE1Q780*? It appears to have severely reduced IRE1 levels when compared to the RAD87-IRE1WT cell line (figS1D). Furthermore, the WT and not the truncated form seems to be predominantly expressed. Intriguingly, XBP1 is still being spliced after tunicamycin treatment in this mutant line. All these results differ significantly from the U87-Q780* cell line originally published by Lhomond et al., 2018. Can the authors comment on these differences? Was there a mixture in cell lines? *

Response 2: We agree with the reviewer that the level of IRE1Q780* expression on RADH87 cells is lower than the IRE1WT expression (Fig.S1D). As observed by this reviewer, XBP1 was still spliced in Q780* cells but XBP1s expression was reduced as shown in Figure S1D. This is mostly due to the ratio between the expression endogenous IRE1 and that of Q780*, which as previously shown (Lhomond et al; 2018) acts as a dominant negative and preempts endogenous IRE1 signaling. The differences observed are also probably due to the methods used, indeed we measured XBP1 and XBP1s mRNA expression in U87 cells (Lhomond et al. 2018), whereas XBP1s protein expression was used with RADH87 cells (introducing the RNA translation parameter that was not monitored in U87 cells). Differences could be also linked to the cell lines as we used the U87 immortalized and RADH87 primary cell lines.

Review#2 point 3: Fig. 1: Impact of IRE1 inhibition on recruitment of myeloid cells to the TME. The experiment in figure 1E-F, which is the only in vivo evidence supporting a role of IRE1 signaling on myeloid cell recruitment, is very hard to interpret. The authors show no evidence that IRE1 is being inhibited under the treatment and if so, up to which extent. Furthermore, what are the cells targeted by MKC in this setting? The differences in the infiltration of PN cells seem very slight, nothing is mentioned regarding the number of mice per group, or the statistical analysis performed. I would suggest performing a simpler experiment to demonstrate an intrinsic effect of IRE1 signaling in GBM cells, comparing the recruitment of myeloid cells in tumors generated by GL261 cells expressing WT vs deficient forms of IRE1.

Response 3: The mouse model used in the paper is fully described in (Le Reste BioRxiv 2020 - doi: https://doi.org/10.1101/841296) and all the details about the procedures can be found in this manuscript. This model was developed to recapitulate in mice the standard of care for GBM patient including surgical resection. In addition, drug delivery in brain tumors is often an issue due to the blood-brain barrier. Therefore, the IRE1 inhibitor was delivered locally after resection of the tumor, exposing both tumor and stromal cells. To quantify the myeloid cell recruitment in Fig1E-F, at least thirty random fields from tumor tissue and at least thirty random fields from tumor periphery were quantified for control (PLUG) and MKC-treated group (2 mice/group). The number of positive cells in tumor tissue and tumor periphery were then pulled together for statistical analyses. The significance of the differences in myeloid cells recruitment between control (PLUG) and MKC-treated group was estimated using unpaired student t-test. At least 8 tumors of each group were analyzed comprising 2 to 3 sections of each and 10 fields per section. In addition, we have also performed the experiments using GL261 cells knockout for IRE1, the data are already available and could be possibly added to the revised manuscript.

Review#2 point 4: Fig. 2: Correlation between IRE1 signature and cytokine/chemokine signature. In the IRE1 signature as determined in the EMBO Mol Med paper (and to which the authors continuously refer) 6 out of 38 (15%) of the genes correspond to cytokines and/or chemokines (Il6, Il1b, Cxcl2, Cxcl5 and Ccl20) (Lhomond et al., 2018). Besides the fact that it is very unclear how this signature was obtained in the first place, it is rather surprising that in the current paper the authors correlate this "IRE1 activity" signature with the same or other cytokines/chemokines mRNA levels and come to the conclusion that there is a high correlation (fig 2A). Isn't this to be expected? Can the authors clearly explain how the IRE1 signature was determined and prove that their "IRE1 signature" is, in fact, representing IRE1 activity? For instance, it is important to cross validate their results by using an independent signature of IRE1 activity (e.g. ChipSeq XBP1s targets, Chen et al., 2014)?

Response 4: We thank this reviewer for asking for precisions about the procedure. The IRE1 signature was fully described in Lhomond et al. 2018 and was obtained from transcriptome datasets obtained from U87 modified for IRE1 activity (Pluquet et al., 2013). IRE1 was validated on GBM patients and appeared as an important tool to evaluate IRE1 activity in tumor specimen not only in GBM but also in other tumor types (Rubio-Patiño C, Cell Metab 2018). Furthermore, IRE1 activity was also directly linked to the pro-inflammatory tumor cell secretome in various studies such as Logue et al. 2018. As indicated by this, some cytokines/chemokines studied in this work were indeed part of the IRE1 signature and correlation between this signature and their expression was indeed expected. However the other main cytokines/chemokines studied here were not present in the IRE1 signature indicating that IRE1 could have been involved in the regulation of their expression. As proposed by reviewer#2, we will include in the revised version of the manuscript the analysis of cytokines/chemokines from the dataset ChipSeq XBP1s targets (Chen et al. 2014), although this study was performed on breast tumors.

Review#2 point 5: Fig 2: XBP1s controlling cytokines/chemokines expression in GBM cells - As suggested by the data on fig1C-D and fig2E, IRE1 appears to be constitutively active in GBM cells, as IRE1 deficiency is sufficient to cause a defect in chemokine production. However, as shown in fig S1D, XBP1s protein was not detected under basal conditions, suggesting that the deficiency in chemokine production in IRE1-deficient cell lines is XBP1s-independent. Can the authors further discuss these results?

Response 5: We thank the reviewer for commenting this point. We think that indeed IRE1 is constitutively active in GBM cells. As we have tested XBP1s protein expression in untreated and tunicamycin-treated RAD87 cells (FigS1D), and we will also provide real time qPCR data to show the presence of basal XBP1s mRNA levels (data already available). We agree that the way we presented the results are misleading and undermine the basal expression of XBP1s. This will be fixed in the revised manuscript.

Review#2 point 6: Fig 3: IRE1/XBP1s/UBE2D3/NF-kB axis - Authors must show the activation status of NF-kB in parental U87 cells (Fig3A), as this is a critical evidence to support that IRE1a-deficient U87-DN cells are defective in chemokine production due to an impairment in NF-kB signaling. In addition, even when tunicamycin treatment induce XBP1s and UBE2D3 (figS2D) it does not induce IkB degradation nor NF-kB phosphorylation in parental U87 and RADH87 cells (figS3C) as one should expect if IRE1/XBP1s/UBE2D3/NF-kB pathway is operating in these cells. How can this be explained? Only after XBP1s or UBE2D3 overexpression, NF-kB signaling appears to be affected.

Response 6: As shown in Fig3A, U87 cells deficient for IRE1 signaling (DN) exhibit decreased NFkB signaling as exemplified by decreased phospho-NFkB and phospho-IkB compared to control U87 cells proficient for IRE1 signaling. In our manuscript, we mainly focused on the activation of the IRE1/XBP1s/UBE2D3/NFkB signaling axis under basal condition. One could speculate that tunicamycin treatment leads to a strong stress response that others mechanisms are activated that overwhelm the IRE1/XBP1s/UBE2D3 pathway we are describing herein. For instance, it has been demonstrated that the IRE1/JNK signaling was linked to NFkB activation upon acute ER stress (Tam et al. PLoS One. 2012;7(10):e45078; Schmitz et al. Biomedicines. 2018 Jun; 6(2): 58.) and furthermore PERK activation upon thapsigargin or tunicamycin treatment was also found to promote NFkB activation (Deng et al. DOI: 10.1128/MCB.24.23.10161-10168.2004; Fan et al. Cell Death Discov. 2018 Feb 12;4:15). We believe that the pathway we describe here might be linked to constitutive activation of IRE1 signaling (proper to tumor cells) rather than acute activation of this pathway and be compatible with sustained proliferation. To further document this point, we have already generated data about the phosphorylation status of NFKB in GL261 cells KO for IRE1 compared to the parental cells (data will be provided in the revised version of the manuscript). In addition we are currently investigating the correlation between IRE1 activity signature and that of NFkB as defined previously (Jin et al. Cancer Res. 2014 May 15; 74(10): 2763–2772.), results should be available shortly and will be added in the revised manuscript.

Review#2 point 7: Fig 4: UBE2D3 and MIB1 – The authors should discuss better what is the possible interaction between UBE2D3 and MIB1. As shown in fig4G, silencing of MIB1 cause a severe increase in UBE2D3 protein levels but this is not commented in the text.

Response 7: We thank the reviewer for this comment. We believe that MIB1 might also controls the expression of UBE2D3. The data are already available and will be included in the revised version of the manuscript.

Review#2 point 8: Fig 6: Chemokines driving recruitment of myeloid cells to UBE2D3 overexpressing tumors. A formal demonstration that GL261-UBE2D3 tumors recruit higher numbers of MM and PNs through an enhanced production of CXCL2, IL-6 and/or IL-8 is lacking. For instance, they could compare the infiltration of myeloid cells in GL261-UBE2D3 vs GL261-UBE2D3-CXCL2KO tumors.

Response 8: To address this point, we propose to test the expression of these cytokines/chemokines in the GL261 tumors after resection using ELISA. These experiments could be carried out in IRE1 KO tumors, in UBE2D3 overexpressing tumors and performed for instance using perfusion of CXCL2, IL6 or IL8 neutralizing antibodies or cells KO for these chemokines. These experiments could be performed but might lead to inconclusive results (not statistically significant) if there is redundancy between the roles of those chemokines. As such, we think that we could provide in vitro information about the respective roles of these chemokines in recruiting MM and PNs but that at present stage the in vivo demonstration is to premature.

Review#2 point 9: Authors must provide replicates of the blots to sustain their claims: FigS1D, Fig3A, Fig3I, Fig4G.

Response 9: Replicates and quantifications are already available and will be provided in the revised version of the manuscript.

Review#2 point 10: The authors should include a better description of the methods regarding bioinformatic analysis. For instance, which genes where used for MM/PN/T cell signatures in fig1A/S1A?.

Response 10: We thank the reviewer#2. This information is available and a complete description will be included in the revised version of the manuscript.

Review#2 point 11: Missing statistical significance on fig 2C and fig 6A to support their claims.

Response 11: Statistical values will be included in the revised manuscript.

Review#2 point 12: Fig2F is presented in the text as mRNA levels but in the figure as protein levels.

Response 12: This point will be fixed in the revised version of the manuscript.

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Reply to the reviewers

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

In this report, van Schaik et al., modified an established CUT and RUN method and combined it with previously used DamID to identify Lamin Associated Domains (LADs) with better temporal resolution. Previous DamID experiments labeled locations where lamin proteins were present within a 5-25 hour window while the new technique, pA-DamID, labels DNA within a 30 minute window providing better temporal resolution. The authors used this technique to identify LADs at multiple stages of the cell cycle and applied this protocol to different cell types. The authors FIND differences when comparing data sets between cell cycle time points and cell lines.

We thank the reviewer for the helpful comments.

**Major points:**

1) The data sets generated and displayed in this manuscript seem incomplete. In Figure 1G, the authors compare lamin B2 vs. lamin B1 generated LADs in HAP-1 cells and lamin A/C vs lamin B2 LADs in hTERT-RPE cells. In figure S4, panel C compares lamin B1 and lamin B2 in K562 cells and lamin B2 and lamin A/C in hTERT-RPE cells. It would have been informative to have a complete dataset for lamin B1, lamin B2, and lamin A/C identified LADs in all cell lines analyzed. The information provided from these datasets would be useful to the scientific community.

We did not think it was necessary to generate every lamin pA-DamID data set in every cell line, given that previous DamID studies indicated that lamins A, B1 and B2 give the same genome-wide pattern {Meuleman, 2013, 23124521; Kind, 2014; 24717229}. However, we agree with the reviewer that the missing data sets lead to a sense of incompleteness and might distract the reader from the main message of the manuscript. We suggest to generate Lamin B1 pA-DamID in hTERT-RPE cells– provided that the current Corona virus shutdown will not prevent us from doing this experiment. Doing so, we 1) have a complete lamin data set in hTERT-RPE cells, which we study in most detail in this manuscript 2) can compare all lamins within the same cell type 3) can compare all Lamin B1 DamID data to the corresponding Lamin B1 pA-DamID data.

2) The authors discovered that LADs reposition during progression through the cell cycle. It would have been interesting to know whether these changes have transcriptional consequences? One could perform RNA-SEQ experiments to discover if LAD occupancy results in transcriptional changes and choose a few genes to confirm the findings with RT-PCR. Is this the same for lamin B1, lamin B2, and lamin A/C occupied LADs? Analyze if there are any genomic features such as CTCF or transcription factor binding sites that correlate with the loss of LADs.

In the first part of this point, the reviewer suggests to look at transcriptional consequences of changes in NL interactions. To address this point, we require some measure of nascenttranscription during the cell cycle, which is not available in any of the studied cell lines. A potential experiment would be to map polymerase occupancy with pA-DamID / CUT&RUN or run-on transcription with any other method at the synchronized time points. However, this experiment is not trivial and we feel that this goes beyond the scope of this manuscript, which focuses on the development of pA-DamID and the m6A-Tracer with a proof-of-principle example of NL binding dynamics during the cell cycle.

In the second part of this point, the reviewer asks whether changes in NL binding correlate with genomic features such as CTCF binding sites or transcription factor binding sites. In the manuscript, we already include correlations with various active features (active gene density / replication timing) (Fig. 3E-G, 4C-E), that generally correlate well with transcription factor binding. We have added CTCF peaks as comparison (Fig. S7F).

3) The authors state that using H3K27me3/H3K9me3 in pa-DamID showed no enrichment. This is surprising considering that both modifications are enriched in heterochromatin and at the nuclear periphery. It appears that the peripheral enrichment is masked by the larger overall internal pool. The authors should discuss this observation and comment on the sensitivity of the method to detect local enrichment versus the global levels of a protein or modification in pa-DamID.

We believe that H3K27me3 and H3K9me3 histone modifications show the expected pattern in their distribution in the nucleus. However, due to the peripheral mask slightly extending beyond the cell boundaries, the calculated peripheral enrichment is underestimated. This has been better described in the figure legend.There is a small enrichment at the nuclear periphery compared to diffuse Dam and untargeted pA-Dam (Fig. 1B/1C/1F). To further support the pA-DamID data quality of these histone modifications, we have added a comparison with ENCODE ChIP-seq data tracks in K562 cells (Fig. S3C).

**Minor points:**

Figure 1: Change colors for Figure 1F and Figure 2D. The colors are hard to discern.

Figure 2B: Please mark which antibody was used for this analysis.

Figure 2C: Please also overlay data from pA-DamID lamin A/C experiments.

Figure 4: Please mention which antibody was used for the pA-DamID experiments used to generate this dataset.

Figure 5: Please mention which antibody was used for the pA-DamID experiments used to generate this dataset.

Figure S5 C and D: Please mention which antibody was used for the pA-DamID experiments.

We have made edits to address the minor comments above. However, we do not have Lamin A/C data in HAP-1 and K562 cells to add to Fig. 2C.

Reviewer #1 (Significance (Required)):

The major contribution of this manuscript is the description of an improved method to map LADs. This is a valuable contribution. By using this new method, the findings of this paper provide some new insight in LAD dynamics throughout the cell cycle although the experiments are largely phenomenological. This is a technically sound study.

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

The paper describes a new method for detecting Lamin associated DNA domains, which allows better time resolution than classical DamId. It is a good idea and its functionality is demonstrated in tissue culture cells. There are minor insights but it is important that we advance the field with new and better technologies, thus this version amply suffices to give evidence of that.

We thank the reviewer for the positive feedback.

Reviewer #2 (Significance (Required)):

The audience is all persons working on chromatin organization in the nucleus, which is a large audience. The data are clear as they basically are proof of principle for a new technique. There is nothing major to request as revision. They might cite papers on damID in worms and tissue specific applications of this in living organisms, as this is likely to be the situation that is most interesting in the long run. The resolution (in bp) would be interesting to know and validate.

We have extended the discussion on new applications of pA-DamID.

We now compare data quality and resolution between DamID and pA-DamID, focusing on the mapping of NL interactions (Fig. S4D-E).These plots indicate similar data quality and resolution between the two methods.

I have no other major revisions to request.

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

In the manuscript by Schaik et al (Van Steensel laboratory) the authors describe a very clever approach to identifying Lamina Associated Domains (LADs) using the principles of the 'cut-n-run' strategy. Specifically, they engineer the Dam methyltransferase used in canonical DamID in frame with a protein A moiety capable of interacting with an antibody (in this case lamins--B1, B2 and A/C). After permeabilization, cells are incubated with antibodies, then pA-Dam purified protein--for a brief time window--is added to mark associated DNA with GmATC. This technique is a valuable contribution to the field, particularly since, as the authors point out,an advantage of pA-DamID is that the labeled DNA can also be visualized in situ using the m6A-Tracer, before this DNA is sequenced. This allows for validation of findings and is highly amenable to cell sorting technologies. In addition, this technology allows for a time-resolved measure of LADs not currently available by standard DamID. The authors apply this technology to four different cell types. They noted that the 'maps' generated by the is technology differed from canonical DamID at very specific regions (small LADs in very localized regions) . They then embark on a series of experiments to show that these differences arise from cell cycle -related differences that are differentially picked up by the methods--with the pA-DamID allowing for dissection of more discrete cell cycle stages/configurations. In general they find an initial preference for sub-telomeric LADs to associate with the nuclear lamina fist, then more centromeric. There is some data suggesting loss/gain of LADs in specific regions/with specific features. The manuscript is well written and the data well presented. However, there are some points that need to be addressed . Overall, there is some oversimplification or omission of previous data in the field, a lack of clarity in how some of the data was interpreted, and some areas where clarification and/or additional analyses would be helpful. I sincerely hope the authors find the following critiques to be useful. Thank you for the opportunity to review your very nice work.

We thank the reviewer for the constructive and very detailed comments, these have been extremely helpful in improving the manuscript.

**Introduction:**

**Microscopy studies found that telomeres are enriched near the NL in early G1 phase, leading to the hypothesis that telomeres may assist in NL reassembly onto chromatin [13].**

● There have been numerous studies identifying the timing and disposition of INM proteins and Lamins at m the end of mitosis (during NE reformation). Why are you citing just this one? (e.g. ​Thomas Dechat et al., 2004; T. Dechat et al., 2000; Ellenberg et al., 1997; Haraguchi et al., 2001​)

We have expanded the introduction to better cover previous work on the reforming NL (paragraph 2) and initial genomic interactions with the NL (paragraph 3).

**Furthermore, during S-phase B-type lamins have been found to transiently overlap with replication foci in the nuclear interior, at least in some cell types [20]**

● While, technically, this has indeed been reported, this study is from 1994 and has not been repeated. The cells used in this study (3T3 fibroblasts) are widely used and others have not noted this phenomenon. Soften this.

**Other studies have indicated that lamins are important for DNA replication [reviewed in 21].**

● Likewise, direct roles for lamins in replication are controversial (acknowledged in the small section of the cited review on the role of lamins in replication).

● Perhaps combine the two sentences above to soften the implication that this is a "known" role of B-type lamins. e.g. "A handful of studies have implicated a role for B-type lamins in replication, but the direct role of the lamina in this process remains unclear. Nonetheless, ......"

This is a very good suggestion by the reviewer. We agree that literature has been controversial and should be approached with care. We have followed the advice and changed this.

**Results:**

**So far, the cell cycle dynamics of genome - NL interactions have primarily been studied by microscopy. While these studies have been highly informative, they were often limited to a ​few selected loci.​**

● Please cite your own study (Kind et al.) and other recent papers (Luperchio et al.-​https://www.biorxiv.org/content/10.1101/481598v1​;; Zhang et al., Nature-​https://www.nature.com/articles/s41586-019-1778-y​;) in which they were either 1) not limited to a few selected loci and/or 2) not microscopy-directed studies? There is an argument to be made here for the resolution (time and b.p.) you have achieved through your studies that these studies did not.

To our knowledge, there have been no high-throughput microscopy studies of many individual loci performed studying this. Microscopy has been performed of collective sequences (i.e. all LADs (Kind, 2013 and indeed Luperchio, 2018)), which provide additional insights but lack sequence information in the images. We have expanded the introduction to better acknowledge these microscopy studies that are not limited to single loci.We feel that observations on LAD domain clustering (Luperchio) and B compartment formation (Zhang) are better suited for the Discussion, given that these observations are not directly related to genome – NL contact dynamics. We already discussed B compartment formation in the discussion, but now also include the observed LAD domain clustering. Also, we have discussed data resolution in more detail in the results (see reviewer #2).

How does this data correlate with TSA-seq, another antibody-based method developed by the Belmont lab, but collaboratively developed for use in identifying LADs (ie Dam alternative) with the Van Steensel group?​ I can imagine there are numerous advantages to this approach (radius of "labeling" being one).

TSA-seq provides a different perspective on genome – NL interactions, given its distance dependence rather than contact. We have added a comparison with TSA-seq to the Discussion.

**When Dam-Lamin B1 is expressed in vivo for 5-25 hours during interphase, LADs that interact with the NL become progressively labeled, eventually resulting in a layer of labeled chromatin of up to ~1 μm thick [8]. This is because LADs are in dynamic contact with the NL. We expected that in pA-DamID this layer would be thinner, because the NL-tethered Dam is only activated for 30 minutes. In addition, permeabilization depletes small molecules including ATP and thus prevents active DNA remodeling in the nucleus [26]. Indeed, pA-DamID yields a m6A layer that is ~2.5 fold thinner than the layer in cells that express Dam-Lamin B1 in vivo (Fig. S2A-C). This is not an artifact due to collapse of chromatin onto the NL caused by the permeabilization, because permeabilization of cells expressing Dam-Lamin B1 in vivo did not significantly reduce the thickness of the m6A layer compared to directly fixed cells (Fig. S2C). The thin layer of labeled DNA obtained by pA-DamID points to an improved temporal resolution of pA-DamID compared to conventional DamID.**

● I think this requires a bit more care. Your previous work clearly demonstrates LADs are dynamic. Others in the field have shown that these domains are also constrained within the larger sub-chromosomal compartment (self-interaction) of LADs (e.g. Luperchio 2018) within a chromosome. So, this is truly a temporal "snapshot" that may miss some regions of LADs that are less directly (or more dynamically) associated with the lamina, but still compartmentalized into the larger LAD sub-chromosomal compartment. It is unclear if the treatment used for this study perturbs these LAD-lamina​ dynamic​ interactions--one can imagine that the LADs are much less mobile generally under the protocol described in your supplemental information. In other words, ​LADs don't collapse, nor do they behave in the same way they would after permeabilization​. The technique has compromised some of that --which is actually fine for most of the purposes in this manuscript, but this needs to be discussed.

As the reviewer points out, there are fundamental differences between DamID and pA-DamID in their m6A deposition that should be clear from the text. We elaborated on this in the comparison between pA-DamID and DamID.

● In addtion, imaging data showing dam-LaminB1/2 plus m6A-tracer is missing (figure S2). This should be included. Is the intensity of the "tracer" similar between conditions? If so, were the exposures kept constant in all images? This is important since the decay rate is highly related to intensity of signal.

We are afraid that this figure has been misinterpreted. We have changed the figure labels and legend to explain it better. The HT1080 Dam-Lamin B1 clonal cells (new clone kindly supplied by Jop Kind) still showed significant variation in m6A-Tracer intensity per cell, suggesting different expression levels of Dam-Lamin B1. To create optimal images for halfway decay estimation, laser settings were changed between images. This has now been mentioned more clearly in the methods.

**In some cell types, especially in​ HCT116 and ​hTERT-RPE​ cells, we noted local discrepancies between the two methods (Fig. 2A,bottom panel). These differences involve mostly regions with low signals in DamID that have higher signals in pA-DamID. However, such differences are not obvious in HAP-1 and K562 cells.**

● Only HCT116 data is shown in the indicated figure. hTERT-RPE cells are shown in the accompanying supplemental figure and use a different antibody (lamin B2) as the target for the pA-Dam.

We have changed the pointer to include the supplementary figure.

(See reviewer #1 for a similar comment.)We agree that the comparison between Lamin B1 DamID and Lamin B2 pA-DamID in hTERT-RPE cells leads to sense of incompleteness and confusion. We suggest to generate Lamin B1 pA-DamID data in hTERT-RPE cells to solve this – provided that the current Corona virus shutdown will not prevent us from doing this experiment.

This brings up another point: the data (log2 ratio schema) shown in figure 2 is for HCT116 lamin B1 pA-Dam. Yet, the subsequent studies for transient/building interactions during G1 and into S (Figure 3) are done in hTERT-RPE cells using lamin B2. To be consistent, data from lamin B2 should be used in both figures (it seems lamin B2 data is available for all cell types). The comparison of Dam-Lamin B1 can be addressed in the Venn overlays (as they are now) and in the supplements. The hTERT-RPE data should be in Figure 2 since it is followed up on in the subsequent figure (ie it fails to meet the definition of being relegated to 'supplemental' data).

As written in the response above, we suggest to generate Lamin B1 pA-DamID data in hTERT-RPE data.This will allow us to make a more consistent story and address these comments.

**suggesting that the separation of LADs and inter-LADs becomes progressively more pronounced after mitosis. Nevertheless....**

● This is overstated, especially given the previously mentioned work (Luperchio, Zhang). More accurate to say LADs ​association with the nuclear lamina becomes more pronounced​. LADs (predominantly B-compartment) and inter-LADs (predominantly A-compartment) show much earlier separation from each other. This may be distinct from association with the lamina. This is an important distinction as it may lead to different hypotheses regarding mechanisms of LAD targeting/association with the lamina.

We agree that this is an overinterpretation of our data. We have changed the phrasing to make it more accurate.

**Progression from prometaphase to late telophase in HeLa cells takes about 1 hour [33], suggesting that this timepoint captures the initial interactions with the reforming NL. Remarkably, the majority of these interactions is shared with later time points, indicating that most LADs can interact with the NL throughout interphase and are defined (and positioned at the NL) very soon after mitosis.**

● There is wide variability in this number, some cells rapidly exit, others take significantly longer. This number is an average (and, for what it's worth, based on a very compromised cancer cell line). The "interactions' mapped are likely reflecting the ensembe measurements of the many cells that have transited into G1. Also, this statement seemingly directly contradicts the premise of many of your following data/interpretations of a sort of step-wise wave or prefered interactions from telomere proximal toward centromeric regions. This also disagrees with your previous work (Kind et al) and more recent work regarding positioning to the NL very soon after mitosis. Again, this is BULK (many cells of a continuum of configurations) versus single cell observations. This is overstated.

We felt that there was a need to explain why we interpret the 1h time point as the initial interactions with the NL and included this reference, but the reviewer is correct that this number can vary greatly between cell types and conditions. We have removed the reference and now include FACS and imaging data supporting this claim directly.

We have changed the phrasing of these results to make our interpretation clearer.

**We next looked into characteristics of the dynamic LADs. At early time points, LADs with decreasing interactions do not have lower pA-DamID scores than stable LADs, suggesting that their ​detachment from the NL is not simply due to weak initial ​binding**

● The methods used here are dynamic proximity measures. Words like "binding" and "attachment" should be avoided (use interacting, associated, etc )

Good point. We have replaced all occurrences of these words.

**LAD dynamics are linked to telomere distance and LAD size in multiple cell types**

● Perhaps I am missing something, but I find relatively little data showing centromere-proximal LADs across cell cycle stages (referring here to Log2 ratio plots similar to what is shown for telomere-proximal LADs, Supplemental figure 6 is the only place where this is obvious.).

To better illustrate the inverse dynamics of telomeres and centromeres in hTERT-RPE cells, we have changed Fig. 3B to a full chromosome overview.

● In addtion, it seems to me that you are arguing in this and the preceding section for the following parameters: intensity of the LAD region. ie small, telomere-proximal, more euchromatic, AND less "intensely" associated.

● What is a "small" LAD? 100 kb or less? In Figure 2 (HCT1016, log 2 ratios), the original observation that leads into a discovery of changing NL associations through the cell cycle, the LAD that changes appears to be at least average size. Perhaps a "small" LAD adjacent to an "average" LAD. Nor do the signals appear to be all that low. There are regions within this sub-chromosomal plot that do appear to be "small" "low intensity" LADs. I am uncertain what parameters are defining these attributes. Are the cut-offs the same between cell types (ie is there a rule here?).

We do not set any cut-offs for any features that we compare with. We took the strategy to define stable and dynamics LADs (Fig. 3C) and ask whether there are differences in feature distributions, including LAD size, replication timing and other features. As you can see in Fig. 3E, LADs with decreasing NL interaction are smaller than stable or increasing LADs. This strategy is consistent between cell lines. To assist the reader in following our reasoning, we have added LAD domains and their differential status to Fig. 3B.

● The rules outlined above seem to break down across the different cell types. In particular, the number of active genes per Mb seems to have very little correlation overall with LADs that change. In addition, it is very unclear if "LAD size" is really a readout of both size AND intensity of interactions (understanding that this is not necessarily a direct quantitative measure of interactions).

This comment reflects our reasoning why we added a comparison between cell types in Fig. 4. Indeed, we find no general trend that active gene density correlates with LADs with decreasing NL interactions in every cell type. In contrast, LADs with decreasing NL interactions are consistently close to telomeres and smaller in size than stable or increasing LADs. We made it clearer that LAD size solely reflects the genomic size in basepairs.

**Correlation of pA- determined LADs that change into G1/S with B-compartment sub-types**

● There is certainly Hi-C data on most (all?) of the cell types analyzed in this manuscript. It would be very useful for the authors to parse out how the gain/loss LADs correlate with the B1, B2. A1, A2 (etc) compartment classifications. This may help to address the point above.

We have now included a comparison with Hi-C sub-compartments (Fig. 4F).

**Nucleosomal pattern of pA-DamID digestion/amplification (figure S3)**

● Onset of apoptosis needs to be ruled out. The nucleosomal (laddering) pattern could be due to DNA getting cleaved through programmed cell death pathways after permeabilization. These fragments could easily be amplified by the subsequent DamID protocol.

Amplification of apoptotic fragments, if present, is visible in DamID assays using the negative controls. Every library preparation, we include one or more negative controls in which we omit DpnI. If apoptotic fragments are present in this negative control, these can ligate to the DamID adapter and result in amplification, which we consistently do notsee. We have added a supplementary figure that shows this (Fig. S3A).

**Definition of 'bulk' assays**

● All of the assays were done in bulk. Some were synchronized, some were not. This is important since the implication is that anything not 'bulk' is single-cell. Throughout the manuscript and in the figures, please refer to the conditions as 'synchronized' versus 'unsynchronized'

The reviewer is correct that our terminology is wrong. We changed all occurrences of “bulk” to “unsynchronized”.

**Much of supplemental Figure 6 should be in a main figure**

● It is puzzling why the first (and most easily seen/interpreted) description of LAD organization relative to telomeres/centromeres after exit from mitosis is relegated to supplemental figures. It is a foundational experiment(s) for the paper.

We have changed the zoomed-in Fig. 3B with a chromosome overview that better captures this main observation. We see the remainder of Fig. S6 as technical controls and details of the experiment that are useful to include but not necessary as main figure.

**pA-Dam is possibly influenced by cell-cycle related chromatin accessibility (particularly at mitotic exit)**

● During the transition from mitosis to early G1, there are dynamic changes to chromatin state that are directly coupled to the cell cycle. A recent report, for instance, highlights that interactions of antibodies (or other proteins) with H3K9me2/3 modifications is likely influenced by phosphorylation of histone tails. The dynamics of histone modification/chromatin state possibly occluding or interfering with the interpretation of the results must be discussed.

Similar to DamID, pA-DamID utilizes a Dam-control to measure DNA accessibility and control for this. We show that a change in pA-DamID score is due to changes in NL reads, while the Dam reads do not change (Fig. S6F). In other words, we find no evidence that a change in chromatin state impacts the accessibility as measured by our Dam-control and thereby influences the results. We now repeat this observation in the discussion.

Reviewer #3 (Significance (Required)):

N/A

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Referee #3

Evidence, reproducibility and clarity

In the manuscript by Schaik et al (Van Steensel laboratory) the authors describe a very clever approach to identifying Lamina Associated Domains (LADs) using the principles of the 'cut-n-run' strategy. Specifically, they engineer the Dam methyltransferase used in canonical DamID in frame with a protein A moiety capable of interacting with an antibody (in this case lamins--B1, B2 and A/C). After permeabilization, cells are incubated with antibodies, then pA-Dam purified protein--for a brief time window--is added to mark associated DNA with GmATC. This technique is a valuable contribution to the field, particularly since, as the authors point out,an advantage of pA-DamID is that the labeled DNA can also be visualized in situ using the m6A-Tracer, before this DNA is sequenced. This allows for validation of findings and is highly amenable to cell sorting technologies. In addition, this technology allows for a time-resolved measure of LADs not currently available by standard DamID. The authors apply this technology to four different cell types. They noted that the 'maps' generated by the is technology differed from canonical DamID at very specific regions (small LADs in very localized regions) . They then embark on a series of experiments to show that these differences arise from cell cycle -related differences that are differentially picked up by the methods--with the pA-DamID allowing for dissection of more discrete cell cycle stages/configurations. In general they find an initial preference for sub-telomeric LADs to associate with the nuclear lamina fist, then more centromeric. There is some data suggesting loss/gain of LADs in specific regions/with specific features. The manuscript is well written and the data well presented. However, there are some points that need to be addressed . Overall, there is some oversimplification or omission of previous data in the field, a lack of clarity in how some of the data was interpreted, and some areas where clarification and/or additional analyses would be helpful. I sincerely hope the authors find the following critiques to be useful. Thank you for the opportunity to review your very nice work.

Introduction:

Microscopy studies found that telomeres are enriched near the NL in early G1 phase, leading to the hypothesis that telomeres may assist in NL reassembly onto chromatin [13].

● There have been numerous studies identifying the timing and disposition of INM proteins and Lamins at m the end of mitosis (during NE reformation). Why are you citing just this one? (e.g. ​Thomas Dechat et al., 2004; T. Dechat et al., 2000; Ellenberg et al., 1997; Haraguchi et al., 2001​)

Furthermore, during S-phase B-type lamins have been found to transiently overlap with replication foci in the nuclear interior, at least in some cell types [20]

● While, technically, this has indeed been reported, this study is from 1994 and has not been repeated. The cells used in this study (3T3 fibroblasts) are widely used and others have not noted this phenomenon. Soften this.

Other studies have indicated that lamins are important for DNA replication [reviewed in 21].

