Hukou: a strict Chinese household registration record that determines access to public services like education, healthcare, and housing based on birthplace

Exemplifies the adaptation of a country driven by culture. The encouragement of this development was driven by the filial piety culture all through out the country. The country feels an obligation to take care of their elderly population.

Demonstrates filial piety through monetary means.

Author(s) note four different outside influences on caregiving in China to set their study into context.

Notes the sample of the study.

Notes where and when research was conducted.

The situation sets up a question for migrant workers. Though they are being paid, there's still complex motivations and feelings towards the action of caregiving itself.

The main point of this article. Filial piety based caregiving vs other perspectives motivating caregiving.

In this context, affect means the feelings that produce behavior and action means the behavior itself.

Textbook chapter 3 defines ethnography as "the in-depth study of everyday practices and lives of a people."

Habituated: a person or animal that has become accustomed to a situation, person, or stimulus through frequent exposure, often making it a regular habit or a routine response

The internal conviction kind of contradicts the filial piety aspect to caregiving. The text is saying in a western point of view, caregiving is done because the individual wants to. In a filial piety point of view, caregiving is done because the caregiver "owes" it.

I can absolutely see why this is a concern in modern times. I think this concern would also be projected on the Chinese diaspora, I wonder if they would "disprove" this concern as well.

Relates to chapter 1 of the textbook's "As powerful as culture is, humans are not necessarily bound by culture; they have the capacity to conform to it or not and even transform it." The author references observed changes in the familial care culture due to outside forces.

Emphasizes the reasoning behind the "need" for caregivers: adult children feel the need to take care of their parents as an act of reciprocity for their parents taking care of them as children. I grew up with this ideology & it's been heavily practiced in my family between the members in Vietnam and in America.

Interesting to frame it this way. To me, I think emotions and personal connections can definitely be exploited. I never thought about it this way but other examples of the commodification of intimacy are companies advertising "personal AI robots" to prey on loneliness. I think caregiving can definitely reflect the idea of "commodification of intimacy" as people tend to seek caregivers for their family members that fit into their family. A level of trust and connection typically are sought out when hiring staff for these purposes.

One of the purposes of this piece is to further "prove" that caregiving can contribute to and shape culture in an anthropological lense.

Patrilineal norms: social systems tracing kinship, inheritance, and succession exclusively through the male line

Patriarchal patriliny: a social system combining male authority (patriarchy) with descent and inheritance traced through the male line (patrilineality)

Kinship practices: the social, cultural, and behavioral systems that define relationships, responsibilities, and care

Kinship: the state of being related, typically through blood, marriage, or adoption

Salience: the quality by which an item, stimulus, or idea stands out from its surroundings, catches attention, and appears prominent or important

Notes writing on the complexity and fluidity of relationships.

Basically caring for the elderly or family members doesn't necessarily require a blood relation, the care can create familial-like bonds between caregiver and care reciever.

I think this is also in part to the collectivist culture in Asia, we see all elders as "ours." Korea uses the word "uri" as a possessive meaning "we" or "ours" instead of "i" or "my" which reflects this deeply engrained collectivist culture that's shared in many Asian cultures.

Filial heart: a deep, loving, and obedient heart towards one’s parents. Or, in this case, towards the elders that caregivers are taking care of.

Filial piety: a Confucian virtue emphasizing deep respect, obedience, honor, and care for one’s parents, elders, and ancestors.

The reason for these migrant caregivers is because Chinese (and many other Asian cultures) emphasize taking care of your family. You can often find multigenerational households across the Asian diaspora. However, the need for the caregivers is because adult children are often too busy with work to fully take care of their parents as they become elderly.

The studied population is various migrant care workers in Shanghai

"Emic perspectives refer to descriptions of behaviors and beliefs in terms that are meaningful to people who belong to a specific culture," (Chapter 3 of the textbook)

Sometimes we forget this.

We cannot forget this. When planning our lessons, it is important to know what our students know. Connecting the new learning to their background knowledge is key.

Something we need to remember. Learning is interactive.

We need to connect with students, value and include them. This is the best way in which students will be ready to learn.

Very important for learning. Feeling safe is key.

We need to have this in mind to support our students learning. Making these connections is important for learning.How do we help our students? What is the best way?

This is important to have in mind. This is the part of the brain that manages our executive functions. The question is how do we help our students and ourselves to develop thispart of the brain.