● Likewise, direct roles for lamins in replication are controversial (acknowledged in the small section of the cited review on the role of lamins in replication).

● Perhaps combine the two sentences above to soften the implication that this is a "known" role of B-type lamins. e.g. "A handful of studies have implicated a role for B-type lamins in replication, but the direct role of the lamina in this process remains unclear. Nonetheless, ......"

Results:

So far, the cell cycle dynamics of genome - NL interactions have primarily been studied by microscopy. While these studies have been highly informative, they were often limited to a ​few selected loci.​

● Please cite your own study (Kind et al.) and other recent papers (Luperchio et al.-​https://www.biorxiv.org/content/10.1101/481598v1​; Zhang et al., Nature-​https://www.nature.com/articles/s41586-019-1778-y​) in which they were either 1) not limited to a few selected loci and/or 2) not microscopy-directed studies? There is an argument to be made here for the resolution (time and b.p.) you have achieved through your studies that these studies did not.

● How does this data correlate with TSA-seq, another antibody-based method developed by the Belmont lab, but collaboratively developed for use in identifying LADs (ie Dam alternative) with the Van Steensel group?​ I can imagine there are numerous advantages to this approach (radius of "labeling" being one).

When Dam-Lamin B1 is expressed in vivo for 5-25 hours during interphase, LADs that interact with the NL become progressively labeled, eventually resulting in a layer of labeled chromatin of up to ~1 μm thick [8]. This is because LADs are in dynamic contact with the NL. We expected that in pA-DamID this layer would be thinner, because the NL-tethered Dam is only activated for 30 minutes. In addition, permeabilization depletes small molecules including ATP and thus prevents active DNA remodeling in the nucleus [26]. Indeed, pA-DamID yields a m6A layer that is ~2.5 fold thinner than the layer in cells that express Dam-Lamin B1 in vivo (Fig. S2A-C). This is not an artifact due to collapse of chromatin onto the NL caused by the permeabilization, because permeabilization of cells expressing Dam-Lamin B1 in vivo did not significantly reduce the thickness of the m6A layer compared to directly fixed cells (Fig. S2C). The thin layer of labeled DNA obtained by pA-DamID points to an improved temporal resolution of pA-DamID compared to conventional DamID.

● I think this requires a bit more care. Your previous work clearly demonstrates LADs are dynamic. Others in the field have shown that these domains are also constrained within the larger sub-chromosomal compartment (self-interaction) of LADs (e.g. Luperchio 2018) within a chromosome. So, this is truly a temporal "snapshot" that may miss some regions of LADs that are less directly (or more dynamically) associated with the lamina, but still compartmentalized into the larger LAD sub-chromosomal compartment. It is unclear if the treatment used for this study perturbs these LAD-lamina​ dynamic​ interactions--one can imagine that the LADs are much less mobile generally under the protocol described in your supplemental information. In other words, ​LADs don't collapse, nor do they behave in the same way they would after permeabilization​. The technique has compromised some of that --which is actually fine for most of the purposes in this manuscript, but this needs to be discussed.

● In addtion, imaging data showing dam-LaminB1/2 plus m6A-tracer is missing (figure S2). This should be included. Is the intensity of the "tracer" similar between conditions? If so, were the exposures kept constant in all images? This is important since the decay rate is highly related to intensity of signal.

In some cell types, especially in​ HCT116 and ​hTERT-RPE​ cells, we noted local discrepancies between the two methods (Fig. 2A,bottom panel). These differences involve mostly regions with low signals in DamID that have higher signals in pA-DamID. However, such differences are not obvious in HAP-1 and K562 cells.

● Only HCT116 data is shown in the indicated figure. hTERT-RPE cells are shown in the accompanying supplemental figure and use a different antibody (lamin B2) as the target for the pA-Dam.

● This brings up another point: the data (log2 ratio schema) shown in figure 2 is for HCT116 lamin B1 pA-Dam. Yet, the subsequent studies for transient/building interactions during G1 and into S (Figure 3) are done in hTERT-RPE cells using lamin B2. To be consistent, data from lamin B2 should be used in both figures (it seems lamin B2 data is available for all cell types). The comparison of Dam-Lamin B1 can be addressed in the Venn overlays (as they are now) and in the supplements. The hTERT-RPE data should be in Figure 2 since it is followed up on in the subsequent figure (ie it fails to meet the definition of being relegated to 'supplemental' data).

suggesting that the separation of LADs and inter-LADs becomes progressively more pronounced after mitosis. Nevertheless....

● This is overstated, especially given the previously mentioned work (Luperchio, Zhang). More accurate to say LADs ​association with the nuclear lamina becomes more pronounced​. LADs (predominantly B-compartment) and inter-LADs (predominantly A-compartment) show much earlier separation from each other. This may be distinct from association with the lamina. This is an important distinction as it may lead to different hypotheses regarding mechanisms of LAD targeting/association with the lamina.

Progression from prometaphase to late telophase in HeLa cells takes about 1 hour [33], suggesting that this timepoint captures the initial interactions with the reforming NL. Remarkably, the majority of these interactions is shared with later time points, indicating that most LADs can interact with the NL throughout interphase and are defined (and positioned at the NL) very soon after mitosis.

● There is wide variability in this number, some cells rapidly exit, others take significantly longer. This number is an average (and, for what it's worth, based on a very compromised cancer cell line). The "interactions' mapped are likely reflecting the ensembe measurements of the many cells that have transited into G1. Also, this statement seemingly directly contradicts the premise of many of your following data/interpretations of a sort of step-wise wave or prefered interactions from telomere proximal toward centromeric regions. This also disagrees with your previous work (Kind et al) and more recent work regarding positioning to the NL very soon after mitosis. Again, this is BULK (many cells of a continuum of configurations) versus single cell observations. This is overstated.

We next looked into characteristics of the dynamic LADs. At early time points, LADs with decreasing interactions do not have lower pA-DamID scores than stable LADs, suggesting that their ​detachment from the NL is not simply due to weak initial ​binding

● The methods used here are dynamic proximity measures. Words like "binding" and "attachment" should be avoided (use interacting, associated, etc )

LAD dynamics are linked to telomere distance and LAD size in multiple cell types

● Perhaps I am missing something, but I find relatively little data showing centromere-proximal LADs across cell cycle stages (referring here to Log2 ratio plots similar to what is shown for telomere-proximal LADs, Supplemental figure 6 is the only place where this is obvious.).

● In addtion, it seems to me that you are arguing in this and the preceding section for the following parameters: intensity of the LAD region. ie small, telomere-proximal, more euchromatic, AND less "intensely" associated.

● What is a "small" LAD? 100 kb or less? In Figure 2 (HCT1016, log 2 ratios), the original observation that leads into a discovery of changing NL associations through the cell cycle, the LAD that changes appears to be at least average size. Perhaps a "small" LAD adjacent to an "average" LAD. Nor do the signals appear to be all that low. There are regions within this sub-chromosomal plot that do appear to be "small" "low intensity" LADs. I am uncertain what parameters are defining these attributes. Are the cut-offs the same between cell types (ie is there a rule here?).

● The rules outlined above seem to break down across the different cell types. In particular, the number of active genes per Mb seems to have very little correlation overall with LADs that change. In addition, it is very unclear if "LAD size" is really a readout of both size AND intensity of interactions (understanding that this is not necessarily a direct quantitative measure of interactions).

Correlation of pA- determined LADs that change into G1/S with B-compartment sub-types

● There is certainly Hi-C data on most (all?) of the cell types analyzed in this manuscript. It would be very useful for the authors to parse out how the gain/loss LADs correlate with the B1, B2. A1, A2 (etc) compartment classifications. This may help to address the point above.

Nucleosomal pattern of pA-DamID digestion/amplification (figure S3)

● Onset of apoptosis needs to be ruled out. The nucleosomal (laddering) pattern could be due to DNA getting cleaved through programmed cell death pathways after permeabilization. These fragments could easily be amplified by the subsequent DamID protocol.

Definition of 'bulk' assays

● All of the assays were done in bulk. Some were synchronized, some were not. This is important since the implication is that anything not 'bulk' is single-cell. Throughout the manuscript and in the figures, please refer to the conditions as 'synchronized' versus 'unsynchronized'

Much of supplemental Figure 6 should be in a main figure

● It is puzzling why the first (and most easily seen/interpreted) description of LAD organization relative to telomeres/centromeres after exit from mitosis is relegated to supplemental figures. It is a foundational experiment(s) for the paper.

pA-Dam is possibly influenced by cell-cycle related chromatin accessibility (particularly at mitotic exit)

● During the transition from mitosis to early G1, there are dynamic changes to chromatin state that are directly coupled to the cell cycle. A recent report, for instance, highlights that interactions of antibodies (or other proteins) with H3K9me2/3 modifications is likely influenced by phosphorylation of histone tails. The dynamics of histone modification/chromatin state possibly occluding or interfering with the interpretation of the results must be discussed.

Significance

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** The work reports finding a molecular genetic basis for individual differences in behavior in different strains of outbred mice, even including individual behavioral differences between mice of the same inbred genetically isogenic strain. The authors were able to measure copy numbers for the tandemly repeated intronic snoRNA clusters SNORD115 and SNORD116 and found correlation with measures of anxiety in open-field test and elevated plus maze. Expression data for previously proposed targets of these snoRNAs are also provided.

We note that this description represents only part of the achievements in our paper. The key of our paper is that we have not only used "different strains of outbred mice", but in addition one very different species of mouse (Apodemus) and a Guinea pig species. We believe that the test in very different species with very different genetic backgrounds is the crucial proof for the specificity of the effect.

**Major comments:** 1.The techniques to measure copy numbers are challenging and the authors' conclusion that ddPCR was their method of choice is convincing. They were able to obtain limited optical mapping (Bionano zephyr) data, only for SNORD116 and only in mouse, but these data are useful to corroborate those obtained with ddPCR. 2.Figure 3 reports single copy numbers for individuals that are presumably heterozygous. Do we have to assume that the numbers reported represent the larger alleles since the ddPCR method does not allow to distinguish two different size alleles, as was shown for optical mapping?

The numbers are derived from the whole genome DNA, i.e. represent the cumulative copy number of both alleles. We have updated the text to make this clear.

3.The analyses reported do not take into account the specific parental origin of the alleles used in the regression analyses. Since PWSCR-specific SNORDs are only expressed from the paternal chromosomes, this generates some uncertainty about the whole dataset.

We had explained why it is not possible to distinguish the two alleles with the current technology. Hence, it is evidently also not possible to determine which allele comes from the paternal side. In fact, given that we showed that copy numbers can change every generation, even the knowledge of which chromosome is the paternal one would not predict its copy number. Accordingly, it lies in the nature of the whole phenomenon that this uncertainty is given. It is therefore just the more surprising that we still can observe correlations that are much stronger than has been shown for natural alleles of any genetic locus implicated in behavioral traits so far.

4.Lines 353-365: The ankrd11 exon-specific RNAseq data are confusing and too preliminary. More work needs to be done to resolve the splice variants in this region and their relationship to SNORD116 copy numbers. Alternatively lines 356-361 should be deleted.

We have included the data to show that the mechanism must be different from the one that is seen for Htr2c. This difference is clearly documented and we should therefore like to retain it. What is missing is to show the actual mechanism by which SNORD116 causes the alternative splicing. This will require more biochemical approaches that go beyond the current study.

5.In all tested rodents, higher SNORD copy number was correlated with higher relative anxiety score. In the human samples, however, higher anxiety scores were associated with lower copy numbers. These apparently contradictory results are not mentioned in the abstract, and are not satisfactory explained in the text.

We have decided to leave the human data out from the current manuscript. First, the behavioral tests for the rodents are indeed not directly comparable with the questionnaire scores in humans. Second, in human genetics one usually asks the results to be confirmed in an independent study. hence, we plan to extend the human data, but to present them eventually in a follow-up paper.

6.Extension to other species would be desirable but was limited by availability of genomic data: Results are presented for wood mouse only for SNORD115 and for the guinea pig for SNORD116.

Given that we show a strong correlation between SNORD115 and SNORD116 copy numbers for those species where the information is available for both loci, we do not think that this is a major limitation of our study.

**Minor comments:** 1.The authors present skull shape data related to SNORD116 copy numbers, but fail to consider how these data are relevant to the craniofacial abnormalities reported in an ankrd11 mutation. Barbaric et al (2008) reported a dominant ENU- induced mutation caused shortened snouts, wider skull, deformed nasal bones, reduced BMD, reduced osteoblast activity and reduced leptin levels. This phenotype was traced to a heterozygous missense mutation (conserved glutamate to lysine change) in an HDAC binding site. They postulated that the mutation fails to recruit HDACs to a transcription complex and to inhibit hormone-receptor activated gene transcription. What is the postulated link between this mechanism and the here reported skull shape data correlated with SNORD copy number variation?

The described missense mutation is located in the differentially spliced exon, i.e. a direct functional link is given. We have added this information to the text and compared the direct phenotypic effects from their study and our study.

2.The observed co-variation of copy numbers between the two SNORD clusters could indicate a duplication involving the entire region, This could be tested by determining the dosage of IPW, UBE3a and Snrpn genes.

While this is a theoretical possibility, it was not described in the literature before. Also, in our systematic survey of copy number variation in mouse populations (Pezer et al. 2015) we did not find a deviation of these genes from expected diploid copy number in any of the populations analysed.

3.Line 129 "the RNA coding region" and Line 148 "snoRNA coding parts" (and elsewhere) does seems correct, as by definition, this is non-coding RNA. The region they are referring to could be called the "processed C/D box snoRNA". The mechanism that generates these C/D box snoRNAs is well established: the "genes" are located in introns of host genes - and after transcription - the spliced out introns are exonucleolytically trimmed to the functional sizes. Both SNORD115 and 116 clusters are within a large transcript that originates from the SNRPN promoter of the paternal allele.

We adjusted the wording to make clear that we refer to the mature RNAs.

4.Figure 2 does not show data on skull shape as claimed in the legend.

We apologize - this was a carry-over from an older version of this figure. The skull shape analysis had been moved to a new figure in the current version of the manuscript.

5.S1 Figure: Snprn should be Snrpn

Thank you for spotting the error - we have corrected this

Reviewer #1 (Significance (Required)): This provocative work proposes the regulation of behavioral variance by dosage changes of a regulatory RNA. The dosage changes are apparently caused by dynamic and frequent alteration in copy number. This is a novel concept and worthy of publicizing. Extensive data documentation is provided for others to analyze and possibly replicate. The data potentially throw light on the function of the tandemly repeated imprinted snoRNA clusters in the PWS critical region. Novel aspects of this work include the discovery of copy number variation of these snoRNAs; and validation of a target of SNORD116: Ankrd11 is one of many potential targets of SNORD116 that was previously computationally predicted, this paper reports experimental evidence for this interaction. The work would be of interest to researchers in behavioral evolution, non-coding RNA function, epigenetics and overall genome evolution. Define your field of expertise with a few keyword: Molecular genetic disorders, Prader-Willi syndrome, mouse models

Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Summary** Maryam Keshavarz et al. aimed at seeking the molecular basis underlying individual behavioral variance within populations. Focusing on the Prader-Willi Syndrome (PWS) gene complex, which has been well recognized being associated with neurodevelopmental disorders, anxiety and metabolic issues, the authors found that the levels of PWS region's small nucleolar RNAs SNORD 115/116 of individual animals correlated with these individuals' behavioral test scores. The variations in transcript processing of some anxiety-associated target genes also revealed correlation with SNORD 115/116 copy numbers. Authors implicated that the copy numbers of SNORD 115/116 within PWS plausibly influenced behavioral variances among individuals. • Authors first validated that the droplet digital PCR (ddPCR) was suitable for quantifying variations in copy numbers of genomic clusters. Their ddPCR data showed confident correspondence with reads calculation of whole-genome-seq dataset. Also, ddPCR showed good replicability and congruent tissue-to-tissue similarity. • Authors found the ddPCR-measured SNORD copy numbers from several mice populations showed significant regression with SNORD RNA levels, respectively. Also, the anxiety profiling using Open Field Test and Elevated Plus Maze test indicated a significant regression between SNORD copy numbers and anxiety profiling scores, namely individual mouse with higher copy numbers received higher relative anxiety scores. Some other representative genes outside PWS, such as Sfi1 and Cwc22, failed to show such copy number-anxiety score regression. • Authors applied RNA-seq of individual mice with different SNORD 115/116 copy numbers and analyzed potential target gene regions. They found the level of alternative splice-resulted exon Vb of gene Htr2c, a serotonin receptor, was positively correlated with SNORD 115 copy number. Also, an alternative splicing product of gene Ankrd11, a chromatin receptor regulating GABA receptor, was found to positively correlated with SNORD 116 copy number. Positive correlation to SNORD copy numbers also occurred to some Htr2c and Ankrd downstream genes. • Authors used a landmark-based analysis to score mice craniofacial features and found the scores were in relationship with SNORD 116 copy numbers. • Authors also found significant regression between SNORD copy numbers and behavioral evaluations in other rodents. In humans, the Tridimensional Personality Questionnaire (TPQ) based evaluation also showed correlation with SNORD 115 and 116 copy numbers. **Major comments** The study mainly revealed important correlations between copy numbers of 2 small nucleolar RNAs and cognitive behavioral variance of different individual animal. Although very useful and important findings, the study did not provide any evidence about the causality between SNORD 115/116 and the observed behavioral phenotypes. For instance, • #1: the behavioral observations (i.e. anxiety) may not be merely regulated by the PWS gene complex.

It is already well understood that the respective behavioral observations have a polygenic basis. But our data show that the SNORD copy numbers act as major modulators of the behavior.

• #2: the paper did not show if manipulations on mouse SNORD 115/116 could affect target genes as well as the consequential behavioral phenotypes.

A direct interaction between SNORD115 and its target gene HTr2c has previously been shown in cell culture experiments. Further, we show that the commonly used inbred mouse strain C57Bl6 carries already different copy number alleles that would not be different from artificial manipulation of the copy number. There is a long tradition in mouse genetics to accept also spontaneous alleles as genetic proof, not only the alleles that were created by artificial intervention.

Further, as also pointed out in response to reviewer 1, in the absence of the possibility to do a direct genetic manipulation in a given genetic background, we use the comparative analysis between different genetic backgrounds to prove causality.

Reviewer #2 (Significance (Required)): Authors provided a potential molecular basis regulating the PWS region, which is a genomic imprinted gene complex and related to many neurodevelopmental diseases in mammals, including humans. Considerably cost-saving than whole-genome deep-seq, the application of droplet digital PCR on copy number (esp. in stretching regions) measurement can overcome some technical difficulties, for example, qPCR has limit in resolution when differentiating subtle variance in copy numbers; the Nanopore seq and current mapping algorithm show difficulties when placing the internal repeats also. Authors proposed SNORD copy number as a potential explanation to the individual-to-individual variance within the same species or even the same population.

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

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Reply to the reviewers

Reviewer #1:

(Evidence, reproducibility and clarity (Required)):

This study aims to develop tools for yeast researchers to automatically segment and classify yeast colonies. The machine learning method enables rapid classification compared to manual counting.

\*MAJOR CONCERNS:***

Please include additional details about the types of images that must be captured for segmentation and categorization. It is important to provide details of what level of magnification might be needed during image capture. We anticipate that providing clear protocols for altering thresholds to classify colonies might be one way to overcome this challenge

That’s correct. Details on image acquisition, such as the level of magnification, are important to obtain accurate results.

To address this, we provide a detailed protocol in our companion article on ProtocolExchange: https://protocolexchange.researchsquare.com/article/nprot-7305/v1

We have updated the manuscript to include this link.

While the program crops colonies and segments them accurately, there is no spatial information of where these colonies are located in the image. This loss of spatial information limits the ability to use this platform to identify colonies of interest following experiments such as a genetic screen.

In principle it would be possible to retain the location of each cropped colony in the form of (x,y) coordinates in pixels. This could be included in a future release. However, we doubt the utility of such information for a genetic screen, unless identification of a positive hit could be linked to robotic picking of the identified colony (which would certainly go beyond the scope of this work). In reality, researchers will pick positive hits manually anyways, making our pipeline superfluous for such an application. We emphasize that we have developed our pipeline for large-scale quantification of red/white color assays. Here the pipeline makes a huge difference, as compared to manual counting.

The inability to accurately recognize sectored colonies as sectored (rather than red) is a significant limitation to the usage of this program for quantitative assays. While differentiating between red and white colonies is useful, the conclusion by the authors about its value for quantitative assays is limited unless variegation can be accurately defined. The authors should either soften this conclusion or qualify what quantitative measurements might mean given the limitations of their classification program. This somewhat diminished our overall enthusiasm.

The reviewer correctly points out that our algorithm shows lower accuracy when differentiating between red and variegating colonies than when differentiating between white and non-white colonies (including red, variegating and pink). Given this observation, we initially focused on predicting white vs. non-white colonies with our tool. However, the output of our pipeline also includes more granular predictions of numbers of white, red, pink and variegating colonies. We therefore leave it up to the user to decide which level of granularity is more appropriate, taking into account the tradeoff between granularity and accuracy. In particular, we note that for the colonies we tested, splitting the non-white category of predictions into red, variegating and pink resulted in a decrease of sensitivity from 0.98 for the non-white category to 0.86-0.88 for the individual categories, while the corresponding specificity showed a smaller reduction from 1.0 to 0.97-0.98. Considering that a lack of any predictions for the red, pink, and variegating categories effectively prohibits the researcher from detecting them at all, even a reduced sensitivity may be better than nothing and therefore acceptable in this case. In order to make this clearer in the text, we provide a more detailed comparison of performance metrics between levels of prediction, which may help to guide the user’s decision.

This program must be benchmarked with other colony classifiers. Cell Profiler is an example of a popular yeast colony segmentation program. How does this machine learning based tool compare with other colony segmentation and categorization programs. One possibility is to include an additional figure that compares their program with clear benchmarks. The outcome of effort based on benchmarking is not as important since we believe it is useful to have many alternatives for yeast segmentation and categorization. We think this revision would be essential to the manuscript and would add significant value.

We have used other approaches and were not satisfied with the outcomes. Hence, we developed our own pipeline, specifically designed to accurately distinguish red from white colonies and quantify such assays at a large scale.

When using CellProfiler we could not reliably distinguish variegating, pink, and red colonies. White colonies show up in the Red, Blue and Green channel, Red colonies mainly in the Red Channel. Therefore, variegating, pink and red colonies can be distinguished from white by reduced Blue and Green values, which is indirect and caused several issues. One of the problems was reflection of the flash during image acquisition, giving two reflective white patches on each colony that differed in pixel size depending on the magnification and colony size. We tried to prevent reflection with a ‘tent’, which reduced but could not eliminate the reflection. Therefore, the MaxIntensity of the Green/Blue channel was always the same of each colony, impeding classification. Furthermore, most red/pink colonies had a slim white rim, which was sometimes bigger/smaller and the relative area of rim to colony depends on the colony size, which made it impossible to tell a bit variegating from red by the output values from CellProfiler.

If deemed useful by the editors, we will be happy to mention this in the manuscript. A systematic comparison with other classifiers seems to be a bit of an overkill though. As stated by this reviewer, the outcome of such comparison would not matter much. It is important that the community has several approaches to choose from, so that the best solution can be found for each specific application.

\*MINOR CONCERN***

The program currently saved cropped images of each segmented colony. This takes up a lot of storage space. It might be useful to provide an option to save or not save these cropped images. This flexibility will be valuable for users but does not detract from the major conclusions of the manuscript.

While we appreciate that the need to save individual images of cropped colonies may be a drawback for some users, in the current implementation it is not possible to avoid this step. One could imagine a scenario in which all cropped images were stored in RAM prior to classification rather than written to a computer’s disk; however, we believe that most users would have more limitations on the availability of RAM than on disk storage, therefore making this option also not feasible.

The authors have provided excellent examples of colonies they believe are red, white or sectored. More accurately defining a pink colony would be valuable for users of this program. How much of red is classified as pink by this program?

As the reviewer points out, it is difficult to give an objective definition of a pink colony. In this case, we relied exclusively on subjective expert annotations to define which colonies were pink (as well as for all other categories).

We acknowledge that this may introduce some error into the model, as there may be some overlap between red and pink colonies or between pink and variegating colonies; however, this problem also exists in the case of manual annotation. As shown in Figure 1d, for the colonies we tested, 4 out of a total of 55 colonies annotated as red by an expert were predicted as pink by our algorithm. We would like to emphasize that our pipeline alleviates biases between different researchers who would be annotating colony color manually, therefore improving reproducibility. Such biases could be subjective or objective, such as different monitors used to inspect the images.

Providing an example data set with the protocol would be helpful for users with limited Python experience. In combination with their protocol on Protocol exchange, this would serve as a valuable resource for novices in programming.

We agree with the reviewer’s suggestion and will be happy to provide an example dataset used in the manuscript. We will defer to the journal’s guidelines as to the best way to share these raw images.

One technical issue of the program is that the program tries to open all files in the specified folder even if they aren't jpg. This causes problems if there are additional or hidden files in the folder and the program cannot process the additional files.

We appreciate the reviewer pointing out this issue and have fixed it in a new version of the code.

Reviewer #1 (Significance (Required)):

This manuscript describes a machine learning approach to segment and categorize yeast colonies based on a red/white selection assay. The approach has been implemented using Python which makes this widely accessible to many researchers. Their detailed protocol on Protocol Exchange is a valuable resource which made it possible for us to evaluate its performance. The program meets its goals of reducing user time via manual counting. It is also reasonably accurate in discriminating between red and white colonies based on our initial tests. However, there are several important concerns that the authors will need to address before this manuscript can become a valuable resource for the yeast community. It is important to note that our framework is one where we have a great interest in quantitative yeast genetics but cannot evaluate the strengths and weakness of the computational approach. So much of the review is focussed on what would be needed to make this tool more user appropriate.

Reviewer #2

(Evidence, reproducibility and clarity (Required)):

\*Summary:***

Carl et al present an application of a deep learning-based image analysis able to segment and classify individual yeast colonies by their phenotype in a special plate. They evaluated the method and show that it provides the accuracy similar to the one achieved by experts' manual classification.

\*Major comments:***

The key conclusions are convincing. The evaluation is performed on 3 datasets showing different properties (strong presence of phenotype, almost lack of the phenotype, gradual change of the phenotype).

The claims are carefully formulated. The deep learning methodology (training, validation, using modern technologies such as transfer learning, Unet, augmentation) is carefully designed and carried out. The evaluation is sound. The limitations are discussed.

For a short paper as it's formulated currently, no additional experiments are necessary.

The methods are implemented and are available on GitHub.

However, I'd strongly recommend to share also the data used in the paper, to make possible the reproduction of the results as well as to be used as examples for future users.

As stated above, we agree with the reviewer’s suggestion and will be happy to provide an example dataset used in the manuscript. We will defer to the journal’s guidelines as to the best way to share these raw images.

No replicates are provided unfortunately. The manuscript would benefit from showing results from replicates, especially because they should be easily obtainable.

It is not clear to us to which experiment the reviewer is referring. All of the results presented in Figure 2 did include replicates, as detailed in the figure legend.

\*Minor comments:***

I'm not familiar with the state of the art to judge on whether prior studies are referenced.

The text and fitures are very clear and well formulated.

Reviewer #2 (Significance (Required)):

Despite the conceptual innovation is average, the method is well-developed and seems to be very useful for yeast analysis.

I'm not an expert in the application area to judge the state of the art. The carried out deep learning methodology is top notch.

The manuscript can be interesting and useful for experts using the described assay for yeast.

My expertise is in omics, image analysis, and machine learning.

Super. Clearly defined research focus, although a massive topic in terms of scope and scale. Some thoughts.

(1) Might not be a bad idea to revisit some of the traditional development economic works on rural to urban migration, although the push / pull factors you are identifying are much more regional or global in nature. Two good ones to look at are Todaro:

https://www-jstor-org.proxy.wm.edu/stable/1807860?seq=1#metadata_info_tab_contents

and Sir Arthur Lewis' Economic Development with Unlimited Supplies of Labour. Please let me know if you can't find a copy of his 1954 essay that earned him the Nobel Prize.

(2) I'm wondering a bit about which data science methodology is best to investigate further with regard to your research topic. Argument could be made for thinking about the application of a hierarchical bayesian model with the survey data you have identified. There are also arguments in favor or gravity type models with the CDR data you are considering.

(3) Migration patterns are such a big topic that it can be difficult to know where to start in terms of geospatial data, data science / machine learning methods / models etc…A common starting point for thinking about migration is in terms of push factors and/or pull factors, more traditionally within the context of rural to urban migration. The scope and scale of what you are investigating is of a much greater magnitude and complexity than these traditionally considered foundations from development economics. Additionally, thinking of migration of this magnitude in terms of only human movement and behavior, likely over simplifies the problem. For example, we could use CDR data to infer the origins and destinations of where people are coming from and where they wish to go as part of a transportation system model (even if it is by foot or mixed mode). I think you need something that seeks to accomplish far more than simply transport behavior and movement, because you also wish to understand the motivations behind why people choose to leave (push factors) as well as the reasons they are attracted to some location (pull factors). I think looking into agent based models as a data science method for describing, analyzing could also be a promising path forward. A simulation system, such as an ABM, may incorporate different model that address factors at the origin as well as at the destination.

Will be very interested to find out more about the direction you have selected moving forwards! Just keep going!

Really good stuff. Your research focus is very clearly defined. Some thoughts.

(1) Migration patterns are such a big topic that it can be difficult to know where to start in terms of geospatial data, data science / machine learning methods / models etc...A traditional starting point for thinking about migration is in terms of push factors and/or pull factors, more traditionally within the context of rural to urban migration. The scope and scale of what you are investigating is of a much greater magnitude and complexity than these traditionally considered foundations from development economics. Additionally, thinking of migration of this magnitude in terms of only human movement and behavior, likely over simplifies the problem. For example, we could perhaps use CDR data to infer the origins and destinations of where people are coming from and where they wish to go as part of a transportation system model (even if it is by foot or mixed mode). I think you need something that seeks to accomplish far more than simply transport behavior and movement, because you also wish to understand the motivations behind why people choose to leave (push factors) as well as the reasons they are attracted to some location (pull factors). I think looking into agent based models as a data science method for describing, analyzing and potentially predicting migration throughout North America might be a good place to start. A simulation system, such as an ABM, may incorporate different model that address factors at the origin (crime as a push factor) as well as at the destination (income differentials, as discussed by Todaro). There is simply a lot to think about here.

(2) It might be helpful to reflect back on some of the historical references. Todaro is an often cited source.

https://www-jstor-org.proxy.wm.edu/stable/1807860?seq=1#metadata_info_tab_contents

Another good one to consider is Sir Arthur Lewis and his Economic Development with Unlimited Supplies of Labour. Please let me know if you can't find a copy of his 1954 essay that earned him the Nobel Prize.

Another good source, is WIEGO.

https://www.wiego.org

Just keep going!

This may be a pet peeve, but I feel many in the the discipline fail to think sociologically about these kinds of questions, and focus far too much on what is right in front of their noses - language, assessment, feedback, etc. Consider, for example, Ira Kaznelson's When Affirmative Action Was White, and Rothstein's The Color of Law. They describe how the huge, transformative public policy programs of the New Deal and Fair Deal, as well as areas like housing policy, lending, zoning, etc. gave enormous, staggering, transformative boosts to white Americans, while systematically excluding people of color. The largest middle class the world has known was created through a slew of programs that people of color were excluded from, and a whole set of policies, esp, housing and lending and zoning and penal, made sure to engrave racism in our schools, housing, urban geographies, prisons, and in wealth accumulation (most family wealth is generated via the transfer of housing value from one generation to the next). Obviously many other forms of racism were important, but this huge framework of laws, regulations, and policies both reflected racism, and were absolutely crucial in reproducing it, with affects the have an enormous impact today.

Yes racism is overdetermined. I'm just skeptical that how teachers assess or give feedback to students in first year university writing classes matters much. I say that (I hope) not defensively or out of a sense of guilt or to deflect blame. I just think we could change everything about how we give feedback and assess writing (we could even follow Inoue's "labor based" model of assessment) and this would change...nothing.

Furthermore, from my perspective, the great strains and stresses on universities that I see doing the most to undermine education come largely from the neoliberalization of higher ed - the relentless defunding of public ed and shifting of costs to students, in order to pay for tax cuts for the wealthiest; casualization of labor, deprofessionalization, adjunctification, the explosion of class sizes to save costs, etc etc. Those things drive pedagogy in many ways. Most of our writing teachers teach 5 sections of 30 students, and sometimes other sections at community college. They work 70-80 hours a week. Their feedback and assessment practices are driven largely by these material considerations. In such situations poor students, first gen students, students of color, may suffer most as they don't have all the support and familiarity with university that other students do. These kinds of issues seem largely ignored by Inoue, although I imagine he must be aware of them.

This is a really interesting set of sentences. It echoes the intro to George Yancey's Backlash: What Happens When We Talk Honestly About Racism in America. Yancey's book emerged from his "Dear White America" columns for the NYT, and the ferocious, staggering racism with which these columns were received by some white Americans. The book asks white readers to think honestly and deeply about impulses to deny, ignore, or get angry when racism is discussed. But I have several reactions to Inoue's framing. 1) It could be a way of asking white readers to do the kind of careful introspection Yancey describes.2) it could be a way of framing things that constructs in advance objections or challenges as "hurt" and resentment at hearing Inoue's "honesty," his revealing of racial wounds. It also frames things in terms of love/hate, honesty/dishonesty, wounding/healing. In this scenario, if I disagree with Inoue am I necessarily accusing him of doing something "wrong," or refusing racial healing? This second framing seems to move things away from the space of argument and debate and into some other space. Perhaps that space is valuable in some ways. But this framing carried into the later online discussions of Inoue's text, and was used by some to dismiss critique as refusal to acknowledge racial injury or refusal to acknowledge the reality if racism in the U.S.

Quarterly Journal of Speech Vol. 95, No. 1, February 2009, pp. 43􏰀65 Bordering the Civic Imaginary: Alienization, Fence Logic, and the Minuteman Civil Defense Corps D. Robert DeChaine

Current figurations of the ‘‘immigration problem’’ in the United States challenge our understanding of the rhetoricity of contemporary bordering practices. The public discourse of the Minuteman Civil Defense Corps serves to chart the alienization of undocumented migrants and the enactment of alien abjection on the U.S.􏰀Mexico border. Alienization promises an antidote to majoritarian anxieties regarding national disunity in the form of a shoring-up of cultural boundaries that border-crossing subjects render troublesome. Ultimately, the fence logic engendered by groups such as the Minutemen reveals how struggles over the boundaries of citizenship both enable and limit an affect-charged civic imaginary.

Keywords: Borders; Alienization; Minuteman Civil Defense Corps; Citizenship; Social Imaginaries

The specter of the border haunts the language of social relations. At present, some 12 million undocumented migrants reside temporarily or permanently in the United States.1 Many of them entered the country across the border with Mexico, a frontier spanning nearly 2,000 miles from the Californian Baja through Texas and to Tamaulipas. These border-crossing individuals, the majority of whom venture to the United States in search of work and enhanced economic opportunities, are not recognized by the U.S. government as citizens, defined in legal terms as native or naturalized rights bearers. Nor, for the most part, are they considered legitimate in the broader sense of what Charles Taylor terms ‘‘recognition’’*that is, they are not viewed as valued and respected members of the national community.2 Indeed, upon D. Robert DeChaine is Associate Professor of Communication Studies and Liberal Studies at California State University, Los Angeles. An earlier version of this essay was presented at the 2007 National Communication Association convention in Chicago. The author would like to thank Editor John Louis Lucaites, Mike Willard, Michelle Ladd, Scott Rodriguez, and two anonymous reviewers, each of whom made decisive contributions to the quality of the essay. Correspondence to: Department of Communication Studies, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8111, USA. Email: ddechai@calstatela.edu ISSN 0033-5630 (print)/ISSN 1479-5779 (online) # 2009 National Communication Association DOI: 10.1080/00335630802621078

44 D. R. DeChaine close examination, the cultural politics of recognition reveals a telling link between state legitimacy and public morality. As Kent Ono and John Sloop note in their analysis of the role of media rhetoric in the passage of California’s Proposition 187 in the 1990s, [T]he contemporary citizenship narrative casts immigration in moral terms: Those who abide by U.S. laws and procedures for how to become U.S. citizens are cast as good and moral citizens; those who do anything but systematically follow expectations of U.S. government officials and their supporters are seen as bad and immoral ‘‘illegals.’’3 The transgressive act of unauthorized border crossing thus produces a double exclusion: it renders migrant persons both legally and morally abject. Given the subordinate status of undocumented migrants in the United States today, what is particularly interesting regarding the ‘‘immigration problem,’’ as it is typically invoked in public discussion, is that it is not framed predominantly in terms of the migrants themselves. After all, the United States is a nation composed of immigrants, so the oft-repeated narrative goes, and its promise as a land of opportunity for hard-working, law-abiding citizens continues to be exalted as among its preeminent gifts. Rather, the problem is most often cast in terms of a lack of border integrity: its leakiness; the ease with which undocumented migrants are able to slip across it unnoticed; and the inability or unwillingness by the U.S. and Mexican governments to seal, secure, and protect the national frontier and its stark line of demarcation. Notwithstanding the nativism and xenophobia that have historically shaped and continue to shape popular attitudes toward undocumented migrants in the United States, the problem of immigration, it seems, lies not with the migrant, but with the border.4 The prevalent characterization of the immigration problem as primarily a concern about the border gestures toward a number of longstanding assumptions regarding what a border is and how it functions. According to the traditional geography-based logic, a border exists as a given entity whose contours can be cleanly and clearly recognized, measured, and mapped.5 A border’s givenness and mappability implies its stability as a resource for delineating spatial territory. Despite its tenacity, the traditional border logic has increasingly been subjected to critique. This scrutiny is owing in part to the emergence and development of postmodern theories of the social character of space beginning in the 1960s, and to a subsequent proliferation of spatial metaphors endemic to human social life in a globalized world.6 Against the traditional view of borders as given and stable entities, the countervailing claim is that whatever form they may assume, all borders are socially motivated constructs.7 They are bounding, ordering apparatuses, whose primary function is to designate, produce, and/or regulate the space of difference. Thus conceived, borders simulta- neously shore up insides and mark off outsides while establishing the terms of their relationality. They perform both division and containment functions, differentiating the self from others, one culture from another, desirable elements from undesirable ones, and, often enough, ‘‘us’’ from ‘‘them.’’ Moreover, according to the revisionist

account, a border’s contingent configuration is often masked by certain political and economic discourses that labor diligently, sometimes feverishly, to maintain the semblance of stability, integrity, and reality. Recent scholarship in communication studies, chiefly in the area of immigration discourse, has begun to attend to the subject of borders and their cultural significance. A majority of this work aligns with critical scholarship that ascribes a truth function to the border, focusing on its constructedness and on the ambivalent identities of border(ed) subjects.8 Thus considered, a border operates as an inducement to action, deployed by agents in specific contexts to warrant claims to both unity and division.9 Circulating as a robust spatial metaphor, the figure of the border functions as a prevalent organizing doxa in a group’s collective vocabulary. Such assumptions, theorizations, and criticism regarding the constitution and function of the border beckon a considera- tion of two interrelated questions, each of which holds epistemological and ontological significance. What kinds of operations are at play in the construction of borders? And once entwined in the cultural fabric of a political community, how do constructed borders shape human values, attitudes, and actions? The aim here is to focus on the rhetoricity of contemporary bordering practices by charting the ‘‘alienization’’ of undocumented migrants in the United States, and to explicate the operations by which alienizing discourse is enacted on and around the U.S.􏰀Mexico border.10 Alienization, I contend, is a bordering project that draws force from a variety of common linguistic and nonlinguistic resources to render individuals and groups abject and unassimilable*irredeemable others whose putative exclusion from the national body is virtually absolute. Alienization materializes as a reactionary rhetoric goaded by a profound anxiety of incompleteness, an anxiety akin to what Arjun Appadurai refers to as a ‘‘fear of small numbers’’ engendered by a minority population whose alterity is perceived by a majoritarian national ethnos as a threat to its unity.11 Thus conceived, alienization promises an antidote to disunity and incompleteness in the form of a shoring-up of cultural boundaries that border-crossing migrant subjects reveal to be troublesome. Moreover, as a form of world making, alienization shapes a population’s collective attitudes toward and practices of citizenship. I share Robert Asen’s view of citizenship as a discursive mode of public action that is ‘‘always conditioned by social status, relations of power, institutional factors, and material constraints’’; as such, citizenship enactment necessarily involves hegemonic struggles over the very meaning of the term ‘‘citizen’’ in a multipublic sphere.12 Evoking the concept of the ‘‘social imaginary,’’ I examine how struggles over the boundaries of citizenship both enable and limit an affect-charged ‘‘civic imaginary.’’ An analysis of alienization and its bearing on the constitution of the civic imaginary requires a shift in focus away from the consideration of borders as physical, geographical entities to consideration of their instrumentality as performative, sociocultural productions. It places emphasis on the process, practice, and affect of bordering, and on the effects, both material and discursive, of border rhetorics on particular social collectivities. My chief ambition, then, is to throw light on the relationship between physical and social bordering practices, and to identify the very human implications of those practices for the shaping of civic culture. In order to lend historical specificity to the Bordering the Civic Imaginary 45

46 D. R. DeChaine discussion, I focus attention on the crafting of public appeals in support of the Border Fence Project, an initiative launched by the Minuteman Civil Defense Corps (MCDC) in 2006. Originally created in 2005 by former elementary school teacher, military veteran, and California resident Chris Simcox in response to a perceived ineffective- ness of U.S. governmental action, MCDC proclaims itself to be ‘‘the country’s largest volunteer grassroots border security advocacy group,’’ boasting more than 350,000 constituents.13 The professed aim of the MCDC’s Border Fence Project is ‘‘to secure America’s sovereign territory against incursion, invasion, and terrorism’’ through the construction of a steel security fence along the U.S.􏰀Mexico border.14 The MCDC and its Border Fence Project has garnered considerable attention from popular media, and support from prominent members of the U.S. government. Drawing on a range of textual evidence including websites, speeches, visual and print media, an online discussion forum, and interviews with Simcox and other MCDC members, I show how the group’s public advocacy of the border fence reflects an articulation of economic, racist, and nationalist narratives that together produce a border rhetoric in which the alienized subject becomes both figurally and literally fenced out of the sacrosanct space of U.S. citizenship. The MCDC’s Border Fence Project presents a timely example of a symbolic enactment of alienization that fuels public sentiment and influences policy regarding undocumented migrants in the United States. Rather than dismiss the organization as extremist, as popular U.S. media often do, or consider its views to be peripheral to dominant attitudes toward immigration, I argue that a close examination of the MCDC identifies its reliance on symbolic strategies and commonly shared values that are anything but radical. In the analysis, I hope to illustrate the ease with which physical and geopolitical borders map onto cultural and ideological borders. Against the popular argument that the immigration problem is preeminently about the integrity of the border rather than about the qualities of the migrant as a human being, I contend that their relationship is in fact profoundly fraught. ‘‘Suspect bodies,’’ asserts Lisa Flores, ‘‘carry the border on them.’’15 As a transgressive, racialized subject, the alienized migrant literally embodies the border, rendering problematic any tidy relationship between physical and social space. Crafting the Abject: The Border(ing) Project of Alienization An examination of the rhetorical dimensions of the border requires a shift in focus from borders to bordering, from a consideration of static entities to analysis of a dynamic practice. As a social ordering practice, bordering produces and enforces spaces of identity and difference, defining terms of identification and exclusion. As such, it influences a community’s ways of seeing and experiencing itself, its members, and those deemed to be outside or unworthy of membership. In this section, I outline a project of cultural and political abjection, whose symbolic form operates according to an affective logic of alienization. My intention is to identify the characteristic features of alienization, to explain its motivations and functions, and to suggest some of its implications for its bordered subjects. My discussion is informed by the

complementary projects of critical rhetoric and cultural studies, each of which claims a stake in uncovering operations of power that shape human experience.16 While I would propose a certain degree of applicability to non-U.S. contexts, my focus is on the specific formation of alienization in post-9/11 U.S. society. Alienization in the United States did not materialize full-blown in the present social-political moment. Indeed, its current manifestation attests to a historical record of laws, policies, and practices reflecting longstanding attitudes about immigration and migrants. As Flores contends, ‘‘Contemporary images of immi- grants, such as that of the illegal alien, do not emerge in a vacuum. Instead, they are part of our nation’s history of immigration, race, and nation; they bring with them varied meanings, reflecting their origins and uses.’’17 This history has been steeped in nativism, an ideology based on a systematic exclusion of designated others, that can be traced back to at least the nineteenth century. Driven by the twin discourses of racism and capitalism, nativism has contributed to the construction of narratives that cast the migrant as culturally different or inferior, a necessary source of labor, and a drain on U.S. government resources.18 Its early manifestations are evidenced in the Chinese Exclusion Act of 1882, which denied migrants citizenship on the basis of national origin, and the Immigration Acts of 1921 and 1924, which placed numerical limits on immigration while entrenching for decades ‘‘a global racial and national hierarchy that favored some immigrants over others.’’19 During the Great Depression, as demand for labor diminished and job competition increased, nativism was reinvigorated in the form of voluntary repatriation campaigns that resulted in the deportation of a half a million Mexican undocumented migrants, U.S. citizens of Mexican descent, and permanent legal residents. During World War II, the U.S. government, presuming that all U.S. citizens of Japanese origin were racially inclined to disloyalty, incarcerated 120,000 persons, effectively nullifying their citizenship status. After the war, with the increased demand for labor, the government instituted the Bracero program, a contract labor policy for Mexican migrants that Mai Ngai describes as ‘‘America’s largest experiment with a ‘guest worker program’’’ to date.20 Arcing into the present, nativism has endured in actions such as 1954’s Operation Wetback, which led to the forcible deportation of more than a million Mexican migrant laborers, efforts in the 1980s to militarize the U.S.􏰀Mexico border, California’s Operation Gatekeeper, Propositions 187 and 209 in the 1990s, state laws mandating English-only policies, and recurring efforts to pass an English Language Amendment to the U.S. Constitution. As such historical patterns of response to migrants underscore, an account of the present conjunctural formation necessarily includes the history of nativist-born attitudes and practices that have designated immigration as a problem and the migrant subject as an undesirable outsider to the American civic community. In its most elemental sense, alienization is a form of otheringa way of seeing and not seeing, of experiencing and not experiencingthat conditions modes of human subjectivity and action. Rather than considering alienization primarily as a condition, or as a terminal state, or in terms of a culmination of effectsalthough it is certainly experienced on each of these registersI propose that it is most usefully understood Bordering the Civic Imaginary 47

48 D. R. DeChaine as an unstable hegemonic process that must constantly adjust the character of its form if it is to win the consent of a national community guided by properly American liberal democratic values. As a hegemonic process, alienization operates as a fundamentally rhetorical mode of action. Invoking Kenneth Burke’s formulation, it functions terministically to direct and reflect the experiential reality of symbol users.21 As a shaper of collective attitudes, it provides a national community with a repertoire of symbolic resources for naming and thus bringing into being its valuative structure. These resources include an array of metaphors ascribed to alienized persons, as well as linguistic and visual figures that serve both to condense and to amplify public values. As I elaborate below, for example, the metaphoric construction of an alien invasion, and inverted visual images such as an upside-down American flag or a photograph of a breached border fence, can provide powerful symbolic grist for mobilizing public sentiment against those perceived as threatening to the sanctity of American values and national security. Moreover, alienizing rhetoric operates according to an essentialist logic based on negative linguistic difference. As Mark Lawrence McPhail explains, ‘‘The socio-political realities of negative difference are products of a language defined in essentialist terms as much as they are a reflection of self-evident realities that exist independent of the languages we use to construct them.’’22 Viewed in this light, alienization operates as a moralizing discourse that tells the truth about undocumented migrants and the essential nature of their otherness. The terministic and ideological functions of language and imagery are integral to the development of a hegemonic border logic that negatively posits absolute identities onto other(ed) migrant individuals and groups. Alienization signifies a context-specific conjuncture of discourses that interanimate and mutually reinforce one another. In its present configuration, it is enacted as an expression of racial, ethnic, and national identity that categorizes and differentiates Americans from non-Americans*or, more accurately, un-Americans. It demon- strates the intimate social and political linkage of race and nation in directing racism as an expression of xenophobic attitudes toward inferior and undesirable constituents of the national body.23 As a bordering practice, alienization shares much in common with what Howard Winant describes as ‘‘racialization,’’ a process involving attempts to fix identities provisionally in accordance with particular social, historical, and political attitudes about race.24 Furthermore, racializing rhetoric is readily linked with economic arguments that reinforce cultural and political attitudes toward migrants. In their analysis of the rhetoric of California’s Proposition 187, for example, Ono and Sloop describe the racialization of the migrant as the discursive production of an ambivalent subject who is both desired and reviled, necessary for both capitalist production and the object of ‘‘loathing of the laborer who does anything other than work specific jobs associated with facilitating the interests of efficient capital processes.’’25 Arguments about the migrant’s relative economic value are tethered to ideologically charged prescriptions of racial and ethnic identity. The undocu- mented migrant becomes both integral to and separated from proper capitalist relations of production; as such, alienization bears more than a passing resemblance to Karl Marx’s conception of ‘‘alienation.’’26

Alienization functions discursively to shape public understandings of social and political identities. However, to gauge its influence only in terms of discourse misses a great deal about what drives it as an expression of national communal values. At the motivational level, alienization operates according to an affective logic similar to that which Appadurai describes in terms of an ‘‘anxiety of incompleteness.’’27 Appadurai accounts for what he takes to be an increasing propensity towards ethnic-based violence committed by national majorities (including liberal democracies) against their minority populations, a tendency he attributes in large part to the anxieties of social life in a globalized world: [W]here the lines between us and them may have always, in human history, been blurred at the boundaries and unclear across large spaces and big numbers, globalization exacerbates these uncertainties and produces new incentives for cultural purification as more nations lose the illusion of national economic sovereignty or well-being.... [T]he tip-over into ethnonationalism and even ethnocide in democratic polities has much to do with the strange inner reciprocity of the categories of ‘‘majority’’ and ‘‘minority’’ in liberal social thought, which produces what I call the anxiety of incompleteness. Numerical majorities can become predatory and ethnocidal with regard to small numbers precisely when some minorities (and their small numbers) remind these majorities of the small gap which lies between their condition as majorities and the horizon of an unsullied national whole, a pure and untainted national ethnos . . . . The anxiety of incompleteness (always latent in the project of complete national purity) and the sense of social uncertainty about large-scale ethnoracial categories can produce a runaway form of mutual stimulation, which is the road to genocide.28 According to Appadurai, it is the national ethnic minority populations who bear the brunt of the uncertaintiesand the violencespurred by globalization and its disjunctive modes of organization. Majorities, goaded by the anxiety of incomplete- ness and a fear of small numbers, find a scapegoat in the figure of the minority, whose presence is both necessary and unwelcome: necessary for dirty work such as fighting wars and shouldering menial labor, but unwelcome for the threats their racial, financial, linguistic, and cultural border-blurrings pose. Above all, Appadurai claims, national ethnic minorities ‘‘blur the boundaries of national peoplehood,’’ a cardinal transgression for which they are not to be forgiven.29 They are the embodiment of the anxiety of incompleteness. Minorities problematize the meanings of ‘‘us’’ and ‘‘them’’*and for that, they must pay. Appadurai’s argument regarding the majoritarian fear of small numbers lends to an understanding of the affective logic of alienization in the United States today. In a globalized world, where both ‘‘vertebrate’’ and ‘‘cellular’’ tendencies of hypercapit- alism move across national boundaries with swiftness and ease, it is the figure of the border-crossing migrant who increasingly signifies the social (dis)ease of the U.S. border problem.30 To be sure, arguments casting undocumented migrants as criminals and threats to national security have long figured in U.S. popular, political, and legal discourse. However, public expressions of anxiety regarding the status of migrants have once again become pronounced in the wake of global free trade policies such as those engendered in the General Agreement on Trade and Tariffs Bordering the Civic Imaginary 49

50 D. R. DeChaine (GATT) and the North American Free Trade Agreement (NAFTA). This has partly to do with the U.S.-led effort to relax economic borders under the auspices of creating an integrated North American market while at the same time resisting an equivalent integration of cross-border labor.31 However, underlying this contradictory policy, I contend, is an affective impulse motivated by fear of losing control. As economic borders loosen, sociocultural borders tighten; as the U.S. economy becomes ever more subject to the disjunctive flows of a global cultural economy, its majoritarian reaction is to allay its anxieties by maintaining control where it canon cultural terrain. In a post-9/11 climate stoked by an omnipresent affect of terrorthe threat of a cellular enemy who is both outside and potentially inside our national borders it is perhaps unsurprising that the population’s fears and uncertainties, as well as its search for enemies, turn inward. Undocumented migrants and their rapidly growing small numbers serve as constant reminders of the incompleteness of a unified, pure American ethnos. Their geographical and cultural border crossings are perceived as challenges, indeed threats, to national sovereignty. Alienization promises an antidote to border anxieties: the present U.S. cultural climate precipitates uneasiness and a compulsion to engage in shoring-up operations, to effect a return to an imagined wholeness, unity, authenticity, and knowability. Thus conceived, alienization is the panicked reaction to the anxiety of incom- pleteness. Coursing through the national body, it casts alienized subjects as abject, inassimilable outsiders to the American community. The abject migrant is not absent from social experienceabjection is not synonymous with absence, as non- recognition is not the antonym of recognition. Rather, because she is both necessary and unwelcome, she is both visible and invisible, both acknowledged and ignored.32 Indeed it is this ambiguous positionality, the troublesome both/and of migrant subjectivity, that compels a national community to search for a means of reconciliation. However, reconciliation entails symbolic violence. In its materializa- tion as a border rhetoric, alienization operates through victimage. Blamed for the ills of a society that proclaims root values of tolerance and pluralism but longs for wholeness, the undocumented migrant fulfills the role of a ‘‘perfect enemy,’’ a vessel for that which the American reviles, disavows, and fears.33 Rhetorical victimage relies on reductive categories and stereotypical modes of representation in its rendering of the subject-scapegoat it designates. Marouf Hasian Jr. and Fernando Delgado note this reductive tendency in their analysis of California’s Proposition 187: ‘‘The typology of illegal immigrant becomes a signifier meaning Mexican (collapsing distinctions among Mexicans, Mexican Americans, Latinos) in a stereotypical manner similar to the social construction of welfare mother as African American female.’’34 Through victimage, the reductive figure of the border-crossing, border-blurring migrant serves as a vessel for containing the threat of an un-American presence from within. As an ideological production that is woven thickly into the fabric of society, alienization is infused in the language and images of everyday communication practices. A reactionary expression of the nativism that has historically shaped the character of U.S. political culture, alienization assumes a more rather than less

ordinary rhetorical form. Nowhere is its manifestation more apparent than in the invocation of ‘‘illegal immigration,’’ a key figure that is rapidly subsuming the normative rhetoric of immigration in U.S. political culture. Its ubiquity in popular media, town hall meetings, public policy analysis, congressional hearings, and political campaign platforms attests to its ascendance as an orienting devil term. And the potency of illegal immigration is nowhere more concentrated than in the signifier for its idealized subject, the ‘‘illegal alien.’’ The illegal alien is the ideal embodiment of illegal immigration precisely because she is always already doubly stigmatized. Through a process of repetition and sedimentation in public culture, the articulation of the designations ‘‘illegal’’ and ‘‘alien’’ gives them the appearance of a singularity, of being inextricably interlocked. More and more, in fact, ‘‘illegal’’ is becoming an implicit modifier, the already- present threat that marks the alienized subject. To invoke the term ‘‘alien’’ is now nearly all that is required. Her illegality is given. Through the alien’s consolidation as the implicit subject-signifier of illegal immigration, alienization is enacted linguis- tically and performatively as a self-evident expression of the abject other. Moreover, through her tacit link with illegal immigration, the alien is made available for identification with a variety of other(ing) signifiers. These have historically included constructions of the alien as ‘‘criminal,’’ ‘‘communist,’’ ‘‘animal,’’ ‘‘dirt(y),’’ and ‘‘disease(d).’’35 Such signifiers call attention to the ultimately unknowable quality of the alien, an ascription that is threatening to the national community since it belongs to the one who seeps through the cracks, gets in and out, and troubles the very discreteness of the national inside/outside binary. Across all of its identifications and significations, it has become clear, ‘‘Whether invoked directly or indirectly, the figure of the ‘illegal alien’ is hauntingly consistent.’’36 Indeed, the totalizing ascription of ‘‘illegal alien’’ to the alienized subject performs an essentializing function that literally denies the possibility of a positive referent. Given her overwhelmingly negative social-cultural status, redemption of the alienized subject is unlikely in the United States today. In order to be refigured as a potential member of the American community, she would need to undergo a radical transformation, entailing both avowed and ascribed identity reformation. In effect, the alien would have to become an altogether different person. It is true that there are legal pathways to U.S. citizenship, although in rapidly restricting forms.37 However, the fact that alienization involves concomitant racialization makes the prospect of redemption difficult, if not doubtful. Not only do border guards check papers, but since the alienized subject carries the border on her back, she is constantly subject to surveillance and search. She may or may not be what she seems; although she is among the community, although she may be naturalized, she is not naturally of the community. As Bhikhu Parekh astutely notes in his discussion of multicultural citizenship, ‘‘Although equal citizenship is essential to fostering a common sense of belonging, it is not enough. Citizenship is about status and rights; belonging is about acceptance, feeling welcome, a sense of identification. The two do not necessarily coincide.’’38 Bordering the Civic Imaginary 51

52 D. R. DeChaine Alienization, I have suggested, is a border(ing) rhetoric par excellence. It operates in U.S. society as a hegemonic project concerned with the forging and maintaining of dominant American civic values. At the structural level, it signifies an alliance of racist, capitalist, and nationalist discourses that materializes rhetorically. At the motivational level, it is goaded by majoritarian anxieties of national incompleteness and a longing for civic communion. Although reactionary in tone, it reflects widely shared values and attitudes. Dogged by the threat of an ambiguous enemy within, the primary labor of alienizing rhetoric is the production of the abject subject, the illegal alien, the embodiment of that which is both necessary and unwelcome in the space of U.S. citizenship. Reviled for her border-crossing and border-blurring behavior, the alien is constructed through symbolic acts of reduction and victimage. With her identity tethered to the negative, racialized figure of illegal immigration, the alien’s prospects for redemption are acutely diminished. Above all, the rhetorical production of the alien illustrates the collective desire for purity, perfection, and order*a compulsion, as Burke insists, that is a hallmark of the human condition.39 Put differently, although alienization appears primarily to be about the imputed qualities of the undocumented migrant, it is also substantially about the fraught logic of the border and the symbolic power that it wields. Pledging ‘‘Eternal Vigilance’’: Rhetorical Alienization in the Minuteman Civil Defense Corps Since its inception under the leadership of its founder Chris Simcox, the Minuteman Civil Defense Corps has been effective in shaping public attitudes about the immigration problem in the United States. Proclaiming itself as ‘‘one of the most important, socially responsible, and peaceful movements for justice since the civil rights movement of the 1960s,’’ Simcox and the MCDC have garnered support from a number of legislators, including Republican representatives Duncan Hunter of California and Tom Tancredo of Colorado.40 Like other contemporary social movement groups, the MCDC is acutely attuned to the politics of the public screen and its power to mobilize popular attitudes.41 In order to promote its mission and its border operations, the organization relies heavily on hypermedia for the dissemina- tion of information, public statements, speeches, fundraising campaigns, and volunteer musters. Its official and unofficial discourse proliferates in the blogosphere, and postings of speeches by Simcox as well as protests against him can be found on popular video sites such as YouTube. Its continued media presence and its often infamous appearances on college campuses have helped the MCDC to maintain a significant profile in the public conversation on immigration reform, while contributing to its ethos as a controversial activist group. A substantial amount of the MCDC’s public advocacy has focused on its effort to secure funding for the construction of approximately 70 miles of fencing along portions of the U.S.􏰀Mexico border in southeastern Arizona. The MCDC has dubbed the initiative the Minuteman Border Fence Project, a campaign that serves as an umbrella for a number of regional fundraising efforts.42 Professing to be ‘‘doing the

job the [U.S.] President and the Senate refuse to do,’’ MCDC-aligned volunteers began constructing bits of the fence themselves in October 2006.43 Referring to the proposed border fence as ‘‘America’s ultimate protection against foreign invasion and terrorism,’’ the MCDC capitalizes on the historical symbolism of the Minutemen of the American Revolution, describing its undertaking as not a solution but an example of what committed AmericansWe the Peoplemust do in order to safeguard the border and the citizenry.44 Through its efforts to rally public support for a security fence along the U.S.􏰀Mexico border, particularly in its crafting of appeals that draw on affect-charged language and images of formative American values, the border(ing) rhetoric of the MCDC demonstrates how public under- standings of national identity, community, citizenship, and ‘‘the other’’ are mutually constructed. Reflecting commonly shared beliefs about migrants and immigration in U.S. political culture, its appeals bespeak a ‘‘fence logic’’ that effectively displaces claims about alien subjectivity and refigures them as claims about conditions said to result from nonsecure U.S. borders. In this way, the group enacts a project of alienization that avoids overt racism by framing the immigration problem as a concern about geographical borders rather than about the alien per se. In all, the MCDC contributes to the production of an anxiety-ridden narrative of citizenship of what it means to be a member of the broadly imagined American Community. Ultimately, its fence logic both shapes and reflects broadly held attitudes about culture and difference in contemporary U.S. society. The MCDC demonstrates its similarity to other social-political movement groups insofar as its public appeals, if they are to be acted on, must successfully construct an orientation toward a situationa compelling exigency, a plausible accounting of involved actors, and a fitting and attainable mode of redress. What most distinguishes the MCDC from other activist groups is its particular crafting of a vision of national community, an understanding of civic identity that seeks resonance with a dominant regime of true American values. In constructing appeals that articulate values of social unity, national security, rule of law, and civic responsibility, the MCDC draws from a common vocabulary of U.S. political culture to advance an alienizing logic that shores up communal boundaries while simultaneously defining terms of exclusion. The MCDC’s casting of the rhetorical situation turns on its characterizations of the duty-bound Minuteman volunteer and the nature of the national security nightmare at the U.S.􏰀Mexico border that impels him to action.45 The United States, the group warns, is a nation in peril. The threat strikes at the heart of the American democratic ethos: the Border Fence website asserts that the country ‘‘is under siege by forces and interests that have the capacity, over time, to destroy our great experiment of responsible self-government.’’46 The sovereignty of the nation and the unity of the American people have become jeopardized as a result of those whose unauthorized movements impinge on them. The danger, the MCDC insists, issues from the current state of unsecured U.S. borders, giving rise to a ‘‘human tsunami’’ of illegal border crossers and the threats they pose to citizen safety, economic stability, and cherished American values.47 Bordering the Civic Imaginary 53

54 D. R. DeChaine The forces and interests that are claimed to undermine American unity and sovereignty are both internal and external. In an address entitled ‘‘A Letter to My Fellow Americans,’’ Jim Wood, Executive Director of the Border Fence Project, cautions, ‘‘[T]he national language, character and culture of our sovereign American people are under attack by ‘multi-culturalism’ run rampant. It’s dangerous and it’s destructive.’’48 Employing violent metaphors and reductive categories to consolidate the gravity of the alien threat, constituents of the MCDC make repeated reference to the imputed ‘‘illegal invasion’’ now said to be underway.49 Such an evocation points to the danger to the American ethnos of an abstract, massified enemy already among us, ‘‘the illegal aliens residing in our midst who have been allowed by a feckless government to violate our laws and mock our sovereignty as a nation.’’50 The fear of increasing numbers of alien noncitizens in the United States and their ability to undermine American unity is further evidenced on the Border Fence Project’s website, which cites California as an example of the disunity produced by an ‘‘increased flux of illegals, who displace legal citizens whose numbers are decreasing there.’’ Moving unchecked across the U.S. border, the alien-enemy is now within, infecting the body politic. It corrupts the government, disunifies its citizenry, dilutes its culture, and precipitates a virulent strain of anti-Americanism: as Simcox stated, in response to May 1, 2007 demonstrations in which ‘‘anti-American extremist militants’’ flew upside-down U.S. flags and flew Mexican flags above the U.S. flag, I think it exposes an anti-American, anti-European sentiment. And we see that on college campuses across the country. The vitriol, the hate. The hate groups that exist on college campuses and then take to the streets are calling for a reconquista of the United States.51 Flags and other visual imagery are routinely employed by the MCDC to dramatize the anti-Americanism fueled by the alien invasion. For example, an image on the Border Fence Project website titled ‘‘Tear for My Country’’ features a close-up photograph of a portion of a human face, eye cast downward, with a tear rolling down its cheek. In the foreground flies a Mexican flag, positioned on its mast above an upside-down American flag. Superimposed at the bottom of the image is an iconic rendering of Uncle Sam (unaccompanied by its familiar ‘‘I Want You!’’ caption) pointing at the viewer, as if to admonish her to take up the pro-American resistance. In public appearances, Simcox and other MCDC members also often display photographs of anti-American migrant protesters, offering them as evidence of an enemy in our midst whose force, like that of a virulent disease or inexorable flood, must be contained. The destructive forces that threaten American unity, sovereignty, and democracy are the byproduct of more ominous threats to national security and public safety that an unsecured U.S. border represents. The MCDC invokes national security as a caution to Americans about the dangers of terrorism, criminality, and violence posed by an illegal invasion across broken borders. The alien-terrorist menace is real. ‘‘We’re being attacked by people from a foreign country,’’ exclaims Simcox; ‘‘Is that not

Bordering the Civic Imaginary 55 terrorism in its own right?’’52 Those who threaten national security, whether wittingly or unwittingly, are culpable: ‘‘If you’re breaking into this country when this country is at war, then you’re a potential enemy of this country, and you should be treated accordingly.’’53 In MCDC discourse, a link between illegal immigration and ‘‘terrorism,’’ another prominent devil term in post-9/11 U.S. society, is forged through an association of the border crosser with crime and violence. Simcox, emphasizing this association, makes reference to ‘‘the violence caused daily by illegal entrants and often violent alien intruders,’’ and advocates ‘‘the feasibility and efficacy of fencing to secure America’s borders from illegal incursion by aliens and international criminal cartels.’’54 And the alien-borne violence is growing.55 As the number of unauthorized border crossings increases, ‘‘More gang violence erupts in our cities by murderous thugs such as MS-13, the ruthless gang of self-professed friends of al Qaeda.’’56 The deadly admixture of crime, violence, and terrorism at the hands of undocumented border crossers is perhaps most dramatically asserted by the Border Fence Project: ‘‘Because illegal aliens murder 5,000 innocent Americans every year and we take a trillion dollar hit overall to our economy, illegal immigration rewards us with a 9/11 or worse every year.’’57 Close-up photographs of decrepit and hole- ridden fencing along the U.S.􏰀Mexico border, and images of aliens individually and collectively breaching the fence, are offered by the MCDC as evidence of the national security nightmare that is claimed to exist. A Minuteman Border Cam with multiple views of the border fence is featured on the MCDC website, so that viewers may witness for themselves the always-looming threat to national security in real time. By playing on national anxieties regarding a terrorist threat both beyond and within U.S. borders, the MCDC collapses categorical distinctions between terrorism, criminality, and alien subjectivity, drawing an abstract figure of the dangerous, already suspect un-American other. The MCDC readily identifies the U.S. government as the primary agency responsible for the immigration problem. Although it is the alien who crosses the border into the United States, she is rarely directly named as the villain. Rather, the group insists, it is the U.S. government (and to a lesser extent, the Mexican government) and its lax attitude toward border enforcement that allows aliens to cross the border. The government’s negligence in upholding its commitment to safeguard the border has ominous consequences. As Simcox fervently warns, The existing border crisis is a dereliction of duty by those entrusted with American security and sovereignty, leaving America vulnerable to terrorist infiltration and an unprecedented crime wave caused by drug smugglers, rapists, thieves, human traffickers and murderers who currently cross our borders at will.58 In its attribution of the problem, the MCDC fuses the values of national security and the rule of law, imputing a causal relationship between lax border enforcement, a permissive welfare state, and the economic and social burdens shouldered by law- abiding U.S. citizens. Placing emphasis on this causal relationship, Simcox calls on fence logic to offer a remedy:

56 D. R. DeChaine When you secure the borders, cut off welfare and social services to illegal aliens at the expense of the American taxpayer, and strictly enforce the laws against hiring illegally, the problem of what to do with the estimated 12-20 million illegal aliens already in this country will drastically shrink in size and scope.59 Political, economic, and social reform such as that proposed by Simcox and the MCDC requires a strong, morally committed U.S. government. Simcox warns, In a time of war, the future of the American republic hangs in the balance . . . . The confidence of we the people can only be restored by those elected officials who will truly act in defense of Americaher liberty, sovereignty, security and prosperity in the face of all foreign threats.60 Taken at face value, the MCDC’s assignation of blame to the U.S. government would seem to belie the main thrust of alienization, the chief operation of which is the rendering of the abject alien subject. However, the displacement of the alien and her deflection away from being the primary agent of blame is key to the crafting of the group’s persuasive appeals. For all its drama and hyperbole, the MCDC’s alienizing, nativist rhetoric appears on its surface to be relatively devoid of overt racism and open hostility toward migrants as persons. Notwithstanding the xenophobic tenor of its appeals, one is hard pressed to locate explicit epithets directed against migrants in its official discourse. It is tempting to attribute this lack of directness to what Stuart Hall terms ‘‘inferential racism,’’ those ‘‘apparently naturalised representations of events and situations related to race, whether ‘factual’ or ‘fictional’, which have racist premises and propositions inscribed in them as a set of unquestioned assumptions.’’61 As its narrative makes clear, inferential racism infuses the group’s language and visual imagery. Be that as it may, the indirectness of the MCDC’s alienizing project is also reflective of its formal construction, which effectively displaces an alien-centered threat and refigures illegal immigration as a border-centered threat. Alienizing rhetoric does not so much actively ascribe an identity quality to the alien subject as to passively describe that which is said to result from the condition of unsecured borders. As an effect of this formal refiguration, ‘‘invader’’ is reinscribed as ‘‘invasion’’; rather than an agent-centered claim that aliens are invading our nation, it is more common in MCDC discourse to encounter condition-centered statements such as ‘‘Our borders are under attack’’ or ‘‘Our borders are being invaded.’’ Likewise, agent-centered references to aliens as terrorists or criminals are more often refigured as statements referring to the threat of terrorism and criminal activity*both claimed to be effects of the condition of unsecured borders. Functioning as the rhetorical form of inferential racism, alienization depersonalizes (and dehumanizes) the alien subject while providing the rhetor, whether an individual or an organization, with an alibi. By establishing a socially acceptable position for anti-migrant sentiment, one that places blame not on a person or an ethnic group but on an impersonal condition, the formal construction of alienization provides an inoculation against charges of racism and scapegoating. Thus conceived, rhetorical alienization allows the MCDC and other alienizers to proclaim unself-

consciously that broken borders, not aliens, are the source of the immigration problem. As the MCDC’s account of the exigency works to underscore, the dire situation on the U.S.􏰀Mexican border demands immediate and decisive action. Against the corrupting forces of an alien invasion and a complicit U.S. government, the group calls on true Americans ‘‘to safeguard our beloved nation from violence, from sedition, from wholesale attack by those who do not love America and wish to see her destroyed or irremediably altered from a nation of liberty, equality, and justice, and a bastion of western civilization.’’62 Such an intervention requires selfless action, honor, respect for tradition, and love of country: moral qualities embodied in the figure of the MCDC citizen volunteer. Deriving ethos from the venerable narrative of the American Revolutionary Minuteman, the MCDC citizen volunteer exemplifies the values of a national polity based on the rule of law and civic responsibility. As Article Four of the Minuteman Pledge attests, ‘‘[a] Minuteman believes in a strong, safe and secure America that begins with borders open only to those who have a legal right to enter, and who have met all the lawful criteria to cross into our territory established by the sovereign American people.’’63 In its appeal to the U.S. citizenry to secure its borders, the MCDC extols individual and collective responsibility, cementing together American virtues of patriotism and duty in taking action against present and future threats of an alien invasion. The U.S. government’s dereliction of duty must be met by the force of duty-bound American citizens*patriotic Minutemen who ‘‘will not sit idly by while we are colonized by another country.’’64 As the Minuteman Border Fence Project proclaims, ‘‘The politicians will talk; the liberal media and the ‘Hate America’ crowd will offer AMNESTY to law breakers. The Minutemen will ACT in defense of America!’’65 The appeal to American responsibility is future oriented: in the fight to secure U.S. borders against the alien invasion, the ‘‘future security and public tranquility for generations of our American posterity’’ hangs in the balance.66 Looking toward the future well-being of the country, the MCDC citizen pledges his commitment to responsibility as an individual, consecrating it in a vow before God and his fellow Americans. Invoking a phrase popularized by Wendell Phillips in a speech to American abolitionists, the Minuteman declares, ‘‘‘Eternal vigilance is the price of liberty . . . ’ And so I will stand watch on America’s borders and in her sovereign interest until relieved from duty by my fellow countrymen.’’67 As movement-oriented action, this commitment to responsibility is not simply individual; it is also collective. The MCDC pledges eternal vigilance, promising to ‘‘continue to stand watch at the border and report illegal activity, build border fencing, urge local and federal officials to enforce the law and push for the enforcement of our laws to keep our country and your families and children safe.’’68 Several Border Fence Project websites feature photographs of groups of MCDC citizen-patriots building, repairing, and guarding the border fence, offering a vivid demonstration of the organization’s collective commitment to civic duty and responsibility. The theme of eternal vigilance recurs Bordering the Civic Imaginary 57

58 D. R. DeChaine regularly in MCDC discourse and serves to codify its sacred pledge to the American people. On the California border, an MCDC-run outpost named Camp Vigilance serves as a base for the group’s semi-annual musters, from whence fence-building teams are organized and deployed. Additionally, the MCDC offers an opportunity to enact eternal vigilance virtually. On joining its ‘‘Become a Cyber Minuteman!’’ campaign, those owning property on the U.S.􏰀Mexico border are provided with motion-sensing surveillance cameras that down-stream video directly to MCDC representatives, local police departments, and U.S. Border Patrol agents.69 What is apparent from this brief examination of the MCDC’s rhetoric is that its impassioned call for greater U.S. border security bespeaks an intense border insecurity. Relying on common tropes of American identity in the crafting of its appeals, the group’s rhetoric reveals a stock of motivations consonant with broadly shared majoritarian anxieties. Through hyperbolic appeals to the oblique yet powerful values of social unity, national security, rule of law, and civic responsibility, the group crafts a vivid human drama in which modern Minutemen take up their historical calling to pledge eternal vigilance, dutifully safeguarding American borders from the threat that lies beyondand, increasingly, within. The current corruption of the U.S. government, its political process, its economy, and its cultural character provides proof that the danger posed by millions of necessary but unwelcome border- crossing aliens is real. The alien invasion strikes at the very heart of national civic identity, threatening its purity. Harboring terror both known and unknown, the invasion must be stopped; the future safety of American citizens and the sanctity of American values are at stake. The fear of small but increasing numbers of illegal aliens combines with anxieties of national disunity and a federal government complicit in a hegemonic project of colonization. Only a counter-hegemonic movement led by a unified corps of committed American patriots can hope to stop the invasion, fence the border, protect national sovereignty, and illuminate a path toward civic restoration. As its appeals make clear, the MCDC relies on a fence logic that underwrites its construction of the immigration problem. In characterizing the threat of an alien invasion as preeminently an issue of border security, the MCDC contributes to an essentializing epistemology of the border. In its public discourse, national borders are static and given; their realness is never in question. The implications of such an epistemology and the worldview it constructs are not only political but profoundly ethical. Fence logic works to (re)inscribe the naturalness of geographical borders, positing the alien as a subject who is naturally out of place. Border security is thus framed as being about legal enforcement, not about wielding power and control. As Joseph Nevins explains, [T]he ‘‘illegal’’ is someone who is officially out of placein a space where he does not belong. Thus, the official relationship of the ‘‘illegal alien’’ to the particular national space in which he finds himself defines his status. The practice of territorialitythe effort to exert influence over people and/or other phenomena by asserting control over a defined geographic areareinforces the designation of the ‘‘illegal.’’ Territoriality helps to obfuscate social relations between controlled and

Bordering the Civic Imaginary 59 controller by ascribing these relations to territory, and thus away from human agency.70 It is not only geographical borders that are naturalized. As a territorializing apparatus, the MCDC’s fence logic also works to (re)inscribe the social-cultural borders as natural. Defining clear and self-evident lines between American citizen and alien invader, as the MCDC does, reveals the ease with which psychic territories and their cultural investments map onto and reinforce physical territories and their geopolitical investments. More importantly, the us/them binary that such bordering practices work to cement into place all but guarantees an irredeemable non-place for the racialized, alienized, border-crossing migrant in the United States today. Ultimately, as the MCDC’s rhetoric illustrates, the naturalization of borders reflects an affect-charged, ideological operation. It provides an anxiety-ridden American ‘‘we’’ with terms for ordera common-sense orientation for gauging the truth about people, places, social statuses, and communal allegiances. Bordering the Civic Imaginary I have argued for the centrality of the figure of the border in contemporary rhetorical culture. In so doing, I have advanced an account of political and cultural abjection that prompts a scholarly reorientation away from a static concept of borders toward a dynamic conception of bordering practices. Proposing alienization as the name for a racializing project that produces an abject migrant subject, I have focused attention on the articulation of discourse and affect and its labor across geopolitical and sociocultural space to subjugate the border-crossing migrant. In particular, I have aimed to convey a sense of the panicked character of alienizing rhetoric, and, through the example of the Minuteman Civil Defense Corps, to illustrate the enduring tenacity of fence logic in a globalized world. Although reactionary in tone, the symbolic strategies that the MCDC uses to craft its appeals, and the attitudes it expresses, are far from radical. The liberal democratic values propounded by the groupunity, national security, the rule of law, patriotism, and responsibilityand the ordinariness of the language and images it mobilizes to advance its cause demonstrate the group’s close affinities with the tradition of U.S. social justice movements. As such, its narrative offers a cautionary tale about the all too easy slide from communal appeals based on majoritarian values to violent ethnonationalist exhortations. Writ large, the fears and anxieties that spur the MCDC’s alienizing practices signal the increasing friction between the denationalizing tendencies of economic and cultural globalization and a redoubling of desire for a singular national community, purity, and civic identitya condition betraying a long history of racial, ethnic, socioeconomic, and gender exclusions in the United States. The alienization enacted by the MCDC and other adherents of fence logic is goaded by an overriding fear of losing control: this fear itself is based on a fictional premise*that the American community (and its putative boundaries of identity and citizenship) was ever in control, secure in a state of order, sovereignty, and wholeness. ‘‘Borders,’’ as Anne

60 D. R. DeChaine Demo reminds, ‘‘function as an index of sovereignty because their very presence (real or imagined) symbolizes claims of authority over a territorial entity.’’71 In this sense, border(ing) rhetorics serve as crucial sites for investigating intersections of community, territory, identity, and power in contemporary society. As the analysis illustrates, rhetorical renderings of the border are instrumental in the crafting of American civic community. In his discussion of borders and power relations, David Newman argues, [T]he stronger the barrier function of the border, the more powerful the imagined, the more abstract the narrative of what is perceived as lying on the other side. Perceptions of borders usually focus on what exists on the other ‘‘invisible’’ side of the line of separation. Borders exist in our mind by virtue of the fear we have of the unknown of the ‘‘there’’ and which, in turn, causes us to stay on our side of the border in the ‘‘here.’’72 Newman’s emphasis on the correlation between the fear of the unknown, the perceived integrity of borders, and the mystification of the other speaks to the current hold that fear has on U.S. civic culture. The oxymoronic alien subjectshe who is both known and unknown, simultaneously inhabiting the ‘‘here’’ and ‘‘there’’ of the national bodyincites a redoubling of political and moral boundaries in an era of an omnipresent war on terror. Burke’s dictum that ‘‘[a] way of seeing is always a way of not seeing’’ aptly characterizes the situation of the always already suspect border- crossing migrant.73 The intensifying compulsion to deal with the immigration problem, a defining exigency of post-9/11 America, underscores the terministic power of alienizing rhetoric to direct and reflect the social reality of the abject alien noncitizen. Put another way: as alienization is enacted, citizenship is enacted. Significantly, the fears and anxieties foregrounded in this discussion also attest to the force of the imagination as an integral agency in human social life.74 The construction of modern social imaginaries, according to Taylor, involves ‘‘the ways people imagine their social existence, how they fit together with others, how things go on between them and their fellows, the expectations that are normally met, and the deeper normative notions and images that underlie these expectations.’’75 Extending Taylor’s definition, my analysis of the excision of the alienized subject from the space of U.S. citizenship lends credence to an equally powerful notion of the civic imaginary as a social horizon for defining the constituents and limits of civic culture. A contested space for adjudicating conditions of citizen identity and inclusion, the civic imaginary is a contentious production with material consequences for those who deliberately or incidentally occupy it. Both real and imagined, performative and affective, it is a consummately rhetorical space where culture and politics converge, identity is shaped, and power is wielded. An adjunct of the public sphere, it proscribes conditions for citizenship enactment and the voices that are to be included in and excluded from deliberation.76 More than anything, the space of the civic imaginary serves as an ethical horizon for the articulation of the citizen as an embodiment of personhood. For alienizers like the MCDC, civic personhood reduces the border-crossing migrant to a racialized juridical subject. Crucially lacking in such a formulation is consideration of the border-crossing migrant as a human being

worthy of recognition, respect, and dignity. For this alone, the present construction of the American civic imaginary warrants concerted critical attention. Alienization continues apace in the United States today, with real implications for those alienized. In 2006, approximately 222,000 undocumented migrants were removed from the United States, roughly a 20 percent increase from the previous year.77 Moreover, as Ngai notes, ‘‘In the aftermath of the terrorist attacks on the United States on September 11, 2001, the Department of Justice used immigration laws to arrest and detain over 1,100 aliens, many without charge and in secret.’’78 Despite resistance in Congress and protests by numerous city mayors and property owners, plans to complete a security fence along the U.S.􏰀Mexico border have been recently stepped up by way of a presidential mandate granting Michael Chertoff, Secretary of the U.S. Department of Homeland Security, supra-constitutional authority to build it. Current U.S. immigration policies and the social attitudes that shape them do more than affect individuals’ legal status; they tear apart families, deny basic services to those in need, and create conditions whereby growing numbers of the U.S. population are demonized as necessary but unwelcome nonpersons. Such attitudes and conditions, as Dana Cloud has convincingly argued, are not new. Indeed, they are entirely congruent with the naturalizing border epistemology of Samuel Huntington’s ‘‘clash of civilizations’’ thesis and its ascendant hegemony.79 What is novel, I have claimed, is the discursive-affective alliance that shapes its present rhetorical form. The ordinariness of alienization as a mode of citizenship enactment beckons scholars to attend closely to the ways in which alienizing rhetoric works its way into the public vernacular. As I have hoped to show, its conception of the American citizen reveals a profoundly immoral discourse that excludes, racializes, and otherizes individuals and groups*a discourse all too readily conscripted for the cause of national unity in troubled times. A vibrant civic imaginary requires an understanding of personhood that moves beyond questions of legal status to include recognition and respect as formative constituents of citizenship. In the name of such a counter- hegemonic project, it is the debunking of alienizing practices that is urgently needed today. This does not mean substituting border logic for a logic premised on a ‘‘world without borders,’’ as some have suggested; to unborder, after all, is still a bordering practice.80 Rather, it entails a collective effort to recognize, make public, and work against alienization and its material and symbolic violence. Such a project, I have insisted, begins by taking borders seriously. In his meditation on the ‘‘unreflective nationalism’’ that an uncritical view of borders is currently fomenting in U.S. civic culture, Robert Chang observes, ‘‘Although the border is everywhere, your perspective may render it invisible. It is through this invisibility that the border gains much of its power.’’81 Working to unmask this invisibility will be vital to a project that aspires to broaden rather than border the horizon of the civic imaginary. Bordering the Civic Imaginary 61

62 D. R. DeChaine Notes [1] Jeffrey S. Passel, ‘‘The Size and Characteristics of the Unauthorized Migrant Population in the U.S.,’’ Pew Hispanic Center, March 7, 2006, http://pewhispanic.org/files/reports/61.pdf/. It must be acknowledged that this is a contested statistic. My choice of ‘‘migrant’’ to characterize undocumented persons in the United States reflects my intention to leave open the variety of statuses, motivations, and modes of mobility of border-crossing individuals. For a sympathetic discussion of the term, see Mae M. Ngai, Impossible Subjects: Illegal Aliens and the Making of Modern America (Princeton: Princeton University Press, 2004), xix􏰀xx. [2] Charles Taylor, ‘‘The Politics of Recognition,’’ in Multiculturalism and ‘‘The Politics of Recognition,’’ ed. Amy Gutmann (Princeton: Princeton University Press, 1992), 25􏰀73. [3] Kent A. Ono and John M. Sloop, Shifting Borders: Rhetoric, Immigration, and California’s Proposition 187 (Philadelphia: Temple University Press, 2002), 26. [4] Ronald Walter Greene, ‘‘Malthusian World(s): Globalization, Race, and the American Imaginary in the Immigration Debates of the Twentieth Century,’’ in Argumentation and Values: Proceedings of the Ninth SCA/AFA Conference on Argumentation, ed. Sally Jackson (Annandale, VA: Speech Communication Association, 1995), 191􏰀95. See also Marouf Hasian Jr. and Fernando Delgado, ‘‘The Trials and Tribulations of Racialized Critical Rhetorical Theory: Understanding the Rhetorical Ambiguities of Proposition 187,’’ Communication Theory 8 (1998): 245􏰀70; Justin Akers Chaco ́n and Mike Davis, No One Is Illegal: Fighting Violence and State Repression on the U.S.􏰀Mexico Border (Chicago: Haymarket Books, 2006). [5] Michael Curry, ‘‘On Space and Spatial Practice in Contemporary Geography,’’ in Concepts in Human Geography, ed. Carville Earle, Kent Mathewson, and Martin Kenzer (Lanham, MD: Rowman and Littlefield, 1995), 3􏰀32; Henk Van Houtum, ‘‘The Geopolitics of Borders and Boundaries,’’ Geopolitics 10 (2005): 672􏰀79. [6] D. Robert DeChaine, ‘‘Imagined Immunities: Border Rhetorics and the Ethos of Sans Frontie`risme,’’ in Interdisciplinarity and Social Justice: Revisioning Academic Accountability, ed. Ranu Samantrai, Joe Parker, and Mary Romero (New York: State University of New York Press, in press). [7] Phil Hubbard, Rob Kitchin, Brendan Bartley, and Duncan Fuller, Thinking Geographically: Space, Theory and Contemporary Human Geography (New York: Continuum, 2002), 33; Joe Moran, Interdisciplinarity (New York: Routledge, 2002), 165. [8] On the rhetorical constructedness of borders, see Lisa A. Flores, ‘‘Constructing Rhetorical Borders: Peons, Illegal Aliens, and Competing Narratives of Immigration,’’ Critical Studies in Media Communication 20 (2003): 362􏰀87; D. Robert DeChaine, Global Humanitarianism: NGOs and the Crafting of Community (Lanham, MD: Lexington Books, 2005). On the ambivalent character of border(ed) subjects, see Gloria Anzaldu ́ a, Borderlands/La Frontera: The New Mestiza (San Francisco: Aunt Lute, 1987); Pablo Vila, Crossing Borders, Reinforcing Borders: Social Categories, Metaphors, and Narrative Identities on the U.S.􏰀Mexico Frontier (Austin: University of Texas Press, 2000). [9] Aimee Carrillo Rowe, ‘‘Whose ‘America’? The Politics of Rhetoric and Space in the Formation of U.S. Nationalism,’’ Radical History Review 89 (2004): 119􏰀20. [10] I borrow the term ‘‘alienization’’ from Joseph Nevins, Operation Gatekeeper: The Rise of the ‘‘Illegal Alien’’ and the Making of the U.S.􏰀Mexico Boundary (New York: Routledge, 2002), 143. [11] Arjun Appadurai, Fear of Small Numbers: An Essay on the Geography of Anger (Durham, NC: Duke University Press, 2006), 51􏰀52. [12] Robert Asen, ‘‘A Discourse Theory of Citizenship,’’ Quarterly Journal of Speech 90 (2004): 204. [13] Minuteman Civil Defense Corps, ‘‘Minuteman Civil Defense Corps Replaces Volunteer Administrators,’’ http://minutemanhq.com/hq/print.php?sid􏰁342/.

Bordering the Civic Imaginary 63 [14] Minuteman PAC, http://www.minutemanpac.com/about.php/. [15] Flores, ‘‘Constructing Rhetorical Borders,’’ 381. See also Robert S. Chang and Keith Aoki, ‘‘Centering the Immigrant in the Inter/National Imagination,’’ California Law Review 85 (1997): 1397; Linda Bosniak, The Citizen and the Alien: Dilemmas of Contemporary Membership (Princeton: Princeton University Press, 2006). [16] On the complementarity of critical rhetoric and cultural studies, see Maurice Charland, ‘‘Rehabilitating Rhetoric: Confronting Blindspots in Discourse and Social Theory,’’ Communication 11 (1990): 253􏰀64; Thomas Rosteck, ed., At the Intersection: Cultural Studies and Rhetorical Studies, Revisioning Rhetoric: A Guilford Series (New York: Guilford Press, 1999), 1􏰀23. [17] Flores, ‘‘Constructing Rhetorical Borders,’’ 363. [18] Pierrette Hondagneu-Sotelo, ‘‘Unpacking 187: Targeting Mejicanas,’’ in Immigration and Ethnic Communities: A Focus on Latinos, ed. Refugio I. Roch ́ın (East Lansing: Julian Samora Research Institute, Michigan State University, 1996), 93. [19] Ngai, Impossible Subjects, 3. [20] Ngai, Impossible Subjects, 129. [21] Kenneth Burke, Language as Symbolic Action: Essays on Life, Literature, and Method (Berkeley: University of California Press, 1966), 44􏰀62. [22] Mark Lawrence McPhail, The Rhetoric of Racism Revisited: Reparations or Separation? (Lanham, MD: Rowman and Littlefield, 2002), 66. [23] Hemant Shah, ‘‘Race, Nation, and Citizenship: Asian Indians and the Idea of Whiteness in the U.S. Press, 1906􏰀1923,’’ Howard Journal of Communications 10 (1999): 251. [24] Howard Winant, Racial Conditions: Politics, Theory, Comparisons (Minneapolis: University of Minnesota Press, 1994), 59. [25] Ono and Sloop, Shifting Borders, 27. [26] See, for example, Marx’s discussion of alienation in ‘‘Economic and Philosophical Manuscripts,’’ in Karl Marx: Early Writings, trans. Rodney Livingstone and Gregor Benton (London: Penguin Books, 1975), 279􏰀400. [27] Appadurai, Fear of Small Numbers, 8􏰀10. [28] Appadurai, Fear of Small Numbers, 7􏰀9. [29] Appadurai, Fear of Small Numbers, 45. [30] Appadurai, Fear of Small Numbers, 21􏰀31. [31] Douglas S. Massey, ‘‘Understanding America’s Immigration ‘Crisis’,’’ Proceedings of the American Philosophical Society 151 (2007): 309􏰀27. [32] It is worth adding that women and men are often differently alienized. As some scholars have noted, the meaning of ‘‘alien’’ is itself gendered, shifting its connotation according to particular sociohistorical exigencies. Hondagneu-Sotelo, for example, notes, ‘‘Contemporary xenophobia targets women and children because it is they who are central to making settlement happen’’ (‘‘Unpacking 187,’’ 93). [33] Burke, Language as Symbolic Action, 18. [34] Hasian and Delgado, ‘‘Trials and Tribulations,’’ 257. [35] Otto Santa Ana, ‘‘‘Like an Animal I Was Treated’: Anti-immigrant Metaphor in U.S. Public Discourse,’’ Discourse and Society 10 (1999): 191􏰀224. See also Sarah Hill, ‘‘Purity and Danger on the U.S.􏰀Mexico Border, 1991􏰀1994,’’ South Atlantic Quarterly 105 (2006): 777􏰀99. [36] Flores, ‘‘Constructing Rhetorical Borders,’’ 363. [37] With a recent increase in applications for U.S. naturalization, and under pressure to err on the side of caution in vetting applicants for potential security threats, federal examiners have increased their rejection rates. In 2007, for example, approximately 12 percent of applications for naturalization were denied. See Julia Preston, ‘‘Perfectly Legal Immigrants, Until They Applied for Citizenship,’’ New York Times, April 12, 2008.

64 D. R. DeChaine [38] Bhikhu Parekh, ‘‘What Is Multiculturalism?’’ Multiculturalism: A Symposium on Democracy in Culturally Diverse Societies 484 (December 1999), http://www.india-seminar.com/1999/ 484/484%20parekh.htm/. [39] Burke, Language as Symbolic Action, 3􏰀24. [40] Chris Simcox, ‘‘About Us,’’ Minuteman Civil Defense Corps, http://www.minutemanhq.com/ hq/aboutus.php/. [41] Kevin Michael DeLuca and Jennifer Peeples, ‘‘From Public Sphere to Public Screen: Democracy, Activism, and the ‘Violence’ of Seattle,’’ Critical Studies in Media Communication 19 (2002): 125􏰀51. [42] The Border Fence Project emerged on the heels of the 2006 Secure Fence Act, authored by California Republican Representative Duncan Hunter, which required the U.S. Department of Homeland Security to build 854 miles of fence along the 1950-mile U.S.􏰀Mexico border. [43] Chris Simcox, ‘‘Senate and President Promote Anarchy at Borders and in American Cities,’’ Minuteman National Blog, April 11, 2006, http://www.minutemanhq.com/b2/index.php/ national/2006/04/11/. [44] Border Fence Project, BorderFenceProject.com, http://www.borderfenceproject.com/. [45] Chris Simcox, ‘‘Minuteman Corps Expands is [sic] Efforts to Secure the Border,’’ Minuteman Border Fence, January 2, 2007, http://www.minutemanhq.com/bf/pl7.php/. [46] ‘‘About Us,’’ Minuteman Border Fence, http://www.minutemanhq.com/bf/about.php/. [47] Simcox, ‘‘Minuteman Corps.’’ [48] Jim Wood, ‘‘A Letter to My Fellow Americans,’’ BorderFenceProject.com, http://www.border fenceproject.com/letter.shtml/. [49] See, for example, Minuteman Border Fence. [50] Chris Simcox, ‘‘Open Letter to the President of the United States,’’ MCDC Forums, June 19, 2007, http://forum.minutemanhq.com/phpBB2/viewtopic.php?t􏰁11793/. [51] Chris Simcox, interview by Trish Hinojosa, NOW on the News, PBS, May 4, 2007. [52] Chris Simcox, interview by Alan Colmes, Hannity & Colmes, Fox News, April 3, 2006. [53] Susy Buchanan and David Holthouse, ‘‘Minuteman Leader has Troubled Past,’’ Southern Poverty Law Center, http://www.splcenter.org/intel/news/item.jsp?aid􏰁149&site_area􏰁 1&printable􏰁1/. [54] Simcox, ‘‘Open Letter’’; ‘‘Donate to the Minuteman Border Fence,’’ Minuteman Border Fence, https://secure.responseenterprises.com/mmfence/?a􏰁571. [55] The MCDC works to document what it perceives as an increase in criminal activity in the United States as a result of unsecured borders. For example, the online MCDC forum includes a main thread titled ‘‘Illegal Alien Crime,’’ with the subheading ‘‘Please post any articles about illegal alien crime and DUI incidents here. Having these in one place will illustrate the tragic consequences of illegal migration.’’ ‘‘MCDC Forums,’’ Minuteman Civil Defense Corps, http://forum.minutemanhq.com/phpbb2/. [56] Simcox, ‘‘Minuteman Corps.’’ [57] ‘‘DIY Border Fence,’’ PirateNews.org, http://www.piratenews.org/newswire/html/. No docu- mentation for either of these statistics is provided on the website. [58] Brian Bonner, ‘‘Minutemen to Build Arizona-Mexico Border Fence,’’ April 20, 2006, http:// bonner.wordpress.com/2006/04/20/minutemen-to-build-arizona-mexico-border-fence/. [59] Chris Simcox, ‘‘Message from Chris Simcox,’’ Minuteman Border Fence, http://www. minutemanhq.com/bf/about.php/. [60] Simcox, ‘‘Open Letter.’’ [61] Stuart Hall, ‘‘The Whites of Their Eyes: Racist Ideologies and the Media,’’ in The Media Reader, ed. Manuel Alvarado and John O. Thompson (London: BFI Publishing, 1990), 13. [62] Minuteman Civil Defense Corps, ‘‘About Us.’’ [63] Minuteman Civil Defense Corps, ‘‘The Minuteman Pledge,’’ http://www.minutemanhq.com/ hq/mmpledge.php/. [64] Wood, ‘‘Letter to My Fellow Americans.’’

Bordering the Civic Imaginary 65 [65] ‘‘Build It Now!’’ Minuteman Border Fence, http://www.minutemanborderfence.com/. [66] Simcox, ‘‘Open Letter.’’ [67] Minuteman Civil Defense Corps, ‘‘Minuteman Pledge.’’ [68] Simcox, ‘‘Minuteman Corps.’’ [69] ‘‘Become a Cyber Minuteman!’’ BorderFenceProject.com, http://www.borderfenceproject. com/cybermm.php/. [70] Nevins, Operation Gatekeeper, 147􏰀48. [71] Anne Demo, ‘‘Sovereignty Discourse and Contemporary Immigration Politics,’’ Quarterly Journal of Speech 91 (2005): 295. [72] David Newman, ‘‘On Borders and Power: A Theoretical Framework,’’ Journal of Borderlands Studies 18 (2003): 20. [73] Kenneth Burke, Permanence and Change: An Anatomy of Purpose (New York: New Republic, 1935), 70. [74] Cornelius Castoriadis, The Imaginary Institution of Society, trans. Kathleen Blamey (Cam- bridge, MA: MIT Press, 1987). See also Arjun Appadurai, Modernity at Large: Cultural Dimensions of Globalization (Minneapolis: University of Minnesota Press, 1996); Charles Taylor, Modern Social Imaginaries (Durham, NC: Duke University Press, 2004); Benedict Anderson, Imagined Communities: Reflections on the Origin and Spread of Nationalism (London: Verso, 1983). [75] Taylor, Modern Social Imaginaries, 23. [76] See, for example, Michael Warner, Publics and Counterpublics (New York: Zone Books, 2005); Dilip Parameshwar Gaonkar and Benjamin Lee, eds., ‘‘New Imaginaries,’’ Special Issue, Public Culture 36 (2002); Robert Asen and Daniel C. Brouwer, eds., Counterpublics and the State (Albany: State University of New York Press, 2001); Craig Calhoun, ed., Habermas and the Public Sphere (Cambridge, MA: MIT Press, 1993). [77] Julia Preston, ‘‘As Pace of Deportation Rises, Illegal Families Are Digging In,’’ New York Times, May 1, 2007. I was unable to locate reliable data regarding 2007 deportation rates. [78] Ngai, Impossible Subjects, 269. [79] Dana L. Cloud, ‘‘‘To Veil the Threat of Terror’: Afghan Women and the BClash of Civilizations􏰂 in the Imagery of the U.S. War on Terrorism,’’ Quarterly Journal of Speech 90 (2004): 285􏰀306. [80] DeChaine, ‘‘Imagined Immunities.’’ [81] Robert S. Chang, ‘‘A Meditation on Borders,’’ in Immigrants Out! The New Nativism and the Anti-Immigrant Impulse in the United States, ed. Juan F. Perea (New York: New York University Press, 1997), 244, 246.

While we may imagine spring as a great season, where life begins to bloom from the earth, who exactly would think the opposite? Why would spring be considered cruel?

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Reply to the reviewers

We are grateful to all three reviewers for their careful analysis of the manuscript, and for their constructive comments. Two common critiques were:

(1) that assaying origin firing via an independent method would strengthen the conclusions, and (2) that additional analysis of ribonucleotide incorporation to exclude the retention of lagging-strand primers would allow us to definitively determine whether Pol ɛ plays a role in lagging-strand synthesis.

We will include experiments to address both critiques in a revised manuscript. To independently verify changes in origin efficiency, we will sequence nascent BrdU-containing DNA across a time course from cells released into S-phase: we will also use the last timepoint of our Okazaki sequencing analysis to control for potential cell-cycle differences. To further test the contribution of Pol ɛ and ascertain whether lagging-strand primers are retained, we will analyze ribonucleotide incorporation in both wild-type and pol2-M644L (Pol ɛ ribonucleotide hypo-incorporating) strains. We address individual specific comments and our planned revisions in more detail below.

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

This study examined the consequences of limiting levels of DNA polymerase d (Pol d) in yeast. The authors first reported multiple genome instability consequences following lowered Pol d level, including defect in S phase progression, growth defect, elevated spontaneous DNA damage, accumulation of ssDNA and activation of replication and DNA damage checkpoint. These observations are solid but not unexpected. By genome wide analysis using the Okazaki fragment (OF) mapping and ribonucleotide mapping (for polymerase usage), the authors claim a few potentially novel and striking observations that lowered Pol d differentially impact efficiencies of early vs. late origins, and that lowered Pol d results in Pol e participating in lagging strand synthesis around late origins. However, I remained unconvinced based on the data presented. These observations need to be further substantiated and alternative interpretations should be considered.

\*Main concerns:** *

One of major conclusions the authors tried to make is that the early vs. late origins are differentially affected by low level of Pol d. First, they used OF mapping data to examine origin efficiency. Asynchronous "Cultures were treated with IAA for 2h before the addition of rapamycin for 1h to deplete DNA ligase I (Cdc9) from the nucleus via anchor-away". IAA concentrations used were of 0, 0.2 mM, 0.6 mM, and 1 mM. The problem is that Figure S1 clearly showed that treating asynchronous cultures with >0.1 mM of IAA for as short as 30 min significantly alters the cell cycle profiles, mainly resulting in accumulation of S phase cells, to different extent. Presumably Okazaki fragments accumulated from these cultures suffering from the synchronizing effect may not be representative of the real change in global replication profile. For instance, it is not difficult to predict that the Okazaki fragments enrichment may be skewed towards late origins if more cells are accumulated in mid S phase following Pol d depletion. For this reason, I don't believe the result is conclusive. The experiment may be re-designed for samples at different time points following release from G1.

We agree that altered cell-cycle profiles might affect the number of Okazaki fragments sequenced in late vs early replicating regions of the genome. As noted by reviewer 3 in cross-commenting, these differences should not affect origin efficiency calculations as these are based on the ratio of reads on each strand (and therefore normalized). To more directly address this question, we will calculate origin firing efficiencies from the final timepoint of the arrest-release experiments shown in Figure 4 as suggested by the reviewer. We will also analyze origin efficiency using BrdU over a time course.

This concern also should preclude the authors from drawing conclusion about Pol e usage on lagging strands based on comparison between HydEn-seq data and OF mapping data shown in Figure 6. In fact, the rNMP incorporation change is very similar between early and late origins. The only evidence that the author rely on is the discrepancy in OF data between the two groups origins, which makes the reliability if origin efficiency measurement the central piece of data in this study. Thus, alternative approaches should also be considered to map origin efficiencies.

As noted above, we agree that an independent method of tracking origin firing efficiencies would be helpful to strengthen our conclusions. To this end, we will analyze time courses of BrdU incorporation from cultures released into S-phase.

Even if Pol e strand bias is lowered at late origins, as the authors tend to believe, there are still alternative models other than Pol e being used for lagging strand synthesis. For instance, if TLS polymerases are used on lagging strands, it could result in more ribonucleotide incorporation on the lagging strand, as they are lower-fidelity polymerases. Alternatively, if Pol d scarcity leads to more Pol e synthesis or lower RNA primer processing, it might also contribute to more apparent ribonucleotide incorporation on the lagging strands.

We feel that the widespread use of TLS polymerases is unlikely, especially given the data in figure 6A that show no growth or viability change upon deletion of all three TLS polymerases in the Pol ∂ depletion strain, even at very low levels of Pol ∂. We agree with the reviewer that our data do not conclusively rule out increased retention of lagging-strand primers – as we state in the text. We aim to test this possibility by analyzing ribonucleotide strand bias in a pol2-M644L strain that incorporates fewer ribonucleotides than the wild-type Pol ɛ. In this case, increased lagging-strand primer retention would lead to a lagging-strand bias of ribonucleotides upon Pol ∂ depletion, while increased Pol ɛ usage would not. An analogous experiment with wild-type POL2 is potentially harder to interpret because the wild-type polymerase is the predominant source of ribonucleotides in a wild-type strain (Nick McElhinny et al, 2010 - PMID:20729855), but we now have the data for this strain in hand and ready to analyze.

In Figure S5, the two HydEn-seq replicates are very different, where replicate1 shows very low strand bias. I suspect perhaps the strain used for replicate 1 does not contain pol2-M644G or rnh202 deletion.

The change in ribonucleotide incorporation is indeed substantially stronger in one replicate than the other. We have additional time-course data from a Pol ∂ depletion showing that ribonucleotide strand bias decreases over time as Pol ∂ is depleted, and will include this in a revised manuscript.

Reviewer #1 (Significance (Required)):

Given that different aspects of Pol d deficiency have been implicated in various human diseases and cancer, this type of analysis is of interest to the field.

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

\*Summary** *

In this manuscript the authors explore the basis of the deleterious effects of reduced levels of the replicative Polymerase delta. This polymerase plays several important roles in the replication process: it synthesizes most of the lagging strand, but also extends the first primer during the synthesis of the lagging strand, and it contributes to the removal of the RNA and most of the DNA synthesized by primase/Polymerase alpha during Okazaki fragment maturation. In this study, the authors systematically analyze the impact of Pol delta depletion in S. cerevisiae. They use a degron-tagged allele to modulate the levels of the polymeraes and apply mainly NGS methods and classical genetics to explore the consequences for survival, checkpoint signalling and replication features such as fork speed, origin firing efficiency and Okazaki fragment length and distribution. They report that Pol delta depletion leads to a checkpoint activation via the Rad9-dependent damage signalling pathway (but not the Mrc1-dependent replisome-associated signalling) and an accumulation of single-stranded DNA. Phosphorylation of histone H2A is taken as a marker of DNA double-strand breaks, and from the observation that deletion of recombination factors, but not end-joining factors aggravate the fitness of Pol delta-depleted cells they conclude that homologous recombination is responsible for the repair of these breaks. Analysis of replication by Okazaki fragment sequencing indicates a slight decrease in the firing efficiency of early/efficient origins, but an increase in the firing efficiency of late origins. They also observe a reduction in fork speed, although they are not able to attribute this to either a globally slower fork movement or an increase in the stalling of individual forks. They find that Pol delta depletion does not change the size of Okazaki fragments, but causes defects in the nick translation during Okazaki fragment maturation. Finally, they use NGS technology to show that the leading strand Polymerase epsilon engages in lagging strand replication particularly at late origins when Pol delta is depleted. From their observations, the authors develop a model where depletion of Pol delta primarily affects late replicating regions. They explain this by invoking a stable association of Pol delta with early replisomes, which sequesters the enzyme, thus causing an under-supply at replisomes that assemble later during S phase. This then leads to the involvement of Pol epsilon on the lagging strand. Based on the observation that fork speed and Okazaki fragment maturation are both affected, they propose that these two reactions normally compete for Pol delta, suggesting that optimal replication would require two molecules of the polymerase per fork.

\*Major comments** *

The experiments shown here are largely clean and well controlled, and the manuscript is written nicely and well-structured.

Compared to the Okazaki fragment analysis, the treatment of double-strand breaks appears somewhat cursory and remains inconclusive. Phosphorylation of H2A seems insufficient evidence for double-strand breaks, as other structures could also give rise to that signal. These lesions should be detected in a more direct manner, e.g. pulsed-field gel electrophoresis. The authors also don't provide a mechanism by which such breaks would emerge. Related to the minor effect of the ku mutant, I am wondering about the altered appearance of the colonies in Figure 2F (concerning both ku70 and rad51) - what is different about these, and could their „denser" appearance explain the slight suppression effect observed?

We agree that our treatment of double-strand breaks is limited: consistent with comments from all three reviewers about which aspects of this work are most novel, we intend to focus as much as possible on replication enzymology here. We will tone down the language around double-strand breaks in the manuscript.

Concerning the damage signalling: it is surprising to see a damage signal at concentrations of IAA that do not lead to a significant depletion of Pol delta yet (0.05 mM). At this point, it is hard to imagine DSBs to form. Could the authors explain this discrepancy?

We note that, as observed in Figure 1A and to a slightly lesser extent in Fig. 2E, Pol ∂ levels are already substantially reduced in 0.05 mM IAA. This reduction appears sufficient to induce damage

The notion that late origin firing is enhanced despite checkpoint activation is counterintuitive. Do the authors think that this effect overcomes the suppression of late origins that is normally associated with checkpoint activation? It would be helpful to test whether abolishing that phenomenon (e.g. by a mec1-100 mutant) would enhance the effect and render late origins even more active.

We thank the reviewer for this excellent suggestion: we will test the effects of a mec1-100 mutant and include the results in a revised manuscript.

It would be important to characterise the fork speed defect better, using alternative methods rather than just relying on Okazaki fragments. A differentiation between slower fork progression and more frequent fork stalling would be relevant and might help to evaluate the contribution of Pol epsilon. This might be accomplished by DNA fibre analysis. Alternatively, BrdU incorporation could serve to observe replication over the entire genome rather than only in the vicinity of replication origins. It would also be important to differentiate fork speeds in early versus late replicating regions - according to the authors' model, the defects should be most obvious in the late regions (Fig. 4 concerns only early origins).

As noted above in our response to reviewer 1, we will use BrdU incorporation to independently verify changes in fork speed and origin firing. Analysis of fork speed in late-replicating regions is challenging regardless of the methodology used, due to contributions from converging forks, but we will try to do this

Figure S3: Considering the differences in cell cycle progression, it would make more sense to compare equivalent stages of the cell cycle / S phase rather than identical time points.

We can include this analysis, although the changes in cell cycle progression and origin firing efficiency make such comparisons somewhat subjective

Considering that the Okazaki fragment analysis was done with non-synchronised cultures, is it possible that the skew in the cell cycle profile could influence the Okazaki fragment pattern?

(copy-pasted from our response to a similar query by reviewer 1)… We agree that altered cell-cycle profiles might affect the number of Okazaki fragments sequenced in late vs early replicating regions of the genome. As noted by reviewer 3 in cross-commenting, these differences should not affect origin efficiency calculations as these are based on the ratio of reads on each strand (and therefore normalized). To more directly address this question, we will calculate origin firing efficiencies from the final timepoint of the arrest-release experiments shown in Figure 4 as suggested by the reviewer.

Would it be possible to monitor not only total Pol delta levels, but also the level of Pol delta bound to the chromatin? It is shown that the level of Pol delta is depleted in the whole cell extracts, but it would be interesting to see how severe the depletion is on the chromatin.

We agree that the relative fraction of chromatin-bound vs free Pol ∂ is an interesting question, and will attempt this experiment. However, we note that extensive depletion of Pol3 makes it hard to detect by Western blot, so the results are likely to be most informative at modest depletion levels. Regardless, these data should give us an idea of the size of the ‘free’ Pol ∂ pool in cells with normal or mildly reduced Pol ∂.

Figure 6 is confusing and should be clarified: - Figure 6B: assigning the Watson and Crick strands in the schematic would make that figure easier to understand; - Figure 6B-C: the axes are labeled as 'Fraction of rNMP on Watson strand', but would it not make more sense if they were labeled 'Fraction of rNMP in Crick strand'? - Figure 6D-E: the right side scale is labelled as 'increase in rNMP on Crick strand' while in the figure legend is says it is 'change in the fraction of ribonucleotides mapping to the Watson strand. That description should be clarified; - Figure 6D: using 'Change in Okazaki fragments strand bias' to label the black line (description in the box above the figure) instead 'Change in Okazaki strand bias' would be more appropriate; - Figure 6F: the authors seem to have subtracted strand bias measured for Okazaki fragments from the strand bias measured for rNMP. It is valid to subtract these biases from each other, considering that the two structures arise independently and with different frequencies?

We can make changes to figure 6 as suggested. Regarding the validity of subtracting strand biases, we think this is sufficient to give at least a semi-quantitative view of Pol ɛ usage since both of our sequencing approaches produce quantitative readouts that directly report on replication direction or polymerase usage, respectively.

\*Minor comments:** *

Can the authors conclude that Pol delta deficiency/ incompleteness of lagging strand synthesis affects the nucleosome deposition onto DNA? (Figure 5-A)

We cannot rule out that this is occurring, and we agree that this is an interesting question for future studies. But the changes that we observe Okazaki fragment terminus location are very similar to our previously published observations from cells lacking Rad27 function, consistent with decreased nick translation.

Why did the authors use rnh202Δ and not a mutant in the catalytic subunit of RNase H2?

Deletion of any subunit of the heterotrimeric RNase H2 complex completely abolishes its function in yeast, so RNH202 was a somewhat arbitrary choice

An extra control might be useful: comparing POL3-AID rnh202Δ with the POL3-AID pol2M644G rnh202Δ triple mutant could further confirm that the observed effect is Pol epsilon-dependent.

We agree (see also our response to reviewer 1). In addition to the wild-type, we will analyze pol2-M644L – a mutant in which Pol ɛ incorporates fewer than normal ribonucleotides. An increase in ribonucleotide density on the lagging strand in pol2-M644L would support increased primer retention on the lagging strand.

Figure 2H: It would be good to see the cell cycle distribution corresponding to the western blot images.

We can include this

Various spelling, grammar or precision of expression issues: - Pg. 4, line 4: endonucleolytically instead of nucleolytically. - Pg. 6, line 10: Remove 'was'. - Pg. 6, line 12: Remove 'in vivo' from the subtitle. - Pg. 6, line 14: 'an C-terminal' should be 'a C-terminal' - Pg 16, line 13: Phrasing implies that the synthesis of both leading and lagging strands by Pol delta in regions in the vicinity of replication origins is essential - please quote any paper testing its essentiality. - Please follow standard yeast genotype nomenclature, remove ';' when listing the yeast genotypes (e.g. POL3-AID mec1Δ sml1Δ instead of POL3-AID;mec1Δ;sml1Δ- example from Figure 2-B). - Concentrations of IAA are missing in few places (e.g. legend of Figure 1-C, page 24). - Figure 1A: add the label 'IAA (mM)' - Figure 2G: pleae provide a shorter exposure of the H4 blot in addition to the one shown. - Figure 6: adding a schematic presenting the events at actively and passively replicating late origins (and the predictions about leading and lagging strand bias) would help to understand the figure. - The format of the references is inconsistent. - 'On Watson/Crick strand' should be replaced with 'in Watson/Crick strand' We will fix typos, etc

Reviewer #2 (Significance (Required)):

This is a nice piece of work that provides in vivo confirmation of several observations that have been made in purified recombinant systems. In that sense, the overall novelty is limited, but this type of study is still important to do, as biochemical assays do not always reflect what is happening in cells, and this study gives insight into basic activities of the replisome. The participation of Pol epsilon in lagging strand synthesis is an interesting observation. Overall, the study will be of interest for the DNA replication field. My own expertise is in replication, predominantly in yeast. I have experience in NGS analysis of replication as well as in genetic analysis of the DNA damage response. I therefore feel competent to evaluate all aspects of the manuscript.

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

This manuscript describes the consequences of reducing the cellular concentration of a pol-delta subunit in S. cerevisiae. Pol-delta plays multiple cellular roles at both the replication fork (it is one of two DNA polymerases responsible for lagging strand synthesis) and during repair synthesis after DNA damage. The authors combine genetic and genomic methodologies to characterise how reduction in pol-delta concentration impacts on cellular fitness and specifically lagging strand synthesis. Overall it is technically a well executed study that is clearly presented and the data are predominantly appropriately interpreted.

\*I have a number of major comments:** *

1) The authors apply OK-seq (a methodology first developed by the senior author and therefore they are clearly experts at this) to study the consequence of pol-delta depletion on genome replication. OK-seq requires isolation of Okazaki fragments and this in turn requires removal of DNA ligase (Cdc9) - the authors achieve this with the anchor away system. My concern is that in these experiments the authors are depleting two factors required for lagging strand synthesis: pol-delta and ligase; it is unclear to me how the authors can determine the relative contribution of each depletion to the observed phenotypes. Could some of the observed phenotypes (e.g. fork slowing, 5' and 3' ends of Okazaki fragments, etc) be a consequence of the double depletion, rather than just pol-delta depletion as concluded by the authors? The authors present this as a method to determine genome replication timing, but really it is an assay to look at fork direction. Given the need for an addition mutation in OK-seq (cdc9), I would encourage the authors to consider a more direct assay for replication dynamics upon pol-delta depletion, such as a copy number measure (or BrdU-ip) of DNA replication or DNA combing - these methods don't require Cdc9 depletion and could therefore ensure that observed phenotypes are a consequence of pol-delta depletion (rather than the double depletion).

As outlined in our response to the first two reviewers, we will do BrdU-IP experiments. We agree that the double depletion may have an effect on fork speed, and that BrdU-IP will allow us to test this possibility. However, we note that our analysis of Okazaki fragment initiation/processing requires the depletion of Cdc9, so for this we are limited to looking at differences between Cdc9 depletion alone vs Cdc9 depletion + Pol3 depletion.

2) One major conclusion reached by the authors is that pol-epsilon can contribute to lagging strand synthesis upon pol-delta depletion (at least during late replication). This conclusion comes from the authors use of HydEn-seq to measure rNTP incorporation from which the contribution of a polymerase (pol-epsilon in this case) to strand synthesis can be determined. In a manner analogous to OK-seq, this requires the introduction of additional mutations (both in the polymerase and by the removal of RNaseH activity). The authors interpretation that pol-epsilon can play a role in lagging strand synthesis is dependent upon there being no temporal change in pol-delta strand-displacement activity, despite continued pol-delta depletion through S phase. It is not clear to me that the data presented in Fig 5 & 6 has the sensitivity to conclude this (and the OK-seq data is also subject to the potential bias of the double depletion of pol-delta and Cdc9). I feel that a necessary control to support this conclusion, would be to undertake the HydEn-seq experiment in the absence of the pol-epsilon mutation (just pol-delta depletion in the absence of RNaseH activity). This would allow the authors to measure any increase is residual rNTPs (likely from pol-alpha primase) on the lagging strand as a consequence of pol-delta depletion and determine whether they are equally likely in early and late S phase.

As discussed in our response to the first two reviewers, we will analyze analogous data from both a POL2 wild-type and a pol2-M644L strain that incorporates fewer ribonucleotides than the wild-type.

\*The following comments are more minor:** *

-for the experiment in Fig S1B, the growth in 1.0 mM IAA is somewhat surprising given how sick the cells appear on equivalent plates. I couldn't find in the methods a description of the experimental conditions.

The cells grow very slowly in 1 mM IAA (doubling time doubles). We think this is quite consistent with the poor growth on plates

-there is considerable variability in the S phase kinetics from bulk DNA analysis (flow cytometry) when comparing Fig 1C, 2D, S3. Fig 1C appears to be the exception, with all the other figures showing poor S phase progress by comparison. It would be useful for the authors to recognise these differences and comment upon them. E.g. they appear to all be identical experiments, but are there experimental differences that could explain the different kinetics?

We see some variability in our release, but generally cells enter S-phase at around 30 minutes. The release in figure S3 was carried out at 25 ˚C rather than 30 ˚C, which accounts for the additional delay in these data

-Fig 2F, why is the rad51-deletion less severe that rad52-deletion - should they not be identical?

We agree that these should logically be very similar: we do not know why the two mutants behave slightly differently at some (but not all) IAA concentrations

-Fig 2H - could the authors show the flow cytometry (in a supplemental figure) for this experiment?

We can show this

-Fig 3B-E: OEM is described as a measure of origin efficiency - how is possible for this to have negative values?

OEM describes Okazaki fragment strand bias around previously identified origins. If such an origin does not fire in our strain background, a negative OEM can result.

-pp9: "Analysis of Okazaki fragment strand bias across the genome suggested that the average direction of replication was relatively similar at most loci across all Pol3 depletion conditions". The authors data is quantitative and they should be able to quantify how similar their data are across the various conditions, rather than making a qualitative statement: "relatively similar".

We apologize and can re-phrase this. The intention of this statement is simply to draw the reader’s attention to the fact that global distributions of Okazaki fragments are not completely altered (e.g. only 1-2 origins per chromosome) as a prelude to the more quantitative analysis that follows in figure 3.

-pp9: "origin firing efficiency in S. cerevisiae correlates strongly with replication timing"; it would be useful for the authors to support this statement with a citation.

We will add 1-2 citations to support this statement

-Fig 4A: it would help the reader if the authors could show 'zoomed in' examples of the points that the authors make (in addition to the whole chromosome view): slowed fork progression, reduced early origin activity, increased late origin activity (e.g. an origin that is normally passively replicated, that upon pol-delta depletion is no longer passively replicated and therefore becomes more efficient), etc.

We agree that this would be helpful, and can add examples in the supplement

-pp11: "An analogous global decrease in replication-origin firing efficiency has been observed in Pol ∂-deficient human fibroblasts" - but the authors are reporting a global increase in origin firing efficiency (Fig 3B).

We can re-phrase this.

-the nucleosomal ladder in Fig 5A is only weakly apparent from the gel and not particularly apparent from the density trace, this makes it's disappearance upon IAA treatment hard to interpret. Is the weak nucleosomal ladder what the authors had anticipated (in the absence of IAA)?

We do not expect a weaker nucleosomal ladder than normal in the absence of IAA. In our experience these gels just sometimes give better ladders than others, and we hope that the traces help with interpretation

-I found the effects being described by the authors in Fig 5B & C difficult to see, particularly for the transcription factors. Furthermore, why are these data differently normalised to those in Fig 4B & C (median vs. maximum)

In figure 4 we normalize to maximum since all DNA should eventually be replicated, and we therefore think that showing coverage relative to a maximum value of 1 is most informative. In figure 5 we compare distributions of termini around obstacles, and therefore feel that normalizing to the median is a more appropriate way to compare enrichment around a given meta-element. The shapes of the graphs would be unchanged by choosing a different normalization point. In order to make changes easier to see, we can make the lines thinner in figure 5 and/or change the y-axis scale.

-the final sentence of the results section returns to an analysis of the OK-seq data and is essentially a temporally segregated analysis (Fig S6) otherwise equivalent to that presented in Fig 5B. Given the importance placed on these data by the authors in the interpretation of the HydEn-seq data, I feel that it would help the reader to have been presented with these data earlier in the results section.

We can move these data up

-p22: OK-seq methods. The authors should indicate the culture conditions for these experiments.

We can include this

-p22: Computational analyses: the authors should indicate which reference genome assembly they used.

We can include this

-Fig 6B & C: the y-axis labels are confusing - do the authors mean Crick strand here?

Oops. Yes, we do. We thank the reviewer for catching this

\*REFEREE CROSS COMMENTING:** *

All three reviewers seems to be in broad agreement about this manuscript. There is one significant concern raised by the other reviewers that I'd missed: that some of the Okazaki fragment analysis was done with non-synchronised cultures. I agree with this concern, however I don't think that there is necessarily a problem with the alternative explanation suggested by reviewer #1 ('Okazaki fragments enrichment may be skewed towards late origins'). While the accumulation of S phase cells might well be expected to lead to a bias towards isolating more Okazaki fragments from around late origins, the authors calculate the fraction of reads (i.e. Okazaki fragments) mapping to each strand. The potential presence of more late S phase cells would give greater sequence coverage over late replicating regions, but alone would not alter the fraction of reads mapping to each strand. However, I agree that interpretation of this experiment is not as simple as suggested by the authors and there may well be alternative explanations along the lines suggested by reviewer #1.

There was a subsequent Okazaki fragment experiment performed with synchronised cells (Fig 4) and it might be possible to use these data to assess any differential impact on early vs late origins.

We agree, and will do this analysis

Reviewer #3 (Significance (Required)):

My expertise is in DNA replication and genome stability, particularly replication timing and replication origin function.

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

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Reply to the reviewers

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

In this article, the authors characterize a complex formed by sec22b-stx1-Esyt2. They investigate how such interactions are involved in the modulation of dynamics of the plasma membrane in the context of neuritogenesis. They conclude that the contact sites between the ER and the plasma membrane, mediated by the afore-mentioned complex, contribute the expansion of the plasma membrane.

**Major comments:**

Overall, the article clearly shows that in mammalian cells there is an interaction between sec22b-stx1-Esyt2 which appears to be important for filopodia formation and possibly neuritogenesis in neurons. However, performing additional experiments to better clarify some links and assumptions made by authors could strengthen the article.

The manuscript relies on work performed either on cell lines (HeLA, PC12) or primary neuronal cultures. Although it is clear the value of the findings obtained using cell lines, they should be seen as a complementary rather than an exclusive approach. This is particularly important as the authors often make claim about neuron-related cellular biology.

For instance, the biochemistry-based findings on the interaction and characterization of the protein complex (Figure 1) are all derived from experiments perfomed in Hela or PC12. As the authors have the capacity to culture and manipulate primary neuronal cultures, such findings should be validates in neuronal cells. The authors could also consider performing biochemical experiments (i.e. co-ip) of the endogenous proteins in neuronal cultures or brain tissue.*

->Endogenous Co-IP has been tried in E18 brain tissue. One experiment using brain tissue demonstrated co-immunoprecipitation of endogenous Sec22b and E-Syt2. Unfortunately, repetitions of this experiment failed due to high background in negative control (naïve Rabbit IgG). We agree with the reviewer that this data is worth trying again. We will carry out this co-immunoprecipitation experiment from cultured neurons to answer the reviewer’s request.

The authors do show some evidence regarding the complex in neuronal cells using PLA (proximity ligation assay, figure 2) or super resolution microscopy, however, these findings should be corroborated by stronger findings targeting interaction and not based on simple proximity.

->We agree with this reviewer that PLA is limited in demonstrating the occurrence of a protein complex. We would like to stress that we have used PLA complementarily to immunoprecipitation and that we already have shown STED super-resolution data (Figure 3). In order to strengthen the STED data, we will include more details in the figure, as a supplementary movie and a supplementary spreadsheet with the quantification of the distance between the E-Syt2/Sec22b clusters to the plasma membrane stained using WGA. The STED data demonstrate that 50% of the clusters are closer than 33.6nm to the plasma membrane, a distance in the range of ER-PM contact sites.

A similar critique regards the experiments using RNA-interference of Figure 4. Performing loss-of-function experiments in neuronal cultures would strengthen and complement the results obtained via over-expression approaches shown in Figure 5.

->The loss-of-function experiments in neuronal cultures using siRNA were attempted unsuccessfully. The three E-Syts have largely different cDNA sequences thus three distinct siRNAs must be transfected in order to silence all three simultaneously. This is quite challenging in neuronal cultures and we were never able to get strong silencing of the three E-Syts. In the following points, we plan to carry out further experiments using expression of a fragment of Sec22b (Longin domain). We are confident that this is a better approach to demonstrate the importance of Sec22b/E-Syt interaction.

*Given that the authors have already in place all the necessary technology for the suggested biochemical and morphological-related experiments, these could be carried out swiftly within 3-4 months.

**Minor comments:**

The manuscript is really technical and at times tough to follow; it could benefit from key sentences to better guide the reader, particularly if not coming from the specialist field, in the appreciation of the experiments and results.

Authors should submit the manuscript to a severe round of proofreading. There are several inconsistencies and sometimes what looks like internal comments: i.e. in the methods "STED Missing" or the fact that "LTP" is used everywhere but not defined and considering that the targeted audience is most likely neuroscience-based could easily lead to confusion.

*

->We fully agree with this reviewer and apologize for leaving behind such errors. We will carefully proofread the revised ms.

*The experiments appear to have been repeated a sufficient number of times and the statistics seem adequate. It would be advisable to show in dot-plots the findings rather than in bar graphs all findings and not just the morphometrics-relative ones.

*

->We will modify the figures according to this reviewer’s suggestion.

Reviewer #1 (Significance (Required)):

*This work closely follows the excellent previous work from the Galli laboratory. As such, it is mostly incremental from a technical perspective and does not present particularly novel findings. An interesting aspect would be in addressing directly the influence of the described interactions in the lipid transfer between ER and the plasma membrane but in that sense the manuscript falls short. Although it is to be appreciated the functional readouts in terms of neuritogenesis, in the present state the manuscript features findings suitable for a very specific audience.

I believe that the appropriate audience for the present manuscript lies within the neuroscientific community interested in development, specifically neuritogenesis, and/or membrane dynamics. Additionally, it might be interesting also for researchers outside of the neuroscience community and interested in the dynamics between ER and plasma membrane.

*

->We are happy to read the comments of this reviewer. Nevertheless, we would like to stress the importance of deciphering precise molecular mechanisms in any biological process. Here, we are the first to demonstrate an interaction between lipid-transfer proteins E-Syts and ER v-SNARE Sec22b. As an example, the molecular mechanism connecting synaptic SNAREs and synaptotagmin has been the topic of more than 500 publications since seminal articles in the early 1990’s. We think that the first article linking E-Syts to SNAREs cannot be considered as a mere increment from our previous work.

The activity of E-Syts to transfer lipids in vitro has been well established __(1–3) In addition, recent work by the De Camilli lab using Origami showed that reducing the distance between liposomes enhanced the lipid transfer mediated by E-Syt2 (3). Therefore, we did not carry out experiments such as combining SNAREs and E-Syt2 in artificial membranes in vitro because we considered that there would not be much more to demonstrate than what has already been done. Furthermore, we considered the experiments in cells, particularly neurons, much more critical at this point. Demonstrating transfer of glycerophospholipid between ER and PM in cells cannot be performed like other lipids’ transfer at other membrane interfaces for the following reasons: phospholipids are very abundant (4) and they are not modified upon transfer (1)__, there are no specific dyes to detect glycerophospholipids (unlike phosphoinositides), and ER and PM are too close to distinguish if a glycerophospholipid is in one or the other membrane. Such a challenging experiment would require the ability to setup a specific biochemical assay circumventing these constraints. We think that this is out of the scope of the present study focused on the role of E-Syt/Sec22b complex.

Nevertheless, in order to get further insights on this question, we will express WT and mutant E-Syt2, purify the PM using the protocol of Figure 4 in Saheki et al __(1)__, followed by lipidomics analysis. We hope that this approach further supports our idea that E-Syts mediate an important lipid transfer mechanism towards the PM.

* Keywords regarding my expertise: Molecular and Cellular Neuroscience, Morphometrics, Dendrite, Neurons, Dendritogenesis, Biochemistry, Imaging, Microscopy.

__

Reviewer #2 (Evidence, reproducibility and clarity (Required)): *This manuscript identifies and characterises a novel interaction between E-Syts and Sec22b and demonstrates that lipid transfer between the ER and PM contributes to the development of filopodia and neuronal expansion. This interaction with E-Syt2 occurs through the Longin domain of Sec22b Sec22b association. The authors suggest a continuum with further interactions with syntaxin1, that mediates neurite outgrowth. Overall I find this study very interesting and convincing. The experimental analysis is well carried out and the claims are well aligned with their results.

I only have minor issues:

Figure 1. Some of the western blots have several bands and it is difficult to know which band is the relevant one. They should be indicated in the fig panel. Further panel E and F are barely readable and should be redrawn with the appropriate line and font size.*

->We will make the changes requested by this reviewer in Figure 1.

• *

Figure 2: is there a difference between the number of dots in axons and dendrites? Can the author elaborate on this aspect as it is not clear from the image presented.

->We could not combine PLA with further staining of MAP2 and TAU. Indeed, to perform PLA, neurons are already double labelled to detect the proteins of interest. At the stage of the neurons used in this study, both axons and dendrites are growing. Therefore, we did not invest in distinguishing between axons and dendrites. Because growth cones are known to be the major sites of membrane growth, we instead distinguished dots within neurites and in growth cones. We will make the other changes requested by this reviewer in Figure 2.

Figure 7: statistical analysis should be indicated from the BoNT/A and BoNT/C as BoNT/A represent an appropriate control cleaving SNAP25 but not Syntaxin.

->We agree with this request and we will add statistical analysis as suggested, using BoNT/A as an additional control.

On top of controlling fusion and neuronal outgrowth, syntaxin has a role in survival and its cleavage leads to neuronal death. Is this pathway mediated by E-Syts interactions?

->We have stated in the ms: “Since exposure to BoNT/C1 at high concentrations and for long incubation periods causes degeneration of neurons in culture __(5,6)__, various concentrations and incubation times were tested, and a 4-hour treatment of neurons with 1nM BoNTs was chosen to avoid such deleterious effects.” Accordingly, we did not see any degeneration in our experimental conditions.__ __

Reviewer #2 (Significance (Required)): This papers identifies the molecular mechanism of neuronal outgrowth. It is highly significant. ->We are very grateful to this reviewer for pointing out the high significance of our article.

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

__*1. The evidence for the claim that the Sec22b/Stx1 complex and E-Syts colocalize in native cells (neurons) and bind in heterologous cells is strong (3 independent lines of evidence: co-immunoprecipitation, Proximity Ligation (PLA) and STED super-resolution microscopy) However, the current title of the paper makes a claim beyond this interaction/proximity, based on evidence that is obtained with E-Syt over-expression in wildtype cells. The physiological relevance of the effects remain elusive with over-expression in wildtype cells only.

Furthermore, it is plausible that overexpression of membrane binding/bending C2-domains promotes neurite outgrowth and ramifications by a non-specific effect (as shown for copine C2 domains, PMID:25450385 and indirect evidence for synaptotagmins1,2,7).*

* This issue is especially relevant in the light of the fact that loss of all 3 Extended Synaptotagmins does not affect normal mouse development and viability (PMID: 27399837)

It would be more appropriate to choose a more descriptive title*

• *

->We agree with this reviewer that the original title may be too strong and are now proposing the following, more descriptive title:

Role of the Sec22b/E-Syt complex in neurite growth and ramification

We are fully aware that proteins harbouring C2 domains could potentially promote non-specific effects when overexpressed. However, we do not think this is the case here because none of the morphological effects of E-Syt2 expression in neurons and HeLa cells were reproduced by mutants lacking the SMP or the membrane-anchoring domains. Based on work on Copine __(7)__, a cytosolic protein, E-Syt2 still containing 3xC2 domains but lacking the membrane-anchoring domain should have shown a morphological effect if non-specific binding to phosphoinositides was the mechanism of action. We will discuss this point in the ms.

• The evidence for the working model that the morphological effects of E-Syt2 depends on the Sec22b/Stx1 complex is not strong. Although plausible, the positive effect on neurite outgrowth (E-Syt2 overexpression) and the negative effects (inhibition by Stx1 cleaveage, Sec22b-Longin or Sec22b extended linker expression) may in fact be independent

  The evidence could be strengthened by PLA measurements in neurons over-expressing Myc-E-Syt2 and Sec22b to assess MSC density. It is predicted that in both conditions, MCS density increases. MCS density by PLA measurements could also be performed in Sec22b-P33 and DLongin overexpressed and BoNT/C1 treated neurons. According to the model, the number of MCS should go down. This is of special interest for BoNT/C1 treatment, as it is important to show that the altered morphology is not purely caused by a pre-state of degeneration that is known to be induced by BoNT/C1. In addition, EM measurements of ER-PM distances might provide an independent line of evidence.*

->We agree with this reviewer that additional experiments could strengthen the description of the molecular mechanism. To this end, we will carry out the following experiments:

1/Co-immunoprecipitation experiments of endogenous Syntaxin, Sec22b and E-Syt2 in cells expressing GFP as control or Longin-GFP to demonstrate that expression of the Longin domain perturbs the association of Sec22b with E-Syt2 and Syntaxin.

2/PLA measuring the association between E-Syt2 and Syntaxin in cells expressing GFP as control or Longin-GFP to demonstrate that expression of the Longin domain perturbs the association between E-Syt2 and Syntaxin using a complementary approach.

Unfortunately, membrane-associated, BoNTC1-cleaved syntaxin corresponds to a short fragment undetectable by available antibodies whereas the fragment detected by the antibody after BoNTC1 cleavage lacks the transmembrane domain (Figure 7a). Therefore, we cannot perform PLA in BoNTC1-treated neurons.

We are confident that further exploring the mechanism of action of the Longin domain, together with the data already in the ms, will make it very clear that the morphological effects of E-Syt2 depends on the Sec22b/Stx1 complex.

1. • Link between neurite outgrowth and lipid transfer is weak. The authors argue that functional E-syt/Sec22b/Stx interaction is important for neurite outgrowth by mediating lipid transfer. The only line of evidence they provide is the absence of outgrowth effects in E-syt mutants lacking SMP or membrane spanning domains. However, from the data it is unclear whether these mutants are correctly folded, expressed and/or localized. Additional ICC stainings of the mutants in neurons are necessary to drive this point home. *

• *

->The mutants and siRNA have been already used and validated in Giordano et al. 2013 __(8)__, therefore we did not carry out experiments aiming at basic characterization of these reagents. To answer this request, we will show images of the subcellular localization by ICC of WT and mutant E-Syt2 in the revised Figure 6 or in a Supplementary Figure.

In addition, the authors might make the link between neurite outgrowth and lipid transfer stronger by examining PM lipid levels and distribution in control, Myc-E-Syt2 and E-Syt2 mutant neurons.

->We agree with this reviewer that this question is of high relevance. In order to answer this request, we will express WT and mutant E-Syt2, purify the PM using the protocol of Figure 4 in Saheki et al __(1)__, followed by lipidomics analysis. We hope that this approach further supports our idea that E-Syts mediate an important lipid transfer mechanism towards the PM.

1. • There is no clear evidence that E-syt first binds to Sec22b, after which Stx1 leaves SNAP25 and joins the interaction. This should be indicated as speculation.

   * ->We will make it clear that our model in Figure 9 is a hypothetical model.

• An apparent discrepancy exists between the TKD E-syts effects (i.e. reduced MSC density, Fig 4) and the lack of neurite outgrowth defects in TKO E-syts. According to the proposed model, the levels of E-syt correlate with the number of MSCs and thereby neurite outgrowth. Furthermore, to knock down E-Syts, single siRNAs against the three E-syts were used in Fig4. Off-target effects are not controlled in this approach. Using multiple siRNAs and/or siRNA resistant rescues would be required for robust conclusions.

  *

->The mutants and siRNA have already been used and validated in Giordano et al. 2013 __(8)__, therefore we did not carry out experiments aiming at basic characterization of these reagents. In addition, we would like to stress the complexity of carrying out a rescue experiment of a triple KD of proteins.

Statistical analysis is incomplete. It is not clear whether statistical assumptions (e.g. normal distribution) were checked before performing the tests, and whether non-parametric alternatives where used if assumptions were not met.

->We thank this reviewer for making this important alert. We would like to stress that we have always checked whether samples followed the normal distribution and made non-parametric tests__. We will include this comment in the methods.__

In Fig4, a T-test is used between multiple groups. This test can only be used when comparing two groups. Number of (independent) measurements is not clear in Fig1, 2, 4

->In all the figure legends the number of repetitions is specified

All figures: display all individual data points in all bar graphs (as shown in 5c)

*

*

\*Minor comments:**

1. Inconsistencies on distances in model. Syts are enlongated proteins and thought to be found in MSCs of ~20 nm (Fernandez-Busnadiego, 2015). Trans-SNARE complexes start to interact when the distance between membranes is ~8 nm (Liu, 2007). In the introduction, the authors suggest that incomplete zippering might occur between Stx and Sec22b, resulting in a distance between 10 and 20 nm, which would allow E-Syt localization. In the discussion, however, the authors suggest a model where Sec22b/Stx interaction is important to bring the membranes in ~10 nm distance to enhance LTP activity. Proof for either model is lacking. Please clarify.*

Fig1A: Please clarify the multiple bands? for Stx3 (anti-eGFP).

• *

->These additional bands are recognised by the anti-GFP antibody, the tag being N-terminal, thus they represent proteolytic fragments. We consistently observe these in our experiments.

Fig2: There is no size marker for panels C1-C6

• *

->We will make the appropriate correction.

Fig3: Both proteins seem to show a diffuse pattern. Please specify the validity of measuring average distance. A higher magnification zoom of staining pattern in the growth cone and visualization of the calculation could benefit interpretation.

• *

->We agree with this reviewer that Figure 3 was not optimal to show all the extent of our STED data. In order to strengthen this part, we will include more details in both the figure and as a supplementary movie and supplementary spreadsheet with the quantification of the distance between the E-Syt2/Sec22b clusters to the plasma membrane stained using WGA. The STED data demonstrate that 50% of the clusters are closer than 33.6nm to the plasma membrane, a distance in the range of ER-PM contact sites.

• E-Syt2 and E-Syt3 are used interchangeably throughout the manuscript and E-Syt1 is left out completely. It would help the reader if the authors could elaborate on their interpretation on the similarities and differences in structure and functionality between the three E-Syts.

1. Why is there a red line in Fig 7b?*

__->We added the red line to highlight the shift of SNAP25 band in BoNTA samples. If misleading, it can be removed

Reviewer #3 (Significance (Required)):__

A growing body of literature recognizes the importance of close proximities between membranes, facilitating direct interaction between organelles (Scorrano et al., 2019). Membrane Contact Sites (MCSs) are shown to be important for a wide range of cellular functions, such as lipid and calcium transfer. E-Syts have been recognized as one of the key players in neuronal MCSs, mediating lipid transport (Fernández-Busnadiego et al., 2015). A study published in 2014 by the authors of the current study revealed another two proteins important for MSCs in neurons (Petkovic et al., 2014). ER protein Sec22b and PM SNARE Syntaxin1 were shown to form a non-fusogenic trans-SNARE complex, important for lipid-transfer mediated neurite outgrowth. Gallo and colleagues have now provided important new evidence that these two components (E-Syts and Stx1/Sec22b) are together and may work together at MSCs.

->We thank this reviewer for stressing the importance of our article and agree with the conclusion of __Fernández-Busnadiego et al. (9) on E-Syts being one of the key players in neuronal MCSs, mediating lipid transport. We think that our work is a further key piece of evidence in the demonstration of the importance of E-Syts in neuronal development.__

Bibliography

Saheki Y, Bian X, Schauder CM, Sawaki Y, Surma MA, Klose C, et al. Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat Cell Biol. 2016 Apr 11;18(5):504–515. Yu H, Liu Y, Gulbranson DR, Paine A, Rathore SS, Shen J. Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites. Proc Natl Acad Sci USA. 2016 Apr 19;113(16):4362–4367. Bian X, Zhang Z, Xiong Q, De Camilli P, Lin C. A programmable DNA-origami platform for studying lipid transfer between bilayers. Nat Chem Biol. 2019 Jul 18;15(8):830–837. Alberts B, Johnson A, Lewis J, Raff M. The lipid bilayer. Molecular Biology of …. 2002; Osen-Sand A, Staple JK, Naldi E, Schiavo G, Rossetto O, Petitpierre S, et al. Common and distinct fusion proteins in axonal growth and transmitter release. J Comp Neurol. 1996 Apr 1;367(2):222–234. Igarashi M, Kozaki S, Terakawa S, Kawano S, Ide C, Komiya Y. Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth. J Cell Biol. 1996 Jul;134(1):205–215. Park N, Yoo JC, Lee Y-S, Choi HY, Hong S-G, Hwang EM, et al. Copine1 C2 domains have a critical calcium-independent role in the neuronal differentiation of hippocampal progenitor HiB5 cells. Biochem Biophys Res Commun. 2014 Nov 7;454(1):228–233. Giordano F, Saheki Y, Idevall-Hagren O, Colombo SF. PI (4, 5) P2-dependent and Ca2+-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell. 2013; Fernández-Busnadiego R, Saheki Y, De Camilli P. Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum-plasma membrane contact sites. Proc Natl Acad Sci USA. 2015 Apr 21;112(16):E2004–13.

I think a lot of the time when we think of resilency we think of the individual person. What are they doing to impact how they approach the challenges and stretches in life? How much of it is attributed to personality?

There are two other factors that our out of the control of the child. As educators, we can do something about these two factors. Our hands are not tied and we can be the reason that a child develops some resiliency. Obviously, connecting and collaborating between home and school is preferable. But we only have control over our classrooms and sometimes that has to be enough.

I appreciate the way Rachel Wagner explains resiliency. I think the rubber band example is a wonderful visual to help explain how every person has different events in their life that may "stretch" them and resilience is being able to healthily cope with the those stretches.

I also appreciate the tips that she discusses in the video. The first thing she brings up is having positive relationships. As educators, we know that building relationships is a key our and their success. I like how Rachel mentions that these positive relationships can be small moments. It does not need to be a grandiose thing but a simple interaction. Rachel also mentions the important to encourage students to figure out how to approach different challenges they may face. An important aspect of this is giving children the space to figure it out. As educators, these are important things to keep in mind.

Rachel also brings the concept of not being able to pour from an empty cup. Before we, as adults, can help our students become more resilient we have to be in a space to give ourselves. This involves setting our own boundaries and practicing resiliency ourselves.

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Reply to the reviewers

Authors’ response to reviewers for manuscript “Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7” (reference #RC-2019-00125)

Our manuscript entitled “Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7” has been reviewed by Review Commons. We thank the referees for their interest in our study and are very pleased that the referees consider our findings novel, important and well-designed. Based on the comments given by the referees, we have revised our manuscript and have included two new experimental figures:

-Experimental data validating that our gating strategy with Sytox blue correlates well with bacterial killing on plate (Fig S1-B)

-Experimental data validating that non-bactericidal MAC complexes damage the bacterial OM (Fig. S1-C).

In the response letter below, we respond to the comments raised by the reviewers and explain how we have revised our paper accordingly. All changes into the revised manuscript are clearly highlighted in yellow.

POINT-TO-POINT REPLY

Reviewer #1

(Evidence, reproducibility and clarity (Required)): The paper by Doorduijn et al. addresses a question rarely touched upon in modern studies of the complement system, namely the stability and time-resolved functions of complement component. It extends two earlier reports from the same laboratory, however, with a clear, novel point concerning especially the function of C7. The study embodies several techniques and modes of investigation. From these experiments, the paper contributes significantly to our understanding of the MAC complex is formed and why some bacteria escape this host defense mechanism. Over all the study is very well performed and written. I have only a few major comments.

Reviewer #1 raises 3 points:

POINT 1. The AFM pictures shown in Fig. 6D are of outstanding quality. However, it is a disappointment that the outcome of complement incubation was shown only for a complement-resistant E. coli strain. Would it be possible to show the location on the bacterial surface of MAC complexes, or holes, on a complement-susceptible strains? Comparing the visual outcome for such bacteria with locally formed MAC versus C7 replenished would be quite interesting and perhaps important.

ANSWER 1. Since Fig. 6D represents AFM images of MAC on complement-susceptible E. coli bacteria, we assume that the reviewer is asking why we did not perform AFM experiments on complement-resistant strains? To address this question, it is important to note that we have thus far not succeeded in robustly visualizing MAC complexes under conditions at which bacteria were not killed by MAC complexes (Heesterbeek et al., EMBO J, 2019). While non-bactericidal MAC complexes are present on the bacterial surface as demonstrated with C9 deposition by flow cytometry, we hypothesize that they are not well inserted into the membrane (demonstrated by sensitivity to trypsin) and therefore difficult to resolve by AFM. This is consistent with previous AFM experiments on related pore-forming proteins (Leung et al, 2014, 2017), in which inserted pores were readily detected on supported lipid bilayers, but mobile, non-inserted pores were harder to resolve due to the invasiveness of the AFM measurement and/or insufficient temporal resolution. In the revised manuscript we now better clarify this in line 298-301.

POINT 2. The flow cytometric analysis of bacterial killing is somewhat simplistic. Usually, staining of BOTH live and dead bacteria is performed. This permits better gating of the relevant populations. Specifically, the gating seems to fit the population in Fig. S1 only poorly, with the gate in some cases simply dividing what otherwise appears to uniform population ("C9 at t=0")

ANSWER 2. In the revised manuscript, we have now included additional data demonstrating that our gating strategy with Sytox blue correlates well with bacterial killing on plate (new Fig S1-B referred to in line 78-79, 92-93, 96-98 and Supplementals text line 21-24 shows cfu data for Sytox data of Fig 2D). These data correspond with our earlier findings showing that cells gated to be positive for Sytox blue are indeed the relevant population of dead cells (Heesterbeek, EMBO J, 2019). We disagree with the reviewer that the use of a ‘live’ stain is of added value here. Because the outer membrane of Gram-negative bacteria is also a permeability barrier for ‘live’ stains like Syto9, MAC-dependent outer membrane perforation also results in increase in ‘live’ stain during the process of bacterial lysis (also described in Stiefel et al, BMC Microbiol, 2015 PMID: 25881030). We have therefore chosen to only use the Sytox stain in this study as this is a very reliable marker for killing.

POINT 3. The cited literature is, in general, pertinent and comprehensive. I was surprised, however, that none of the many contributions to field of MAC formation by AF Esser was cited. For instance, the studies over C9 conformation (PMID: 2475785) seem not far away in topic from some of the points raised in the present paper.

ANSWER 3. The reviewer is correct that the work of AF Esser has indeed focused on the contribution of C9 and C9 polymerization to the lytic activity of the MAC pore. In the revised manuscript, we have therefore now included some of the work done by AF Esser (references 34, 36 and 37) and have discussed this in our discussion (line 305-309). However, it is important to note that much of the work on the importance of C9 polymerization by AF Esser has been performed erythrocytes and single-membrane particles (also the suggested paper by the reviewer). Translation of these studies to the role of C9 conformation and polymerization on bacterial killing is therefore limited, although it does provide clues to what differences might cause the discrepancy observed between lysis of erythrocytes and bacterial killing by MAC pores.

Reviewer #1 (Significance (Required)): Insight into the concept of locally formed MAC complexes is lacking and the paper clearly adds novel and quantitative data to this point. The paper probably mostly reaches out to an audience interested in the complement system and researchers interested in large protein complexes with conformational changes as part of their function. My own interest lies with complement-mediated protection against bacteria with a special focus on pattern recognition and protein-bacterial surface interactions.

Reviewer #2

(Evidence, reproducibility and clarity (Required)): Doorduijn et al. present a study illustrating the importance of rapid C7 interaction with C5b6 for MAC-dependent killing of complement sensitive bacteria. The absence of direct C7 interaction results in a MAC which i) doesn't kill the bacteria, and ii) is sensitive to trypsin. The authors have step by step investigated this issue by using common in vitro-methods with different strains of bacteria, serum, and/or purified complement proteins. Bacterial killing is evaluated by sytox blue influx in flow cytometry. I like this work. The experimental strategy is sound, and the conclusions are convincing are based on the presented data. The data and the methods presented in such a way that they can be reproduced. I have no concerns regarding the design, execution or conclusions.

Reviewer #2 RAISES 3 POINTS

POINT 1. My only criticism is on the number of replicates and following statistical analysis: • Overall, the experiments are conducted only three times. With the, in general, large differenced seen between the condition, this may still be acceptable. However, the statistic testing using only N=3 is of low value.

ANSWER 1. As the reviewer pointed out, with these in vitro studies where the experimental conditions are highly controlled it is common practice to perform three independent experiments when the differences are large.

POINT 2. The authors have sometimes used paired testing, and sometimes unpaired. For example, Fig. 5A-B is based on paired testing, whereas data in C, which are based on A-B, is tested using unpaired testing. Why so is unclear to me.

ANSWER 2. We thank the reviewer for this comment, as also for Fig. 5C we should have used a paired analysis, so we have done so accordingly in the revised manuscript (line 725-726).

POINT 3. Further on in Fig. 5 A-B., ANOVA with Tukey multiple comparison tests is used, which implements testing between all conditions; still, only significance is reported for blue vs. red. If the intention was to only test red vs. blue, a t-test would be better.

ANSWER 3. As with the previous comment, we have now performed a paired t-test since we only intended to compare C7 at t=0 vs C7 at t=60 in Fig. 5A-B (line 725-726). Moreover, for consistency in our statistical analyses we’ve also applied this to Fig. 3C (line 706-707).

Reviewer #2 (Significance (Required)): As far as I understand, the presented data is of high significance for the conceptual understanding of the buildup of MAC for bacterial killing on Gram-negative bacteria. I work partly with complement but is not an expert on the terminal pathway.

Reviewer #3

(Evidence, reproducibility and clarity (Required)): The study is a follow-up on the paper the same group of scientists published in EMBO J last year. That paper showed that rapid interaction between C5b6 and C7 is necessary for effective killing of Gram negative bacteria. The follow-up this paper makes is to make that case for a series of E. coli strains, showing as part of this that strains of clinical isolate E. coli resistant to complement attack prevent the rapid C5b6-C7 interaction. The story goes that C5 convertase engagement on the surface of targeted bacteria is the necessary context for effective C5>C5b conversion and thence interaction with C6 and C7. The rapid interaction with C7 is necessary because it prevents release/shedding of C5b6 from the bacterial cell surface. Overall, the conclusions seem justified - that C5b6 interaction with C7 stabilises its interaction with the surface and is needed to prevent C5b6 shedding. But this observation needs a mechanical or biophysical framework to be understood properly.

Reviewer #3 RAISES 5 POINTS

POINT 1. The authors do not observe non-bactericidal MAC pores/non-lytic MAC by AFM and so I think in this study there is no evidence for their existence. Their depiction in Figure 8b is therefore misleading and I think should be deleted. Indeed, the authors do not know what the structure of the non-bactericidal MAC pores could be, so depicting them in this specific way isn't appropriate. They have no idea what they might be like, if they exist.

ANSWER 1. We agree with the referee that we do not know the structure of a non-bactericidal MAC pore, and have therefore deleted the speculative structures in Fig. 8B (explained in line 784-785). Although we have no structural information, we do think that non-bactericidal MAC pores exist and our revised manuscript now includes new data to better explain this (Fig S1-C). While our initial manuscript showed that a delayed interaction between C5b6 and C7 results in MAC complexes that cannot perturb the bacterial inner membrane, we now show that these MAC complexes effectively damage the outer membrane (evidenced by leakage of mCherry from the periplasmic space (Fig. S1-C, explained in line 97-98 and Supplementals text line 24-31). This leads us to conclude that there are pores formed in the outer membrane that are not capable of damaging the inner membrane. We think that within this context we can name these ‘non-bactericidal MAC pores’.

POINT 2. This brings me to another point: it is really unclear to me from this study how the authors envisage the inner bacterial membrane be damaged by MAC attack. Do MAC pores formed in the OM deliver MAC components to the IM? Or what happens - is the damage to the IM indirect? The reason why this is relevant to the possibility of non-bactericidal MAC pores is that it could be these are inserted just like bactericidal pores into the OM but the IM attack is deficient in some way.

ANSWER 2. Although we agree with the reviewer that exact mechanism by which MAC pores perturb the inner membrane is unanswered, we think this is beyond the scope of this paper which mainly deals with the time-resolved functions of MAC assembly. However, to meet the referees’ critique, we have now more clearly addressed this question in our discussion and speculate on several mechanisms by which the MAC pore could induce bacterial inner membrane damage (line 277 - 288). In short, we hypothesize that OM damage could indirectly trigger IM damage by affecting regulation of osmosis, overall cell envelope stability and/or envelope stress.

POINT 3. (Significance (Required)). I am intrigued by the difference between MAC assembly on erythrocytes and bacteria. What do the authors believe to be the basis of this difference? It would help understanding of the significance of their work if they could make this clear. Without this kind of attempted explanation the results seem phenomenological - an observation has been made but why this observation occurs, what the important environmental difference is between erythrocyte membranes and the outer membranes of Gram negative bacteria is not addressed. I am looking for some kind of biophysical explanation - specific lipid properties, for example.

ANSWER 3. We agree that this is intriguing and in our revised manuscript we have included different hypotheses on why MAC assembly on erythrocytes and bacteria could be different. Although differences in composition between the erythrocyte membrane and outer membrane can definitely play a role, our data suggest that the difference is mainly a consequence of the fact that Gram-negative bacteria have two membranes (the outer and inner membrane). In the revised manuscript, the newly added figure (Fig S1-C) supports this, since this figure reveals that MAC pores generated from C5b6 that is generated in the absence of C7 can still damage the bacterial OM. However, despite observing OM damage by measuring leakage of a periplasmic protein, this does not lead to bacterial killing and IM damage. Since we here observe that rapid interaction between C5b6 and C7 is required for bacterial killing and IM damage, we think that efficient anchoring of C5b-7 is primarily relevant in damaging the bacterial IM and subsequently causing bacterial cell death. Finally, we have also mentioned this more specifically in the discussion of the revised manuscript (line 277-288).

POINT 4. Related, at the end of the Results section the authors say "Altogether, these data indicate that complement-resistant E. coli can prevent complement-dependent killing by MAC pores by preventing efficient anchoring of C5b-234 7 and insertion of MAC pores into the bacterial cell envelope." My immediate response was: 'How? The Discussion needs to consider this.' But it doesn't.

ANSWER 4. In the revised manuscript, we now explain this more extensively (line 322 – 326). In short, we hypothesize that the composition of the OM, mostly in terms of capsular polysaccharides and lipopolysaccharides, could affect this. We have added additional references supporting the role of these components in complement resistance in multiple Gram-negative species (reference 45-48 and 50).

POINT 5. I was confused by the term "metastable lipophilic domain" at line 262 on page 10. Do the authors mean the MACPF domain?

ANSWER 5. We have now more explicitly named this in our discussion as being the MACPF domain and have further elaborated what we meant by metastable (line 263-267).

Emily Messer ENG 491 Bleak House Installment Eleven 02/02/2020 Bleak House Installment Eleven and its Thematic Relevance

Installment eleven of Bleak House by Charles Dickens begins with chapter thirty-three and ends with chapter thirty-five of forty-eight chapters total. There are twenty installments which were released monthly, making Bleak House an almost two-year endeavor. Situated between a case of spontaneous combustion in installment ten and Esther learning the true identity of her mother in installment twelve, installment eleven runs along a track of eventful and emotion packed installments, just after the middle of the novel. Though it does occur more than halfway through the story, a few of the events in installment eleven set up the later events of the novel, all the way up until the ending. Esther’s illness and recovery, which is the driving force of chapter 35, influence multiple plotlines that lie at the heart Bleak House. Similarly, the confession of love for Mr. Woodcourt, which also takes place at the close of chapter 35, is integral to the ending of Bleak House and its emotional weight. Installment eleven begins with the omniscient narrator describing the court directly after Krooks dies of spontaneous combustion. We learn that Krooks was Mrs. Smallweed’s brother, and therefore the Smallweeds want to inherit Krook’s belongings. We remember that Krooks had a stack of Nemo’s letters and wonder if any may have survived. We also meet the Bagnet family and their learn connection to George Rouncewell while Mr. Tulkhinghorn attempts to collect Mr. George’s debt. Then we switch back to Esther’s narrative, where we learn of her illness and her recovery with resulting scars, her absence from Ada, and an admission of her love for Mr. Woodcourt. Esther writes, “And now I must part with a little secret I have thus far tried to keep. I had thought, sometimes, that Mr. Wood Court loved me; and that if he had been richer, he would perhaps have told me that he loved me, before he went away. I had thought, sometimes, that if he had done so, I should have been glad of it. “(pg. 570) The illness that Esther has contacted, which is decidedly smallpox, was passed down from Jo, to Charley, to her. We learn of all three of these illnesses in installment number ten, and their significance and thematic importance comes to light in installment eleven. Jo, who is poor and resides in Tom All Alone’s, a very impoverished and dirty neighborhood in London, gets the disease first. Then Charley, who is still a poor girl and one of the lower-class characters in Bleak House contracts it from caring for Jo. Then, Esther falls ill after caring for both Jo and Charley. From this, there are two thematic takeaways. First, there is the infection of disease moving its way up the social class but ends with the class just below Ada: people whose perceived birth is low ranking. The disease is not allowed to continue spreading into the upper classed members of the character list. This could be read as a social critique of the idea of birth ranks, calling out how lower classed members of society are seen as a kind of self-contained infection. Secondly, this could fit into the novel’s larger conception of charity. Esther and Charley care for Jo out of compassion: a kind of charity. However, this charitable act winds up giving them smallpox. Similar to the portrayal of Mrs. Jellyby’s neglect at the hands of charity and Mr. Jarndyce’s inability to recognize thanks for his charity, the contraction of disease as a result of charity might be another way Dickens conveys that there is no good or pure form of charity - at least not without consequence. In regard to the effects of her illness, Esther writes “” I had heard my Ada crying at the door day and night I had heard her calling to me that I was cruel and did not love her… But I had only said when I could speak, ‘Never my sweet girl never!’ And I had over and over again reminded Charley that she was to keep my darling from the room whether I lived or died. Charley had been true to me in that time of need, and with her little hand and her great heart had kept the door fast.” Beyond the illness itself, the scars that result from the illness do quite a bit of work for the overall plot going forward from installment eleven. In installment twelve, Lady Dedlock reveals to Esther that she is, in fact, her mother. However, no kind of mother-daughter relationship results from that conversation. This could, in part, be explained by Esther’s complete facial change from the scarring that comes from the illness. Esther’s familiarity to Lady Dedlock is tainted by the scarring, and therefore the scarring becomes a symbol for the years of separation and emotional trauma (especially Esther but also somewhat Lady Dedlock as well) endured. Esther’s scars also inform her decision to accept the proposal made to her by Jarndyce in installment number fourteen, for marriage. Esther believes that her beauty has completely left her as a result of the scarring, and when Jarndyce proposes to her, she feels as though that may be her only chance at marriage. Yes, Esther loves Jarndyce almost like a father, but we have never had any inkling that she is romantically interested in Jarndyce. Therefore, when Jarndyce gives Esther a letter asking her to be his wife, we almost expect, or perhaps wish Esther to say something like, “No! I’m a young and beautiful woman and should not give away a chance at a fulfilling relationship to be the pet of an old man!” However, Esther does not think she is beautiful anymore, not after the scars. When speaking of her scars, Esther writes, “I have thought of this very often. I was now certain of it. I could thank God that it was not a shock to me now. I called Charley back; and when she came – at first pretending to smile, but as she drew nearer to me looking grieved – I took her in my arms and said, ‘it matters very little Charley I hope I can do without my old face very well,’” (pg. 558) Another revelation that comes with installment eleven is the admission of love between Alan Woodcourt and Esther. This has been heavily hinted at, but never outright said until this point. We have already had hurdles to a respective relationship introduced earlier in the novel, such as Mrs. Woodcourt's assertion that Allan should not marry a woman who is not of good birth. This, of course, would not apply to Esther after she learns that she is a Deadlock, but at the time is a clear threat. There is also Mr. Woodcourt’s prolonged absence from Bleak House at the hands of his work as a physician. However, none of the other threats seem as concrete and final as the threat that Woodcourt simply could not be with Esther after she has lost her beauty. This thought leaves the reader with a sadness, which is a perfect setup for the close of the novel where Jarndyce releases Esther from her betrothment and allows her to marry Woodcourt. The emotional weight of learning that Esther, a beloved narrator, can finally be happy with a man she could truly love as a husband is only possible through the many chapters of concealment which lead to the admission at the end of instalment eleven along with the seemingly assured end of any hope with the dramatic change in Esther’s appearance. In reference to how her change in appearance will affect a potential relationship with Woodcourt, Esther writes, “What should I have suffered, if I had had to write to him, and tell him that the poor face he had known as mine was quite gone for me, and then I freely released him from his bondage to one and he had never seen!” (pg. 570) Thematically, the illness and scarring work together to draw parallels between infection and poverty, charity and consequences, emotional scars and physical markings, and loss of beauty and strength of love. The preceding events of the installment, such as the introduction of the Bagnet family and the Smallweed’s connection to Krooks have their importance in the plot of the novel, but the larger thematic overtones of those sections pale in comparison to the importance of the illness. While the illness does first appear in an earlier installment, the resulting scarring and connection to the romance between Woodcourt and Esther does not appear until installment eleven, making this thematic and symbolic groundwork anchored in the eleventh installment.

This is where the potential romantic connect between Allan Woodcourt and Esther is brought to light. The interesting part is that this is also the installment where Woodcourt leaves the country on a voyage to China and India on a ship. We haven't seen very much of him before this, but here we are saying goodbye to him. His exit is almost necessary for the future relationship between Mr. Jarndyce and Esther to even have a chance at beginning -- if Woodcourt had been there all along, through everything (like Esther's illness), Mr. Jarndyce may not have even proposed to Esther in the first place. So, moving his piece off the chessboard of this novel's narrative was significant in itself. We also have hints that Esther's relationship with Woodcourt is more significant than she's letting on.

Her narrative voice shows she has trouble looking at Woodcourt in an unbiased way; for example, she says things like:

"I believe -- at least I know -- that he is not rich" as well as "I think -- I mean, he told us -- that he had been in practice three or four years..." (277).

This obvious attempt to remove herself from the narrative is consistent with her character, but it also shows that her first thought is to portray him the way she subjectively views him, and she must force herself to be more removed.

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Reply to the reviewers

We are very grateful to the three reviewers for their useful and constructive comments on our manuscript. All reviewers appreciated that our manuscript provides a good characterization of the KKT2/3 functional domains, especially in solving crystal structures of the KKT2 central domain and revealing the importance of KKT2/3 central domains for their centromere targeting. They also commented that additional experiments (e.g. testing DNA-binding activities using recombinant proteins and examining whether ectopically expressed KKT2 fragments localize at kinetochores transiently) would significantly strengthen the manuscript. In the revised manuscript, we are going to address their comments as follows.

Reviewer #1:

From the information presented, it seems like there are only two possibilities to explain the role of the zinc finger domains in directing centromere targeting. First, this could mediate a protein-protein interaction. The authors attempt to assess this using their mass spec experiments, but this does not absolutely rule this out as this interaction may not persist through their purification procedure (low affinity or requires the presence of DNA, such as for a nucleosome).

Response: We agree with the reviewer’s comment. We will add a sentence to discuss this possibility in the revised manuscript.

Second, this could reflect direct DNA binding by the zinc finger. Although the existing paper is solid and highlights a role for the zinc finger domains in the localization of these proteins, it would be even better if the authors were to at least assess DNA binding in vitro with their recombinant protein. Comparing its behavior to a well characterized DNA-binding zinc finger protein would be powerful for assessing whether direct DNA binding could be responsible for its centromere localization.

Response: We have tested DNA-binding activities for the KKT2 central domain from T. brucei, Bodo saltans, and Perkinsela using a fluorescent polarization assay. We tested three different DNA probes (50 bp each) that were fluorescently-labelled: a 50 bp DNA probe from the CIR147 sequence, which is the unit sequence of centromere repeats of several chromosomes in T. brucei (36% GC content), as well as two random DNA sequences of 25% and 74% GC content. We found that the Perkinsela KKT2a central domain binds these three different DNA probes with similar affinities (Kd ~100 nM), suggesting that the Perkinsela KKT2a central domain binds DNA in a sequence-independent manner. Although we have not been able to obtain reliable results for T. brucei and Bodo saltans proteins thus far (due to quenching of fluorescent signals by these proteins), it is likely that the T. brucei KKT2 central domain also binds DNA in a sequence-independent manner given the similarity of the Znf1 structure/sequence among kinetoplastids. This is consistent with the observation that there is no DNA sequence that is commonly found in the centromere of all chromosomes in T. brucei and other kinetoplastids. We are going to add the DNA-binding assay results for the Perkinsela KKT2a central domain in the revised manuscript. We do not feel it is informative to compare the KKT2 Znf1’s behavior to a well characterized DNA-binding zinc finger protein (that binds specific DNA sequence), because Perkinsela KKT2a binds DNA in a sequence-independent manner.

The code for KKT2 and KKT3 localization is complicated by the multiple regions that contribute to their targeting. This includes both the zinc finger domain that the authors identify here, as well as a second region that appears to act through associations with other constitutive centromere components. Due to this, it feels that there are several aspects of these proteins that are incompletely explored. First, the authors show that the Znf1 mutant in KKT2 localizes apparently normally to centromeres, but is unable to support KKT2 function in chromosome segregation. This suggests that this zinc finger domain could have a separable role in kinetochore function that is distinct from centromere targeting.

Response: We agree with the reviewer that the mechanism of KKT2 kinetochore localization is complicated because there are at least three distinct domains that contribute to its targeting (Figure 2 in the original manuscript), but we showed that the centromere targeting of the ectopically-expressed KKT2 central domain fragment depends on Znf1 (Figure 6B in the original manuscript). Together with the finding that the Znf1-equivalent domain is essential for the localization of the full length KKT3 protein, we think that a function of the KKT2 Znf1 domain is to promote its centromere localization. In the future, it will be critical to understand the molecular mechanism of how the KKT2 central domain localizes specifically at centromeres.

Second, although the authors identify these minimal zinc finger regions as sufficient for centromere localization, they do not test whether this behavior depends on the presence of other KKT proteins. This seems like a very important experiment to test whether recruitment of the zinc finger occurs through other factors, or whether it could act directly through binding to DNA or histones.

Response: We do not have an experimental setup to test whether the centromere localization of KKT2/3 central domains depends on other KKT proteins (i.e. we cannot keep the expression of the central domain to a low level while inducing RNAi constructs at a high level). As an alternative approach, we have been testing the localization dependency of endogenously-tagged full-length KKT2/3 proteins using RNAi against various KKT proteins but our preliminary results have not found any kinetochore protein whose depletion affects the localization of KKT2 or KKT3 at centromeres. Although these results could be explained by inefficient protein depletion, they are consistent with the possibility that KKT2 and KKT3 central domains directly interact with centromere DNA. We could consider adding these data in the revised manuscript, although a significant amount of additional work will be necessary to confirm these results.

• Based on the description of kinetoplastid centromeres that the authors provide, it is actually unclear to whether these are indeed sequence independent. The authors state that "There is no specific DNA sequence that is common to all centromeres in each organism [Trypanosomes and Leishmania], suggesting that kinetoplastids also determine their kinetochore positions in a sequence-independent manner." However, it remains possible that there are features to this DNA that are responsible for defining the centromere. In principle, enriched clustering of a short motif that may elude sequence comparisons could be responsible for specifying these regions. It would be helpful to use caution with this statement, and I would also encourage the Aikyoshi lab to test this directly in future work, such as using strategies to remove a centromere or alter its position. *

Response: We agree with the reviewer that we cannot exclude the possibility that there might be an enrichment of a short motif that promotes the localization of kinetochore proteins. We will discuss this possibility in the revised manuscript.

• It would be helpful to provide a schematic of kinetoplastid kinetochore organization based on their studies to date (possibly in Figure 1) to provide a context for the relationships between the different KKT proteins tested in this paper.*

Response: While we agree with the referee that a model figure would be helpful, we feel that drawing a model for the overall organization of kinetoplastid kinetochores at this stage could be misleading because we still know very little about it. In fact, our published data (e.g. the microtubule-binding kinetochore protein KKT4 localizes at centromeres throughout the cell cycle and has DNA-binding activities) and our unpublished observations suggest that the design principle of kinetoplastid kinetochores may well be fundamentally different from that of canonical kinetochores in other eukaryotes. We therefore would like to obtain more data before drawing a model of kinetoplastid kinetochores. Instead of a model, we are going to include a summary of localization patterns for kinetoplastid kinetochore proteins in Figure 1 to help orient readers.

Reviewer #2: The experiments are in general well presented but some could be better controlled: - localization of KKT2 and KKT3 mutants is never verified to be centromeres, we have to believe the dots in the DAPI region are centromeres.

Response: We have assumed that the KKT2 and KKT3 mutants that had dots very likely localized at centromeres because they behaved similarly to wild-type proteins (i.e. align at metaphase plate in some 2K1N cells and localize at the leading edge of separating chromosomes). We will confirm this assumption by imaging the KKT2/3 mutants with a kinetochore protein marker (e.g. tdTomato-KKT1).

in some cases mutants are made in full-length (FL) background (viability, sometimes localization), but in other cases only in isolated domains. The former should be done for all assays. This is also important to show that central domain of KKT2 and KKT3 is necessary for localization.

Response: It is very laborious to create point mutants in full-length background at an endogenous locus. This is why we first tested a number of mutants in our ectopic expression of truncated (for KKT2) or full-length (for KKT3) proteins to identify the most critical mutations, which were subsequently tested in the endogenous context. Although not included in the original manuscript, we have performed an ectopic expression of additional KKT2 mutants (C597A/C600A, C616A/C619A, C624A/C627A, C640A/C643A, and H656A/C660A) in the full-length protein and found that all of them had apparently normal localization pattern, which is consistent with the results we obtained in the endogenous expression experiments (C576A, D622A, and C640A/C643A: Figure 6c in the original manuscript).

The data of F2 are interpreted to mean that PDB-like domain and middle region get to kinetochores by binding transient KT components, even though KKT2 itself is constitutive. That interpretation would really be strenghthened by showing the KKT2 fragments are now transient also. **

Response: Our observations suggest that these KKT2 fragments indeed localize at centromeres transiently (from S phase to anaphase). We will confirm this result by imaging with a transiently-localized kinetochore protein, KKT1 tagged with tdTomato, and include in the revised manuscript.

The paper could do with some attempts to get to this, based on the presented data. For example, does Znf1 bind centromeric DNA, does it bind nucleosomes, is it essential for recruiting the other KKTs, etc.

Response: As we responded to Reviewer 1, we have found that Perkinsela KKT2a central domain Znf1 has DNA-binding activities. We agree that it will be important to test whether KKT2 binds nucleosomes but it will be necessary for us to reconstitute nucleosomes using recombinant T. brucei histones. It will also be important to test whether KKT2/3 are essential for recruiting other kinetochore proteins but we think that they are beyond the scope of this manuscript.

Reviewer #3: \*Major Comments:** - No page numbers - this makes it difficult to refer to different parts of the text... *

Response: We sincerely apologize for the lack of page numbers in the original manuscript. We will add page numbers and line numbers in the revised manuscript.

Introduction (page 2), fourth-from bottom line: the authors refer here to "regional centromere" but have not defined this term (I assume, as opposed to point-centromeres of budding yeast?). I suggest rephrasing.

Response: We thank the reviewer for pointing it out. We will rephrase it in the revised manuscript.

Page 4, bottom: The discussion of KKT2 kinetochore localization brings up a lot or questions. First, can the authors use an assay like yeast two-hybrid to test for pairwise interactions between KKT2 domains and other kinetochore proteins? This could provide direct functional data on the role of these various domains in kinetochore localization.

Response: Based on the mass spectrometry of immunoprecipitated KKT2 fragments that localized at kinetochores, we are currently trying to identify direct protein-protein interactions between the KKT2 domains and other kinetochore proteins (e.g. does KKT2-DPB directly interact with KKT1, KKT6, or KKT7 proteins?). While we agree that it is important to address these questions, we think that it is beyond the scope of this manuscript because its focus is the characterization of KKT2/3 central domains. As we mentioned in the manuscript, these central domains failed to co-purify with other kinetochore proteins, and the experiment therefore did not give us any clue about how they might localize specifically at centromeres.

Second, if individual domains are being recruited to kinetochores by their non-constitutive binding partners, wouldn't this be evident if the authors looked at localization at different points in the cell cycle, and/or with dual localization tracking the putative binding partners? Could transient localization of some of the domains explain the intermediate localization phenotype observed for some domains in KKT2?

Response: As we responded to Reviewer 2, our observations suggest that these KKT2 fragments indeed localize at centromeres transiently (from S phase to anaphase). We will confirm this result by imaging with a transiently-localized kinetochore protein, KKT1 tagged with tdTomato.

Page 6: The authors note that KKT2 Znf2 bears strong similarity to DNA-binding canonical Zinc fingers, and even note the high conservation of some putative DNA-binding residues. Have the authors tested for DNA binding by this protein?

Response: As we responded to Reviewer 1 and 2, we used a fluorescence polarization assay and found that the Perkinsela KKT2a central domain binds DNA in a sequence-independent manner.

Can the authors at least model DNA binding and see if that would result in a clash, given the packing of Znf2 against the larger Znf1?

Response: As suggested, we superimposed the structure of Bodo saltans KKT2 Znf2 with that of a zinc finger 268 bound to DNA (PDB:1AAY), which shows a possible mechanism by which Znf2 might bind DNA. It also revealed a clash between DNA and Znf1 (in the crystal packing of the solved structure), implying that the position of Znf2 would need to change in order to bind DNA. We will add a supplementary figure showing a hypothetical DNA-binding mechanism by Znf2 and discuss the possibility of a necessary structural change in the Znf1 position to accommodate the DNA binding by Znf2.

\*Minor Comments:** - Page 5: I'm skeptical as to whether these zinc-binding domains, especially Znf1, should really be referred to as "fingers". *

Response: To our knowledge, the word “zinc finger” could be used for any protein that binds one or more zinc ions. Given that we still do not understand the molecular mechanism by which this domain functions, we wanted to use a very general term, Znf1. However, we do appreciate the reviewer’s point that calling this domain as a zinc finger could be misleading, so we will refer Znf1 and Znf2 in the original manuscript as the CL domain (for centromere localizing domain) and a classic C2H2 zinc finger in the revised manuscript.

Page 8: At the beginning of the section describing KKT3 cellular experiments, I think the authors need to make it much more explicit that T. brucei KKT3 shares both Znf1 and Znf2 with KKT2.

Response: We will add the suggested sentence before describing the functional assay for KKT3.

Figure S1A: The gap between lanes in the middle of the major peak is really confusing (it's not even clear that this is two different SDS-PAGE gels next to one another). I initially thought that KKT2 was in both peaks, given the labeling of this figure. I suggest labeling the lanes specifically, or cropping the picture, to avoid confusion.

Response: As suggested, we will prepare an image that shows only those lanes (from two separate gels) that were used for loading protein samples. We also like to retain the whole gel images in the same figure because those gels have rather low background signal (even without any contrast manipulation).

One of the difficulties for choosing this as a discourse community is the idea of shared goals. I think this is important so please do address it in the paper.

I found this essay for you that might help. Please take a look. https://pdfs.semanticscholar.org/dd19/4239a42bb7989fc8b041e599113604e71dbd.pdf

Throughout the rest of the article, the tone shifts majorly. It started off as an informative piece, trying to explain how depression affects people differently and what they go through before making a life altering decision. But now, it is almost like the writer is attacking the people on the other side of the argument. They are not providing explanations of information, just questions that can be taken as offensive. Certainly this does not show professionalism.

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

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Author Comments:

­Response to Reviewers Comments

We would like to thank all reviewers for carefully considering our manuscript and providing useful suggestions/ideas. The general consensus was that our study provides an important conceptual advance that reveals a new way of thinking about kinetochore phosphatases. However, in light of our surprising findings, it was suggested that additional experiments would be required to fully validate our conclusions. In particular, it was seen as important to test whether PLK1 can activate MPS1 from the BUB complex and to confirm that PP1 and PP2A are effectively inhibited in situations where MELT dephosphorylation can occur normally (Figure 3).

In general, we agree with these and the other points raised by the reviewers, therefore we plan to address all comments as outlined in detail below.

The major new additions to the final paper will be the following:

1) Experiments to test how BUB-bound PLK1 affects MPS1 activity.

2) Experiments to determine the efficiency of phosphatase inhibition in figure 3.

3) Experiments to test whether maintaining PLK1 at the BUB complex causes SAC silencing defects

4) Evolutionary analysis demonstrating that the PLK1 and PP2A-binding modules have co-evolved in the kinetochore BUB complex. This analysis, which has been performed already, strengthens our manuscript because it provides additional independent evidence for a functional relationship between PLK1 and PP2A on the BUB complex.

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Reviewer #1 **Minor comments:** 1) The authors propose that PP1-KNL1 and BUBR1-bound PP2A-B56 continuously antagonise PLK1 association with the BUB complex by dephosphorylating the CDK1 phosphorylation sites on BUBR1 (pT620) and BUB1 (pT609). It is therefore expected that converting these residues to aspartate would increase PLK1 recruitment. It would be interesting to verify if this hypothesis fits with the proposed model.

Response: The general idea to maintain PLK1 at the BUB complex is a good one, but unfortunately polo-box domains do not bind to acidic negatively charged residues. Instead we will attempt to maintain PLK1 at the BUB complex using alternatively approaches (as suggested by reviewer 2).

2) In Figure 1E, are the mean values for BubR1WT+BubWT and BubR1WT+Bub1T609 both normalized to 1? If so, this fails to reveal the contribution of Bub1 T609 for the recruitment of PLK1 when PP2A-B56 is allowed to localize at kinetochores.

Response: The values will be updated and normalised to the BubR1WT+BUB1WT control. We have also performed additional experiments already and overall the results reveal a small reduction in kinetochore PLK1 following BUB1-T609A mutation and a larger reduction upon combined BUBR1-T620A mutation.

3) What underlies the increase in Bub1 levels at unattached kinetochores of siBubR1 cells (Figure S1C?) Is this caused by an increase in Bub1 T609 phosphorylation and consequently unopposed PLK1 recruitment, which consequently increases MELT phosphorylation?

Response: We suspect that PLK1 is not the cause of the increased BUB1 levels because PLK1 kinetochore levels are actually decreased in this situation (Figure S1A).

4) Although the immunoblotting from Figure S1D indicates that BubR1T620A and Bub1T609A are expressed at similar levels as their respective WT counterparts, some degree of single-cell variability is expected to occur. As a complement to Figure 1B,C and Figure S1E,F could the authors plot the kinetochore intensity of BubR1 pT620 and Bub1T609 relative to the YFP-BubR1 and YFP-Bub1 signal, respectively?

Response: There is indeed variability in the level of re-expression of BUBR1/BUB1 on a single cell level, which can at least partially explain the variation on BUBR1-pT620 and BUB1-pT609 observed within in each condition. We can upload these scatter plots at resubmission and include in the supplementary, if required.

5) The authors nicely show that excessive PLK1 levels at the BUB complex are able to maintain MELT phosphorylation and the SAC (independently of MPS1) when KNL1-localised phosphatases are removed (Figures 2A,B). However, it should be noted that PLK1 is able to promote MPS1 activation at kinetochores and so, whether AZ-3146 at 2.5 uM efficiently inhibits MPS1 under conditions of excessive PLK1 recruitment should be confirmed. Can the authors provide a read-out for MPS1 activation status or activity (other than p-MELTs) to exclude a potential contribution of residual MPS1 activity in maintaining the p-MELTs and SAC?

Response: This is a good point because although PLK1 can phosphorylate the MELTs it can also activate MPS1, although it is unknown whether it can do this from the BUB complex. We had left a dotted line in Figure 4B to include this possibility, but we will now test this directly with additional experiments.

6) To examine whether PLK1 removal is the major role of PP1-KNL1 and PP2A-B56 in the SAC or whether they are additionally needed to dephosphorylate the MELTs, the authors monitored MELT dephosphorylation when MPS1 was inhibited immediately after 30-minute of BI2356. This revealed similar dephosphorylation kinetics, irrespective of compromised PP1-KNL1 or PP2A-B56 activity, thus suggesting that these pools of phosphatases are not required to dephosphorylate MELTs. To confirm this and exclude phosphatase redundancy, the authors simultaneously depleted all PP1 and B56 isoforms or treated cells with Calyculin A to inhibit all PP1 and PP2A phosphatases. In both of these situations, the kinetics of MELT dephosphorylation was indistinguishable from wild type cells if MPS1 and PLK1 were inhibited together. These observations led to the conclusion that neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead they are needed to remove PLK1 from the BUB complex. This set of experiments is well-designed and the results support the conclusion. However, it would be of value if the authors provide evidence for the efficiency of PP1 and B56 isoforms depletion and for the efficiency of phosphatase inhibition by Calyculin A. An alternative read-out for the activity of PP1 and PP2A-B56 (other than p-MELT dephosphorylation) clearly confirming that both phosphatases are compromised when MPS1 and PLK1 are inhibited together could make a stronger case in excluding the contribution of residual PP1 or PP2A to the observed dephosphorylation of MELT motifs.

Response: This is also a good point. We had attempted many different combinations in Figure 3 to inhibit PP1/PP2A activity as efficiently as possible. This is especially important considering the “negative” results on pMELT are very surprising. However, we will now test how efficiently we have inhibited PP1 and PP2A phosphatase function in these experiments.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Major comments:** 1) In its current state I am not convinced that the key conclusions are fully supported by the experiments and alternative conclusions/interpretations can be drawn. For example the level of MELT phosphorylation will be determined by the balance of kinase and phosphatase activity and if they do not achieve 100% inhibition of Mps1 in their assays then they are not strictly monitoring dephosphorylation kinetics in their assays. If the combination of Mps1 and Plk1 inhibition then more strongly inhibits Mps1 then dephosphorylation kinetics becomes faster. Thus subtle differences in Mps1 activity under their different conditions could lead to misleading conclusions but in its present state a careful analysis of Mps1 activity is not provided. This lack of complete inhibition also applies to the phosphatases and the experiments in Figure 3E indicates that their Calyculin preparation is not really active as at steady state MELT phosphorylation levels are much less affected than in for instance BubR1 del PP2A (Figure 2A as an example). Thus they likely still have phosphatase activity in the experiment in figure 3E making it difficult to draw the conclusions they do. A more careful analysis of kinase and phosphatase activities in their different perturbations would be recommendable and should be possible within a reasonable time frame.

Response: These are good points and we will now more carefully assess MPS1 and PP1/PP2A activities.

2) A more stringent test of their model would also be needed. What happens if Plk1 is artificially maintained in the Bub complex? The prediction would be that SAC silencing should be severely delayed even when Mps1 is inhibited. This is a straightforward experiment to do that should not take too long. If the polobox can bind phosphoSer then one could also make BubR1 T620S to slow down dephosphorylation of this site (PPPs work slowly on Ser while Cdk1 have almost same activity for Ser and Thr).

Response: These are good suggestions and we will try to see if maintaining PLK1 at the BUB complex produces effects on the SAC.

3) Another issue is the relevance of Plk1 removal under normal conditions. As their quantification shows in figure 1D-E (I think there is something wrong with figure 1E - should likely be Bub1) the contribution of BubR1 T620 and Bub1 T609 to Plk1 kinetochore localisation seems minimal. Thus upon SAC satisfaction there is not really a need to remove Plk1 through dephosphorylation as it is already at wild type levels. It is only in their BubR1 and KNL1 mutants that there is this effect so one has to question the impact in a normal setting. This is consistent with the data in Figure S1D showing no phosphorylation of these sites under unperturbed conditions.

Response: The major finding of this study is that kinetochore phosphatases are primarily needed to supress PLK1 activity on the BUB complex and thereby prevent excessive MELT phosphorylation. The relevance of this continued PLK1 removal under normal conditions is clear, because when it cannot occur (i.e. if the phosphatases are removed) then the SAC cannot be silenced unless PLK1 is inhibited. Therefore, whilst it is true that PLK1 localisation to the BUB complex is low under normal conditions, that is because the phosphatases are working to keep it that way. The relevance of that continual removal is an interesting, but in our opinion, separate question that will require a new body of work to resolve. One possibility is that PLK1 recruitment is a continual dynamic process, that is perhaps coupled to a particular stage in MCC assembly. For example, PLK1 could bind the BUB complex to recruit PP2A to BUBR1, before being immediately removed by PP2A. In this sense, PLK1 binding could still be functionally important even if it is only occurs transiently and steady state PLK1 levels are low. We will add a line to the discussion to highlight that it would be interesting to test PLK1 dynamics on the BUB complex in future.

4) They write that in the absence of phosphatase activity Plk1 becomes capable of supporting SAC independently (of Mps1 is implied). They do not show this - only that MELT phosphorylation is maintained. As Mps1 has other targets required for SAC activity I would rephrase this.

Response: Good point, this will be rephrased.

Reviewer #2 (Significance (Required)): The advance is clearly conceptual and provides a new way of thinking about the kinetochore localized phosphatases. These phosphatases and the SAC have been immensely studied but this work brings in a new angle. The discussion would benefit from some evolutionary perspectives as the PP1 and PP2A-B56 binding sites are very conserved but the Plk1 docking sites on Bubs less so. This will be of interest to people in the field of cell division and researchers interested in phospho-mediated signaling.

Response: Since the paper was submitted, we performed evolutionary analysis to examine this point. We discovered that the PLK1 docking sites are surprisingly well conserved and, in fact, they appear to have co-evolved within the same region of MAD/BUB along with the PP2A-B56 binding motif. We believe this new data strengthens our manuscript because it argues strongly for an important functional relationship between PLK1 and PP2A. A new figure containing this evolutionary analysis will be included in the final version.

Reviewer #3 **Major comments:**

1. An important limitation of this study is that KNL1 dephosphorylation at MELT repeats is monitored only by indirect immunofluorescence using phospho-specific antibodies. Thus, reduction of phospho-KNL1 kinetochore signals could be due to protein turnover at kinetochores, rather than to dephosphorylation. This is a serious issue that could be addressed by checking KNL1 dephosphorylation during time course experiments by western blot using phospho-specific antibodies, as previously done (Espert et al., 2014).

Response: This is an important point that we feel is best addressed by examining total KNL1 levels at kinetochores (instead of simply total cellular levels by western blots). The reason is that KNL1 could potentially still be lost from kinetochores even if the total protein is not degraded. In all experiments involving YFP-KNL1 we observe no change in kinetochore KNL1 levels and this data will be included in the final version. We will also perform new experiments to examine total KNL1 levels in the BUBR1-WT/DPP2A situation to test whether KNL1 kinetochore levels are similarly maintained in these cells following MPS1 inhibition.

1. For obvious technical reasons, the shortest time point at which authors compare KNL1 dephosphorylation upon MPS1-PLK1 inhibition is 5 minutes. Based on immunofluorescence data, authors conclude that kinetics of KNL1 dephosphorylation are similar when kinases are inhibited, independent of whether or not kinetochore-bound phosphatases are active. However, in most experiments (e.g. Fig. 3B, 3C, 3E) lower levels of MELT phosphorylation are detected after 5 minutes of kinase inhibition when phosphatases are present than when they are absent, suggesting that phosphatases likely do contribute to KNL1 dephosphorylation. I suspect that differences between the presence and absence of phosphatases might even be more obvious if authors were to look at shorter time points, when phosphatases conceivably accomplish their function. I would therefore suggest that the authors tone down their conclusions, as their data complement but do not disprove the previous model.

Response: We appreciate that small differences can be seen in figure 3B and 3E at the 5-minute timepoint (between the WT and phosphatase inhibited situations). This may reflect a role for the phosphatases in dephosphorylation or in the ability of drugs such as BI-2536 (3B) or Calyculin A (3E) to fully inhibit their targets in the short timeframe. We will perform additional experiments to examine MPS1 and phosphatase activity under these conditions, in response to comments by reviewers 1 and 2. In the final version we will carefully interpret the new and existing data and, if required, modify the conclusions appropriately.

1. In all experiments cells are kept mitotically arrested through nocodazole treatment, which is not quite a physiological condition to study SAC silencing. This could potentially mask the real contribution of phosphatases in MELT dephosphorylation. Indeed, it is possible that higher amounts of phosphatases are recruited to kinetochores during SAC silencing than during SAC signalling (e.g. during SAC signalling Aurora B phosphorylates the RVSF motif of KNL1 to keep PP1 binding at low levels; Liu et al., 2010). What would happen in a nocodazole wash-out? Would phosphatases be dispensable in these conditions for normal kinetics of MELT dephosphorylation and anaphase onset if PLK1 is inhibited?

Response: All SAC silencing assays where performed in nocodazole for 2 main reasons: 1) PP2A-B56, PP1 or PLK1 can all regulate kinetochore-microtubule attachments, and thereby control the SAC indirectly. Therefore, performing our assays in the absence of microtubules allows us to make specific and direct conclusions about SAC regulation; 2) Previous work on pMELT regulation by PP1/PP2A in human cells was also performed following MPS1 inhibition in nocodazole (Espert et al 2014, Nijenhuis et al, 2014). Therefore, we are able to directly compare the contribution of PLK1 to the previously observed phenotypes, which allowed us to conclude that PLK1 has a major influence.

Nevertheless, we appreciate the point that the influence of PLK1 could, in theory, be different during a normal mitosis when microtubule attachment can form. Therefore, we will attempt to address whether PLK1 inhibition can bypass a requirement for PP1/PP2A in SAC silencing during an unperturbed mitosis.

Other data are overinterpreted. For instance, the evidence that CDK1-dependent phosphorylation sites in Bub1 and BubR1 is enhanced when PP1 and PP2A-B56 are absent at kinetochores suggests but does not "demonstrate that PP1-KNL1 and BUBR1-bound PP2A-B56 antagonise PLK1 recruitment to the BUB complex by dephosphorylating key CDK1 phosphorylation sites on BUBR1 (pT620) and BUB1 (pT609)(Figure 1F)". Similarly, the claim "when kinetochore phosphatase recruitment is inhibited, PLK1 becomes capable of supporting the SAC independently" referred to Fig. 2C-D is an overstatement, as residual MPS1 kinase could be still active in the presence of the AZ-3146 inhibitor.

Response: These are good points and the indicated statements will be reworded.

**Minor comments:**

1. In many graphs (Fig. 1A-C, Fig. 2A,C) relative kinetochore intensities are quantified over "CENPC or YFP-KNL1". Authors should clarify when it is one versus the other.

Response: This will be clarified in the axis and in the methods.

1. The drawing in Fig. 1F depicts the action of PP1 and PP2A-B56 in antagonising PLK1 at kinetochores. Thus, the output should be SAC silencing, rather than activation.

Response: The SAC symbol will be removed from the schematic to avoid confusion and because it is not actually the focus of figure 1 anyway.

1. In the Discussion authors speculate that KNL1 dephosphorylation relies on a constitutive phosphatase with unregulated basal activity. Would a phosphatase be needed at all when MPS1 and PLK1 are inhibited? Could phosphorylated KNL1 be actively degraded?

Response: We will insert total KNL1 immunofluorescence quantification so show that KNL1 KT levels are not decreased in this situation. KNL1 remains anchored at kinetochore but the MELTs must be dephosphorylated to remove the BUB complex.

1. What happens to MPS1 when KNL1-bound PP1 and BUBR1-bound PP2A are absent? Do its kinetochore levels increase as observed for PLK1? And what about the kinetochore levels of Bub1 and BubR1?

Response: We have demonstrated previously that BUB1/BUBR1 increase in this situation in line with the pMELTs (Nijenhuis et al 2014;l Smith et al, 2019) – these papers will be referenced in relation to this. We will also address the effect of phosphatase removal on MPS1 activity, in response to comments by reviewers 1 and 2.

This is the extra stuff.

This type of online activity has been around since the beginning. Well maybe not the complete beginning but you understand... The anonymity and falsehoods are not only currently allowed but also sort of asked for by design. That is to say that there will always be con men and there will always be platforms for them to work within. This is just the modern equivalent of forging a check. Regardless, that is not to say that it does not make an impact. It does! But it currently only does so due to our lack of strong digital literacy. We need to teach that now more than ever but I think that in a way we already are. Younger kids these days have a great grasp on what may be real or fake and have even made a sort of entertainment game out of it with online story telling and ARGs. We just need to continue to strengthen our understanding of evolved digital literacy as a form of prevention and truth seeking.

This is so true. We can't expect children to be experts with something they have not had exposure to. Most preschools I think understand this, but as soon as they enter kindergarten this notion is lost.

I feel like we don't give children enough credit in the way that they think. They may not think as deep as we can as adults, but they are not incapable of thinking in a way that is abstract.

50% agree 50% disagree. While scientific papers are a niche, I still think there is great value in the process of learning how to write and present results. That is a skill we cannot lose as scientists. And I don't think this article is strictly suggesting we step away 100% from journal articles, but I do think journals may reach a broader audience? As a student, we read a lot of papers and those papers contribute to our learning and sometimes we just want a quick look at results or methods or simply the introduction. Personally, I don't want to run code or a software program to see results if I'm just looking to write a sentence or two for an introduction or an abstract. Again, I think maybe the shift should be to have these codes / softwares available to complement the paper. Not the other way around.

In addition to what is power, let's say you are at work and you run into the CEO of the company. Instantly, you might get nervous. You act different, you might walk different. The CEO represents a person of "power" so you may quickly think of something nice or professional to say as you walk closer to them. You will act a little different than you would around you other co-workers that you talk to on a daily basis. There are many other examples of this power we encounter everyday.

[Note: this preprint has been peer reviewed by eLife. The decision letter after peer review, based on three reviews, follows. The decision was sent on 19 May 2019.]

Summary

This paper describes five cryo-EM structures of ribosomal complexes apparently representing different stages of RF2-catalyzed translation termination. The novel observations here are that the tip of domain 3 of RF2 undergoes a rearrangement from an a-helical conformation to a b-hairpin conformation during termination that likely facilitates exit of the newly synthesized protein from the ribosomal polypeptide exit tunnel and that the ribosome can undergo two thermally activated, spontaneous conformational changes, a relative rotation of the ribosomal subunits and a swiveling of the 'head' domain of the small subunit, during termination that likely facilitate dissociation of RF2 from the ribosome. These are interesting observations that significantly extend our understanding of how class I RFs and ribosome conformational changes drive important steps during termination and, as such, all three reviewers recommended publication provided the following comments are addressed adequately.

Essential Revisions

1) The maps provided through the eLife system seemed to be unsharpened, as they showed very little detail. However, even after sharpening them with a B-factor of -100A2, they still did not show the expected features for their respective resolutions. My suspicion is that FREALIGN has been used to overfit the data. This should be addressed in the revision. It should be indicated whether gold-standard separation of halves of the data sets were used in the final refinements, or whether those were limited to a specific spatial frequency (like was done in the classifications). If the latter, those frequencies should also be stated in the manuscript, and they should be significantly lower than the claimed resolutions.

In addition: a lot of basic cryo-EM information is missing: the authors should include: a) at least one micrograph image b) some representative 2D class averages c) local resolution maps of the five structures. Also, because the density of important parts of the maps seems to be a lot worse than the resolution claimed, it would be good to explicitly mention the local resolution of the important features discussed in the main text. d) for each structure, some zoomed-in figures with the density on top of the molecular model. These figures should be chosen as to validate the resolution claim. For example, in structures I, II and V, the RNA bases should be well separated (they do so at 3.6A), and in structures III and IV beta-strands should be well separated, and many (larger) side chains should be visible. In addition, some panels with density for the most important features of each structure should be shown. e) FSC curves between the refined PDB models and the cryo-EM maps are missing from the manuscript. These should be included. In addition, to evaluate potential overfitting of the models in the maps, for each structure, the authors should also include the FSC curves between a model that was refined in half-map1 versus half-map1, as well as the FSC curve between _thesame model versus half-map2.

2) There appear to be many self-citations, and there are also a few places where relevant citations are missing or are mis-cited. There are too many to list individually, but, just a few examples: Page 4: the only citation for the phrase "recent biophysical and biochemical findings suggest a highly dynamic series of termination events" is a Rodnina paper. There are many, earlier papers from Ehrenberg, Gonzalez, Puglisi, Green, Joseph, etc. that should be cited here. Page 5: The only citation for the sentence "By contrast, biochemical experiments showed..." is a Green paper. There are earlier papers from Ehrenberg characterizing the effects of the GGQ-->GAQ mutations on the ability of RF3 to accelerate the dissociation of class I RFs from termination complexes that should be cited here. Page 5: There's a sentence that refers to X-ray, cryo-EM, and smFRET studies, but only provides citations to two smFRET studies (Casy et al, 2018 and Sternberg et al, 2009); Page 5: Moazed and Noller, 1989 identified and characterized the P/E hybrid state, but they didn't report that a deacylated P-site tRNA 'samples' the P/E hybrid state 'via a spontaneous intersubunit rotation'--that was later work from Noller and Ha; etc. There are several other instances of missing citations or mis-citations. We would ask that the authors review their citations with an eye for excessive self-citations and for missing citations or mis-citations. In this context, "Ensemble-EM" is also cited as a specific method in the introduction (Abeyrathne et al., 2016; Loveland et al., 2017). However, this method is more commonly known as (3D) classification of cryo-EM images, and there are many older, more appropriate citations.

3) The sample imaged is a model sample generated by in vitro assembly with purified components of a termination complex. In order to mimic a bona fide termination complex, a short messenger RNA with a strong Shine-Dalgarno sequence followed by a start codon and immediately after by a stop codon was used (mRNA sequence: 5'-GGC AAG GAG GUA AAA AUG UGA AAAAAA-3'). Similar constructs were used to crystallize termination complexes in the past and it has been proven by smFRET experiments that, at least regarding ribosomal inter-subunit dynamics, this model sample behaves similarly to a real termination complex with a peptide linked to the P site tRNA. However, the nature of this model sample should be apparent for the non-specialist reader, highlighting its similarities with a real termination complex but also its possible limitations, especially regarding the "artificial" nature of having a stop codon so close to the Shine-Dalgarno sequence, a situation that never happens in real mRNAs. The authors should explicitly acknowledge this and discuss its implications in the main text.

4) The authors set up a couple of somewhat 'strawman' arguments in claiming that: (i) there are discrepancies in the X-ray, cryo-EM, and smFRET literature with regard to whether ribosomes can undergo intersubunit rotation while bound to class I RFs or whether the non-rotated conformation of the ribosome is stabilized by bound class I RFs and (ii) class I RFs are able to terminate translation and dissociate from the ribosome without the aid of RF3. In the case of (i), it is obviously possible for class I RF-bound ribosomes to undergo intersubunit rotation while still favoring the non-rotated conformation of the ribosome. Moreover, there are enough differences between the cited studies, both in terms of the experimental conditions as well as the technical limitations associated with the various experimental techniques, that it is easy to rationalize differences with regard to whether the class I RF-bound ribosomes would be expected to undergo intersubunit rotation and/or whether the researchers would have been able to capture/observe intersubunit rotation. In the case of (ii), decades of biochemistry from Buckingham, Ehrenberg, Green, and others had already demonstrated that class I RFs are able to terminate translation and dissociate from the ribosome without the aid of RF3, and that the role of RF3 in termination is to accelerate the spontaneous dissociation of the class I RFs. If the authors want to highlight discrepancies in the literature, they should frame them in the context of differences between the studies, experimental design, limitations of the approaches/techniques in the cited papers that might account for such discrepancies. Re-writing this paragraph also in the light of addressing the missing citations and mis-citations pointed out under (2) will further help in toning these arguments down, which would strengthen the manuscript's scholarship.

5) Class I RFs are post-translationally methylated at the Q residue of the GGQ motif of domain 3 and Buckingham, Ehrenberg, and others have shown that this methylation accelerates and/or facilitates class I-catalyzed termination both in vitro and in vivo. Nonetheless, Svidritskiy et al do not report whether and to what extent their RF2 is methylated. Was RF2 overexpressed in a manner that ensured homogeneous methylation or lack of methylation? If they are overexpressing prfB and not overexpressing prmC, it is likely that they have a mix of methylated and unmethylated RF2. Assuming they are using the wt E. coli prfB gene, then the residue at position 246 is a T, rather than an A or S, and Buckingham has shown that, in the wt T246 background, a lack of methylation at Q252 is either seriously detrimental in richer media or lethal in more minimal media. It was felt that a discussion of this issue was not needed in the main text, but that it would be helpful if the authors would include the important/relevant experimental details in the Methods section, for example, did they use the T246 wt E. coli variant of RF2; and did they overexpress prmC along with prfB?

6) Structure I is denoted and treated as a pre-termination complex, but that does not seem at all possible given that the sample was prepared by incubating a pre-termination complex for 30 min in the presence of excess RF2, conditions that Figure 1-Figure Supplement 3 suggest results in robust termination. Structure I is more likely the non-rotated conformation of a post-termination complex that is in equilibrium with its rotated counterpart, Structure V. Based on my reading of the manuscript, it is likely that the authors understand this point, but are nonetheless using this structure as a mimic/analog of a pre-termination complex. If so, I think this is fine, but the authors should explicitly state that this is what they are doing. Related to this, the authors should clarify the description of their activity assay, show the raw data, and/or report 'Released [S35]-fMet (%)' instead of 'Released [S35]-fMet, CPM' on the y-axis of Figure 1-Figure Supplement 3; as the activity assay is currently described, reported, and plotted, it is impossible to determine whether RF2 is 1% or 99% active in termination.

7) The final sentence of the manuscript reads: "Translation termination and recycling of the release factors and the ribosome therefore rely on the spontaneous ribosome dynamics, triggered by local rearrangements of the universally conserved elements of the peptidyl-transferase and decoding centers". There are a couple of problems with this sentence as written. First, smFRET experiments by Gonzalez, Puglisi, and Rodnina have previously shown that "Translation termination and recycling of the release factors and the ribosome therefore rely on the spontaneous ribosome dynamics" and the relevant articles should therefore be cited here. Moreover, given the data are static structures solved using a sample that is at equilibrium, it is not clear how the authors determined that these spontaneous ribosome dynamics were "triggered by local rearrangements of the universally conserved elements of the peptidyl-transferase and decoding centers". Isn't it equally possible, given the data presented, that the local rearrangements were triggered by the ribosome dynamics?

I find it pretty compelling the way the speaker spends the entire poem dismissing the concept of love as something essential to life, and yet in the end she still states that she would probably not trade love for nourishment. She shows how often, we may be well aware that our emotions are not completely rational, and yet we still struggle to control them.

i would not agree to this framing...

i would put it this way... researchers, just like any other humans , are not immune to rewards and punishment, (or STRUCTURE) of their professions...

if we (the PUBLIC) wish researchers to pursue scientific (however defined) interest, then the structure must be modified accordingly...

who created the current structure? public and private elites... in democratic states, the public certainly have power over public elites, so they should pressure these public elites to change the structure...

now, why is the public concerned with science and scientific advancement? to my understanding, this concern is often expressed in state's constitution, which created the public in each particular state...

then we come to the issue what if the public interest is sometimes against scientific interest? well, this is where (democratic) negotiation takes place... scientists must convince the public that science is useful for them, in all its seeming 'uselessness'...

many times the scientists only need to convince the public elites for this, not the public as a whole... but i think it's good if scientists are always in touch with the public as a whole with regards to their work if they use public funding...

in conclusion, i had fun reading the comments, both at the original site and ea forum, because some commenters 'pierced the veil of ideology' behind the original article, and some of the back-and-forth were hilarious with my knowledge of 'money and the state'... would love to expand on this later when i have time... :)

I think that this line can be described as an indirect foreshadow of future events. Othello in the end believes that Desdemona betrayed him even though we the audience know what really went on.

This is something that I always though was true, I remember there would be times were I would be so stressed because I did not know if what I was writing was correct, as I was always focusing on what the right answer was for the question being asked or prompt.

This seems like an important piece of the work to me as it shows a major theme in British Literature. Deception is something that I have found to be predominant in many works that we have read. I also think this becomes a major turning point in the story where the plot thickens and the main conflict will occur.

Will technology preserve or enhance human connection in the classroom is by far a great question. Technology does make it easier to connect you leave an imprint everywhere you go online. I think as much as it enhances communication it also hinders it.

Below him are his friends that are now dead, this supports that he is writing a poem of loneliness. BUT, what I find from this poem is that, even thought dying is undeniably depressing, NOT dying is even MORE depressing when you are of a certain age. This is almost reassuring, for no one like to think about when they are going to leave this planet, worried that we will kicking and screaming as death takes us away; but here we get a sense that this feared experience may never come. This poem is of a man that is depressed not because death is near, but because death has yet to come.

Artist's Statement: Their Eyes Were Watching God Project

While there are many themes, ideas, and messages within Janie as a character and Their Eyes Were Watching God as a book but perhaps the most prevalent throughout is the focus on Janie's journey for independence and how it shapes her from the start to the beginning. Over time her character develops massively and it is often times very hard to encapsulate a development like that in a single drawing without multiple separated parts. I wanted my project to embody this idea of a journey for independence and change in both of those (the very start and the very beginning) points in the novel, with only one image, the tree.

At the start of Janie's journey in Their Eyes Were Watching God she is alone, but far from independent. Her life with her grandmother is Uncomfortable to say the very least and she longs for an escape from the horrible monitnous torture that is everyday life. Eventually, her grandmother decides to marry her off to a man named Logan Killicks who she believes will be able to provide for her and give a stable living situation. This is all out of Janie's control and she has no say in it at all. After forcing herself through the relationship some time , Janie eventually just up and leaves and runs off with Jody Stark. So, if you look at the image as the first part of the book it may look like a beginning. The tree is in bloom and the serene setting begs for something to happen and for the energy of the “too good to be true” calm to be broken by a storm (woooooo foreshadowing). The background depicts an unknown sprawling landscape ready to be conquered and explored by whoever dares to do so. But the start of her adventure is only the tip of the iceberg. After going off with Jody and eventually discovering that he is nothing near the man she thought him to be and quickly she becomes unhappy. Instead of addressing this, Jody attempts to keep her in a box. As tensions build between the two they have a massive fight and Jody ends up dying as Janie is berating him. Soon after this Janie decides to move on in her life. After Jody dies she seems to be more content with herself. Standing up for herself against his abuse gave her some sense of control and this is where we start to see her mindset begin to change and she becomes more independent. Not too long after her period of mourning is over she ends up meeting Tea Cake. At this point in the story, Janie dislikes the idea of being controlled or held back by a relationship but she still longs for one. When she and Tea Cake meet she realizes that he is the one for her. Janie is liberated by the fact that even though the townspeople gossip and talk behind her back about her relationship, she feels comfortable and happy for the first time in a very long time. However, after a long calm a storm strikes. Literally. During an enormous hurricane which destroys Tea Cake and Janie’s house, Tea Cake is bitten by a rabid dog and becomes incredibly sick. They survive the hurricane but due to the Rabies, Tea Cake becomes very paranoid and begins to think that Janie is cheating on him. After a confrontation, Tea Cake becomes violent and attempts to shoot Janie, in self defense Janie kills Tea Cake and thus begins the final phase of the story. After some time of mourning Janie returns to Eatonville with a new sort of resilience. The things which Janie had endured by the end of the novel strengthened her to a point where she doesn't care what people think. As she returns to Eatonville people gossip and talk behind her back but she stands strong. She becomes content with being alone and this very new kind of independence which feels full and liberating. As opposed to being viewed as the earlier parts of the novel the tree viewed as the end of it is calm and serene in a way that symbolizes solemnity and her final independence. Looking at the drawing as the end of the book the clouds and background are less important and more so work as a backdrop to bring out the tree and show how her environment and experiences shaped her independence. Overall, the fact that the tree is the same in both images but can be extremely different depending on how it is viewed shows that her independence and comfort with being independent was within her the whole time and all she needed was to find it herself in order for it to come out. The journey which she took allowed her to find this confidence in herself.

The Color Purple Analytic Essay “Trauma and Essence” by Bodhi Liveright

Alice Walker wrote a story about a woman named Celie and the history of her trauma. The story is a journey from near death to a full life earned through hardships and self-discovery. Most people might easily lose themselves completely as the character Sofia almost does after her jail sentence. But even after years of hiding it to keep herself safe Celie finds her essence. The single most important thing Walker identifies for us is the idea that our essence can always be accessible. Essence is the part of every individual thing that cannot be broken, a light that always shines. Celie realizes her essence in the simple things and it’s one of the contributing factors to keeping her alive. Celie’s statement “I'm pore, I'm black, I may be ugly and can't cook, a voice say to everything listening. But I'm here.” shows her strength and the fact she is able to say something like this is proof that her inner voice had not been lost after years of traumatic experiences. She sets an example for people who struggle hearing that voice in reality. Celie’s world is horrible to imagine. As she lies in bed with Albert on top of her night after night, she is able to disassociate. Her soul escapes from the harsh reality of living with a serial rapist who had captured her to be his wife. Yet, that is the only thing she knows how to do. Even the hope that one day she can be with her beloved sister Nettie slips away as time passes.“Time moves slowly, but passes quickly.” Before Sofia get arrested for hitting a white man and is sent to jail, she was in touch with her essence. “All my life I had to fight. I had to fight my daddy. I had to fight my brothers. I had to fight my cousins and my uncles. A girl child ain't safe in a family of men. But I never thought I'd have to fight in my own house. She let out her breath. I loves Harpo, she say. God knows I do. But I'll kill him dead before I let him beat me.” Celie then advises Harpo to beat his wife Sofia and Sofia’s life begins to change. This is the life Celie knows, it’s not her fault. She’s involved in a pattern of violence. Sofia eventually leaves with her kids but eventually she falls victim to the culture of submission and racism for Black women. She is a broken person when she’s released from prison and Celie takes notice. “What will people say, you running off to Memphis like you don't have a house to look after? Shug say, Albert. Try to think like you got some sense. Why any woman give a shit what people think is a mystery to me. Well, say Grady, trying to bring light. A woman can't git a man if peoples talk. Shug look at me and us giggle. Then us sure nuff. Then Squeak start to laugh. Then Sofia. All us laugh and laugh.” Shug turns out to be a life line for Celie and she seemed to be the same for Shug. They took care of each other. Shug found redemption with her father through her voice and wouldn’t have been able to without her connection to Celie. This sisterhood became another life-line, a reason for living. Finding out Nettie was alive set everything into motion for true freedom from the hell she experienced for so many years with Albert. For Celie it often came down to simple things, by simply remembering running through the fields with her sister, she transported herself to a place of essence. Celie never lost her relationship with her imagination. Some people get stuck in the place of trauma, like Sofia did. For some reason Celie was able to avoid getting stuck, always able to rise above the awful things happening to her. This is a testament to her ability to find her way back to her essence. We learn how to live in the world based on the situation we’re born into. Celie had it rough and had to adapt to her treacherous environment. Having a father who raped her and then giving birth to two children by him is an almost unbearable story to read. Yet, what struck me the most about her story is how she survived it. Celie found essence in everything. Maybe she was luckier than most people. Maybe she was able to sit down and look at a flower and disappear into it. My dad told me this story about sitting down and watching three stalks of grass. It’s part of a poem he sent me the other day. “I watch three stalks of grass shake in the breeze. More like a shimmy shake. To the left. To the right. And then the wind stops, so they stop. And when the wind blows harder these three begin to dance!” We can overcome certain experiences and maybe change our relationship to trauma by acknowledging our essence. Sometimes it’s seeing the divine in the little things like a stalk of grass. Or a good meal. Or a great movie. Or a walk in the park with your friends. Or simply to lie down in a field of purple flowers and daydream. Humans have unique brains that can remember the past and plan for the future. Yet it occurs to me that we’re always in the present, no matter how hard we try not to. It's unclear why it’s easier for some people than others. Maybe it’s because we are all experiencing things differently from each other.

This thought from Loris has helped me think about my use of nicknames for children, especially non-verbal, and my tendency to use them in my practice. During an anti-bias workshop last year, we began our work by sharing our personal name stories with the group. A majority of my colleagues had described a varying level of turmoil that came with nicknames they had been given. I began to reflect on work with young children and how I had given children nicknames. As a tried to justify my why, I realized it felt like it was coming from a place of humor or joy or playfulness. But, the child couldn't advocate for themselves to accept or deny this nickname. I decided to ask the families, at home visits, about nicknames. This has allowed me to understand where I am coming from as an educator with a new lens. There is an internal image, even if it may feel joyful for you, it may evoke a different feeling for families and children.

Robinson Crusoe is trying to explain his decision to disobey his father and leave home. As Crusoe reflects on this conversation, where his father begs him to stay home, he is aware that the correct choice was to obey his father, because if he had, he would not be stranded on an island. However, it is difficult for Crusoe to sympathize with his former self. Crusoe claims that he “was sincerely affected” and “resolv'd not to think of going abroad any more,” which is clearly not true as he immediately contradicts himself with his declaration to go away anyway. David Hume would have argued that Crusoe’s decision to go abroad came from an inability to sympathize his father because his imagination was set on the adventure he would have sailing abroad. Hume argues that “‘tis certain we may feel sickness and pain from the mere force of imagination,” but because the young Crusoe “would be satisfied with nothing but going to Sea,” his imaginative capacity was too occupied to sympathize with his father's fear. But now that he has experienced sailing away, his imagination is no longer occupied with the thought of leaving, allowing him to agree much more easily with his father as he reflects on himself in this passage.

When we think of a lobster, we think of a hard shell. It is rare that we consider what is under the shell, and how it may feel for the animal to be boiled alive.

Is this what man is made up of... I’m confused

I think the dilemma is basically focus on the cultural relativism. People should consider a kind of cultural object in a general way, not just in your own background. The usage of language varies from people by people, and we should not regard this as a symbol to judge them. Apparently technology cannot do this, which is to distinguish languages with emotion and logic. It would be easily to turn to a new problem among races and geography. Casestudy

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

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Reply to the reviewers

Response to Reviewers Comments

We would like to thank all reviewers for carefully considering our manuscript and providing useful suggestions/ideas. The general consensus was that our study provides an important conceptual advance that reveals a new way of thinking about kinetochore phosphatases. However, in light of our surprising findings, it was suggested that additional experiments would be required to fully validate our conclusions. In particular, it was seen as important to test whether PLK1 can activate MPS1 from the BUB complex and to confirm that PP1 and PP2A are effectively inhibited in situations where MELT dephosphorylation can occur normally (Figure 3).

In general, we agree with these and the other points raised by the reviewers, therefore we plan to address all comments as outlined in detail below.

The major new additions to the final paper will be the following:

1) Experiments to test how BUB-bound PLK1 affects MPS1 activity.

2) Experiments to determine the efficiency of phosphatase inhibition in figure 3.

3) Experiments to test whether maintaining PLK1 at the BUB complex causes SAC silencing defects

4) Evolutionary analysis demonstrating that the PLK1 and PP2A-binding modules have co-evolved in the kinetochore BUB complex. This analysis, which has been performed already, strengthens our manuscript because it provides additional independent evidence for a functional relationship between PLK1 and PP2A on the BUB complex.

Reviewer #1

Minor comments:

1) The authors propose that PP1-KNL1 and BUBR1-bound PP2A-B56 continuously antagonise PLK1 association with the BUB complex by dephosphorylating the CDK1 phosphorylation sites on BUBR1 (pT620) and BUB1 (pT609). It is therefore expected that converting these residues to aspartate would increase PLK1 recruitment. It would be interesting to verify if this hypothesis fits with the proposed model.

Response: The general idea to maintain PLK1 at the BUB complex is a good one, but unfortunately polo-box domains do not bind to acidic negatively charged residues. Instead we will attempt to maintain PLK1 at the BUB complex using alternatively approaches (as suggested by reviewer 2).

2) In Figure 1E, are the mean values for BubR1WT+BubWT and BubR1WT+Bub1T609 both normalized to 1? If so, this fails to reveal the contribution of Bub1 T609 for the recruitment of PLK1 when PP2A-B56 is allowed to localize at kinetochores.

Response: The values will be updated and normalised to the BubR1WT+BUB1WT control. We have also performed additional experiments already and overall the results reveal a small reduction in kinetochore PLK1 following BUB1-T609A mutation and a larger reduction upon combined BUBR1-T620A mutation.

3) What underlies the increase in Bub1 levels at unattached kinetochores of siBubR1 cells (Figure S1C?) Is this caused by an increase in Bub1 T609 phosphorylation and consequently unopposed PLK1 recruitment, which consequently increases MELT phosphorylation?

Response: We suspect that PLK1 is not the cause of the increased BUB1 levels because PLK1 kinetochore levels are actually decreased in this situation (Figure S1A).

4) Although the immunoblotting from Figure S1D indicates that BubR1T620A and Bub1T609A are expressed at similar levels as their respective WT counterparts, some degree of single-cell variability is expected to occur. As a complement to Figure 1B,C and Figure S1E,F could the authors plot the kinetochore intensity of BubR1 pT620 and Bub1T609 relative to the YFP-BubR1 and YFP-Bub1 signal, respectively?

Response: There is indeed variability in the level of re-expression of BUBR1/BUB1 on a single cell level, which can at least partially explain the variation on BUBR1-pT620 and BUB1-pT609 observed within in each condition. We can upload these scatter plots at resubmission and include in the supplementary, if required.

5) The authors nicely show that excessive PLK1 levels at the BUB complex are able to maintain MELT phosphorylation and the SAC (independently of MPS1) when KNL1-localised phosphatases are removed (Figures 2A,B). However, it should be noted that PLK1 is able to promote MPS1 activation at kinetochores and so, whether AZ-3146 at 2.5 uM efficiently inhibits MPS1 under conditions of excessive PLK1 recruitment should be confirmed. Can the authors provide a read-out for MPS1 activation status or activity (other than p-MELTs) to exclude a potential contribution of residual MPS1 activity in maintaining the p-MELTs and SAC?

Response: This is a good point because although PLK1 can phosphorylate the MELTs it can also activate MPS1, although it is unknown whether it can do this from the BUB complex. We had left a dotted line in Figure 4B to include this possibility, but we will now test this directly with additional experiments.

6) To examine whether PLK1 removal is the major role of PP1-KNL1 and PP2A-B56 in the SAC or whether they are additionally needed to dephosphorylate the MELTs, the authors monitored MELT dephosphorylation when MPS1 was inhibited immediately after 30-minute of BI2356. This revealed similar dephosphorylation kinetics, irrespective of compromised PP1-KNL1 or PP2A-B56 activity, thus suggesting that these pools of phosphatases are not required to dephosphorylate MELTs. To confirm this and exclude phosphatase redundancy, the authors simultaneously depleted all PP1 and B56 isoforms or treated cells with Calyculin A to inhibit all PP1 and PP2A phosphatases. In both of these situations, the kinetics of MELT dephosphorylation was indistinguishable from wild type cells if MPS1 and PLK1 were inhibited together. These observations led to the conclusion that neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead they are needed to remove PLK1 from the BUB complex. This set of experiments is well-designed and the results support the conclusion. However, it would be of value if the authors provide evidence for the efficiency of PP1 and B56 isoforms depletion and for the efficiency of phosphatase inhibition by Calyculin A. An alternative read-out for the activity of PP1 and PP2A-B56 (other than p-MELT dephosphorylation) clearly confirming that both phosphatases are compromised when MPS1 and PLK1 are inhibited together could make a stronger case in excluding the contribution of residual PP1 or PP2A to the observed dephosphorylation of MELT motifs.

Response: This is also a good point. We had attempted many different combinations in Figure 3 to inhibit PP1/PP2A activity as efficiently as possible. This is especially important considering the “negative” results on pMELT are very surprising. However, we will now test how efficiently we have inhibited PP1 and PP2A phosphatase function in these experiments.

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

Major comments:

1) In its current state I am not convinced that the key conclusions are fully supported by the experiments and alternative conclusions/interpretations can be drawn. For example the level of MELT phosphorylation will be determined by the balance of kinase and phosphatase activity and if they do not achieve 100% inhibition of Mps1 in their assays then they are not strictly monitoring dephosphorylation kinetics in their assays. If the combination of Mps1 and Plk1 inhibition then more strongly inhibits Mps1 then dephosphorylation kinetics becomes faster. Thus subtle differences in Mps1 activity under their different conditions could lead to misleading conclusions but in its present state a careful analysis of Mps1 activity is not provided. This lack of complete inhibition also applies to the phosphatases and the experiments in Figure 3E indicates that their Calyculin preparation is not really active as at steady state MELT phosphorylation levels are much less affected than in for instance BubR1 del PP2A (Figure 2A as an example). Thus they likely still have phosphatase activity in the experiment in figure 3E making it difficult to draw the conclusions they do. A more careful analysis of kinase and phosphatase activities in their different perturbations would be recommendable and should be possible within a reasonable time frame.

Response: These are good points and we will now more carefully assess MPS1 and PP1/PP2A activities.

2) A more stringent test of their model would also be needed. What happens if Plk1 is artificially maintained in the Bub complex? The prediction would be that SAC silencing should be severely delayed even when Mps1 is inhibited. This is a straightforward experiment to do that should not take too long. If the polobox can bind phosphoSer then one could also make BubR1 T620S to slow down dephosphorylation of this site (PPPs work slowly on Ser while Cdk1 have almost same activity for Ser and Thr).

Response: These are good suggestions and we will try to see if maintaining PLK1 at the BUB complex produces effects on the SAC.

3) Another issue is the relevance of Plk1 removal under normal conditions. As their quantification shows in figure 1D-E (I think there is something wrong with figure 1E - should likely be Bub1) the contribution of BubR1 T620 and Bub1 T609 to Plk1 kinetochore localisation seems minimal. Thus upon SAC satisfaction there is not really a need to remove Plk1 through dephosphorylation as it is already at wild type levels. It is only in their BubR1 and KNL1 mutants that there is this effect so one has to question the impact in a normal setting. This is consistent with the data in Figure S1D showing no phosphorylation of these sites under unperturbed conditions.

Response: The major finding of this study is that kinetochore phosphatases are primarily needed to supress PLK1 activity on the BUB complex and thereby prevent excessive MELT phosphorylation. The relevance of this continued PLK1 removal under normal conditions is clear, because when it cannot occur (i.e. if the phosphatases are removed) then the SAC cannot be silenced unless PLK1 is inhibited. Therefore, whilst it is true that PLK1 localisation to the BUB complex is low under normal conditions, that is because the phosphatases are working to keep it that way. The relevance of that continual removal is an interesting, but in our opinion, separate question that will require a new body of work to resolve. One possibility is that PLK1 recruitment is a continual dynamic process, that is perhaps coupled to a particular stage in MCC assembly. For example, PLK1 could bind the BUB complex to recruit PP2A to BUBR1, before being immediately removed by PP2A. In this sense, PLK1 binding could still be functionally important even if it is only occurs transiently and steady state PLK1 levels are low. We will add a line to the discussion to highlight that it would be interesting to test PLK1 dynamics on the BUB complex in future.

4) They write that in the absence of phosphatase activity Plk1 becomes capable of supporting SAC independently (of Mps1 is implied). They do not show this - only that MELT phosphorylation is maintained. As Mps1 has other targets required for SAC activity I would rephrase this.

Response: Good point, this will be rephrased.

Reviewer #2 (Significance (Required)):

The advance is clearly conceptual and provides a new way of thinking about the kinetochore localized phosphatases. These phosphatases and the SAC have been immensely studied but this work brings in a new angle. The discussion would benefit from some evolutionary perspectives as the PP1 and PP2A-B56 binding sites are very conserved but the Plk1 docking sites on Bubs less so. This will be of interest to people in the field of cell division and researchers interested in phospho-mediated signaling.

Response: Since the paper was submitted, we performed evolutionary analysis to examine this point. We discovered that the PLK1 docking sites are surprisingly well conserved and, in fact, they appear to have co-evolved within the same region of MAD/BUB along with the PP2A-B56 binding motif. We believe this new data strengthens our manuscript because it argues strongly for an important functional relationship between PLK1 and PP2A. A new figure containing this evolutionary analysis will be included in the final version.

Reviewer #3

Major comments:

1. An important limitation of this study is that KNL1 dephosphorylation at MELT repeats is monitored only by indirect immunofluorescence using phospho-specific antibodies. Thus, reduction of phospho-KNL1 kinetochore signals could be due to protein turnover at kinetochores, rather than to dephosphorylation. This is a serious issue that could be addressed by checking KNL1 dephosphorylation during time course experiments by western blot using phospho-specific antibodies, as previously done (Espert et al., 2014).

Response: This is an important point that we feel is best addressed by examining total KNL1 levels at kinetochores (instead of simply total cellular levels by western blots). The reason is that KNL1 could potentially still be lost from kinetochores even if the total protein is not degraded. In all experiments involving YFP-KNL1 we observe no change in kinetochore KNL1 levels and this data will be included in the final version. We will also perform new experiments to examine total KNL1 levels in the BUBR1-WT/DPP2A situation to test whether KNL1 kinetochore levels are similarly maintained in these cells following MPS1 inhibition.

1. For obvious technical reasons, the shortest time point at which authors compare KNL1 dephosphorylation upon MPS1-PLK1 inhibition is 5 minutes. Based on immunofluorescence data, authors conclude that kinetics of KNL1 dephosphorylation are similar when kinases are inhibited, independent of whether or not kinetochore-bound phosphatases are active. However, in most experiments (e.g. Fig. 3B, 3C, 3E) lower levels of MELT phosphorylation are detected after 5 minutes of kinase inhibition when phosphatases are present than when they are absent, suggesting that phosphatases likely do contribute to KNL1 dephosphorylation. I suspect that differences between the presence and absence of phosphatases might even be more obvious if authors were to look at shorter time points, when phosphatases conceivably accomplish their function. I would therefore suggest that the authors tone down their conclusions, as their data complement but do not disprove the previous model.

Response: We appreciate that small differences can be seen in figure 3B and 3E at the 5-minute timepoint (between the WT and phosphatase inhibited situations). This may reflect a role for the phosphatases in dephosphorylation or in the ability of drugs such as BI-2536 (3B) or Calyculin A (3E) to fully inhibit their targets in the short timeframe. We will perform additional experiments to examine MPS1 and phosphatase activity under these conditions, in response to comments by reviewers 1 and 2. In the final version we will carefully interpret the new and existing data and, if required, modify the conclusions appropriately.

1. In all experiments cells are kept mitotically arrested through nocodazole treatment, which is not quite a physiological condition to study SAC silencing. This could potentially mask the real contribution of phosphatases in MELT dephosphorylation. Indeed, it is possible that higher amounts of phosphatases are recruited to kinetochores during SAC silencing than during SAC signalling (e.g. during SAC signalling Aurora B phosphorylates the RVSF motif of KNL1 to keep PP1 binding at low levels; Liu et al., 2010). What would happen in a nocodazole wash-out? Would phosphatases be dispensable in these conditions for normal kinetics of MELT dephosphorylation and anaphase onset if PLK1 is inhibited?

Response: All SAC silencing assays where performed in nocodazole for 2 main reasons: 1) PP2A-B56, PP1 or PLK1 can all regulate kinetochore-microtubule attachments, and thereby control the SAC indirectly. Therefore, performing our assays in the absence of microtubules allows us to make specific and direct conclusions about SAC regulation; 2) Previous work on pMELT regulation by PP1/PP2A in human cells was also performed following MPS1 inhibition in nocodazole (Espert et al 2014, Nijenhuis et al, 2014). Therefore, we are able to directly compare the contribution of PLK1 to the previously observed phenotypes, which allowed us to conclude that PLK1 has a major influence. Nevertheless, we appreciate the point that the influence of PLK1 could, in theory, be different during a normal mitosis when microtubule attachment can form. Therefore, we will attempt to address whether PLK1 inhibition can bypass a requirement for PP1/PP2A in SAC silencing during an unperturbed mitosis.

Other data are overinterpreted. For instance, the evidence that CDK1-dependent phosphorylation sites in Bub1 and BubR1 is enhanced when PP1 and PP2A-B56 are absent at kinetochores suggests but does not "demonstrate that PP1-KNL1 and BUBR1-bound PP2A-B56 antagonise PLK1 recruitment to the BUB complex by dephosphorylating key CDK1 phosphorylation sites on BUBR1 (pT620) and BUB1 (pT609)(Figure 1F)". Similarly, the claim "when kinetochore phosphatase recruitment is inhibited, PLK1 becomes capable of supporting the SAC independently" referred to Fig. 2C-D is an overstatement, as residual MPS1 kinase could be still active in the presence of the AZ-3146 inhibitor.

Response: These are good points and the indicated statements will be reworded.

Minor comments:

1. In many graphs (Fig. 1A-C, Fig. 2A,C) relative kinetochore intensities are quantified over "CENPC or YFP-KNL1". Authors should clarify when it is one versus the other.

Response: This will be clarified in the axis and in the methods.

1. The drawing in Fig. 1F depicts the action of PP1 and PP2A-B56 in antagonising PLK1 at kinetochores. Thus, the output should be SAC silencing, rather than activation.

Response: The SAC symbol will be removed from the schematic to avoid confusion and because it is not actually the focus of figure 1 anyway.

1. In the Discussion authors speculate that KNL1 dephosphorylation relies on a constitutive phosphatase with unregulated basal activity. Would a phosphatase be needed at all when MPS1 and PLK1 are inhibited? Could phosphorylated KNL1 be actively degraded?

Response: We will insert total KNL1 immunofluorescence quantification so show that KNL1 KT levels are not decreased in this situation. KNL1 remains anchored at kinetochore but the MELTs must be dephosphorylated to remove the BUB complex.

1. What happens to MPS1 when KNL1-bound PP1 and BUBR1-bound PP2A are absent? Do its kinetochore levels increase as observed for PLK1? And what about the kinetochore levels of Bub1 and BubR1?

Response: We have demonstrated previously that BUB1/BUBR1 increase in this situation in line with the pMELTs (Nijenhuis et al 2014;l Smith et al, 2019) – these papers will be referenced in relation to this. We will also address the effect of phosphatase removal on MPS1 activity, in response to comments by reviewers 1 and 2.

Michelle brings about a great point in how photography has become a book of scenes. We look back and see photographs and remember the times that we had past. We look at experiences we went through and times that have changed for the better. Michelle says, “Pieces of who we are through time. I agree with that I look through pictures and it reminds me of times in my life when I wasn't the person I wanted to be”. I agree that pictures are pieces of us that are expressed through time. As Michelle says that now in this time she does not want to hide herself anymore. That’s what pictures really are you if you think about it. They hide our true selves and she no longer wants to be that person anymore. “They want you to buy into their reality and it can be just be a facade”, Michelle says. I love this quote because people can shame you. They can act like their life might be better based on a photo. A photograph can be a false photograph. People can easily manipulate people into thinking that’s the way they live. Little do people know it can be a fantasy and not a reality. Not saying all pictures are false because I would be lying if this was true. I’m just saying that pictures can be easily a false photograph that may or may not be true. Michelle explains that a photograph explains how something did exist. This is also true because whether it is in the past or not, it is something that did happen and now is part of the past.

Design thinking 在不同领域的应用。

This passage struck me differently because of the allusion of machinery as something that should be praised. If you think about it there is some truth to it. While we may not openly worship the technology we have, we definitely rely on it enough. Theres a resemblance of reliability that is apparent in technology that hasn't been discussed much before.

Wow, the internet is such a powerful tool when you think about it, it can be used for many reasons, which is also a reason why its important to teach students about information they may encounter

This catches my attention as it is very unfortunate that a Mother and Father know that there is a solution to their daughters crisis and they are unable to get it due to the rules in Canada, I do understand all of the precautions but I could only imagine what the family is feeling. I like how the Father has brought it to social media to try and spread awareness. We are so lucky as Canadians to have free healthcare that you would think they would want to act on this fast and get it into the country. They even made a short film on Cystic-fibrosis and it blew up and received many awards, this is a very strong subject that a lot of people are diagnosed with, so the fact they may have found a cure is amazing. I would like to know if any additional personnel has been contacted based off of Chantelle's story and if anyone has reached out to the family with helpful details or assistance. I really feel this story should be shared as Chantelle is still in the hospital.

This reminded me of the part in the podcast where Dash mentions that in his mind computers are a tool to create, not consume. This was a very interesting statement because most people see technology as a commodity that is good to get our needs met, but not all of us see technology as something used to build more things. This makes me think that some of us may appear entitled to this technology and the commodities it gives, without taking into consideration the amount of work that is put into making such technologies possible. I think when it comes to technology and ethics it is important to understand everything that goes on behind the scenes when creating new technology instead of only seeing what is obvious and right in front of us. We need to dig deeper.

I think this is huge because the youth for the most part knows how to navigate the web. But actually understanding and forming ideas form a post or something they may not fully understand. Everything on the web isnt true so I think it is huge for them to actually understand and recognize things on the web.

Good conclusion, but I think you could re-write this to be more concise. Using both liberty and independence may be a little redundant.

In Fowkes, the 'form of appearance' or the Erscheinungsform.

Exchange value is the 'form of appearance' of something contained in it, yet distinguishable from it--this 'third thing' will turn out to be 'socially necessary labor time'.

Book Two of Hegel's Science of Logic, the Doctrine of Essence, begins with a chapter on 'Der Schein,' which appears in A.V. Miller's translation as "Illusory Being" (Hegel, Science of Logic, trans. by A.V. Miller, pp. 393-408).

Here, Hegel describes "schein" as "reflected immediacy, that is immediacy which is only by means of its negation and which when contrasted with its mediation is nothing but the empty determination of the immediacy of negated determinate being," (p. 396).

Hegel goes on to remark that "Schein" is "the phenomenon [Phänomen] of skepticism, and the Appearance [Erscheinung] of idealism," (p. 396).

In describing exchange value as the 'Erscheinungsform' of 'something contained in it, yet distinguishable from it'--which will be labor--Marx is clearly flirting with the terminology surrounding "Illusory Being" in the Science of Logic, which suggests labor as the 'thing-in-itself' of the exchange value. Exchange-value is the reflected immediacy that conceals the congealed labor that it is its essence.

The passage as a whole is suggestive of how exchange value will wend its way through Marx's demonstration, unfolding from itself determinations of itself.

Before presenting a long, difficult quotation from Hegel, I think the most straightforward way to present this reference to Hegel is to say present the argument as follows:

In Kantian idealism, we find that the 'thing-in-itself' cannot become an object of knowledge; consciousness only ever has immediate access to the form of appearance, the 'sensible form' of a 'thing-in-itself' which never presents itself to consciousness. In referring to the value form as the 'form of appearance' of something else which does not appear, Marx is saying that just as idealism subordinates the objectivity of the world to its appearance for consciousness, exchange-value represents immediately an essence that it suppresses, and implicitly, denies the possibility of knowledge of this essence.

Hegel writes, "Skepticism did not permit itself to say 'It is'; modern idealism did not permit itself to regard knowledge as a knowing of the thing-in-itself; the illusory being of skepticism was supposed to lack any foundation of being, and in idealism the thing-in-itself was not supposed to enter into knowledge. But at the same time, skepticism admitted a multitude of determinations of its illusory being, or rather its illusory being had for content the entire manifold wealth of the world. In idealism, too, Appearance [Erscheinung] embraces within itself the range of these manifold determinateness. This illusory being and this Appearance are immediately thus manifoldly determined. This content, therefore, may well have no being, no thing or thing-in-itself at its base; it remains on its own account as it is; the content has only been transferred from being into an illusory being, so that the latter has within itself those manifold determinateness, which are immediate, simply affirmative, and mutually related as others. Illusory being is, therefore, itself immediately determinate. It can have this or that content; whatever content it has, illusory being does not posit this itself but has it immediately. The various forms of idealism, Leibnizian, Kantian, Fichtean, and others, have not advanced beyond being as determinateness, have not advanced beyond this immediacy, any more than skepticism did. Skepticism permits the content of its illusory being to be given to it; whatever content it is supposed to have, for skepticism it is immediate. The monad of Leibniz evolves its ideas and representations out of itself; but it is not the power that generates and binds them together, rather do they arise in the monad like bubbles; they are indifferent and immediate over against one another and the same in relation to the monad itself. Similarly, the Kantian Appearance [Erscheinung] is a given content of perception; it presupposes affections, determinations of the subject, which are immediately relatively to themselves and to the subject. It may well be that the infinite obstacle of Fichte's idealism has no underlying thing-in-itself, so that it becomes purely a determinateness in the ego; but for the ego, this determinateness which it appropriates and whose externality it sublates is at the same time immediate, a limitation of the ego, which it can transcend but which has in it an element of indifference, so that although the limitation is in the ego, it contains an immediate non-being of the ego." (p. 396-397).

In Lenin's notebooks on Hegel's Science of Logic, these sections provoke a considerable degree of excitement. Lenin's 'Conspectus of Hegel's Science of Logic' can be accessed via Marxists.org here:

https://www.marxists.org/archive/lenin/works/1914/cons-logic/ch02.htm

Standard smear piece from a subhuman lefty that want to control people. They'll never be taken seriously since their ideology is dependent on violence and being an entitled little shit.

While I understand the appeal of ring someone who can both do the research and discover the questions that need to be asked, I can't help but question the logic of eliminating the people who may be able to ask better questions. I don't think we should spread people so thin.

When I was in Chemistry 1 alongside people who would one day graduate as engineers and scientists, the running joke was "it doesn't matter how bad my report is, everyone knows engineers can't write." The professors did their best to encourage good writing habits, but their lectures fell on deaf ears, and some of their misguided attempts to instruct me on writing led to an awful habit of passive-voice that I still struggle to eliminate. The idea of eradicating humanities majors in favor of these students, without proper instruction, makes me shudder.