This is definitely interesting to remember how our brains are always on alert.

Be prepared for a class discussion

Globalization is the increasing connections of different parts of the word

This is definitely present in our schools. We are still fighting this in my school. Some teachers make some comments about families and students that are hurtful. Some teachers do not believe that students can learn because of their way of living.

We are definitely failing our students. I see this in some of our students. there are some students who have made so much growth and we are confident that they will be soon at grade level.

I was really surprised to learn about this some years ago. It is sad that we define a student's future by 3rd grade.

This can be evident in the classroom. Also, we need to think about our students' learning styles and personalities.

I think we need to change our mindset in order to support our students better.

Students need to feel safe to take risks and feel part of th community.

Pert of creating a safe environment is to hold students accountable to high expectations.

This is part of getting to know our students but also to be aware of our own biases and the position we take in this world.

We all need to connect with our students in order to get to know them. This way, we can support them better while taking advantage of the knowledge they bring.

do some qualitative validation!

This preprint has now been published here:

https://doi.org/10.1161/CIRCULATIONAHA.125.077665

Tardif JC, et al. Circulation. 2026;153(8):610–612.

Im kind of obsessed with this idea of re-voicing... there is something here for a project.

Vertical alignment problem

Grammatical error - you need the word "and" instead of a comma for a list with two items

typography doesn't need to be capitalized

Why is this paragraph numbered?

Colons are not necessary in headings.

grammatical error (should be highlighting)

grammatical error

• Minimal Caloric Increase: Intense mental effort only increases energy expenditure by about 5% above baseline (approx. 100–200 kcal per day).
• Maintenance vs. Thought: Most of the brain's energy is consumed by basic biological maintenance rather than the active thinking process itself.
• Cognitive Fatigue & Performance: Mental exhaustion can reduce physical performance by roughly 15%.
• Perceived Exertion: This performance drop is caused by an increased subjective feeling of effort, linked to adenosine accumulation in the brain.
• Impact on Training: After heavy mental work, physical exercise feels harder, leading to earlier fatigue and lower intensity, which can hinder physical adaptation and results.

"most uncertain" is stated a little bit too strongly here. .. Perhaps "Arguably the most uncertain "

Double check: is this the only solution, or are there other approaches?

This is likely very oversimplified and presents concepts that are already well known to most workshop participants, but some may be less familiar with the full process.

• The Experiment: The author, struggling with loneliness after college, challenged themselves to talk to one stranger every day for a month at the gym to overcome social anxiety.
• The Approach:
  • Waited for people to finish their sets to avoid being intrusive.
  • Used a standard opener: "Hey, I see you here all the time. You’re pretty strong. What’s your split?"
  • Transitioned to more personalized openers (e.g., asking about a sports hat or specific equipment) as they grew more comfortable.
• Key Results:
  • 35 Strangers: Talked to a wide variety of people, including medical students, engineers, and retirees.
  • Social Connections: Most interactions were positive. Several led to fist bumps and "gym-nod" friendships, while two resulted in off-site dinners and deeper friendships.
  • Anxiety Reduction: The author realized that the "terrifying" social barrier was largely internal; most people were happy to chat or at least polite.
• Major Takeaway: Consistency is key. By treating social interaction like a gym workout—showing up and doing the "reps"—the author significantly improved their social life and mental well-being.

Hacker News Discussion

• Genuine Appreciation: Many commenters praised the author for giving sincere compliments without a hidden agenda. They referenced Dale Carnegie’s How to Win Friends and Influence People, emphasizing that "radiating happiness" creates a "feeling that glows" even if the interaction is brief.
• The "Cringe" Barrier: A popular sentiment in the thread was that "the only path to cool is through cringe." Users discussed the necessity of enduring awkward first attempts to develop social skills.
• "Genuinely Caring" vs. Small Talk: There was a deep debate on whether small talk is "fake." Some argued that showing curiosity about a stranger is a form of "genuine care" for their well-being, while others found the American style of polite inquiry to be performative.
• Gym Etiquette: The discussion touched on the unwritten rules of the gym. While some Redditors (as noted in the article) want to be left alone, HN users generally agreed that waiting for the end of a set and keeping it brief makes social interaction acceptable.
• Friendship After College: Users identified with the author's struggle, noting that modern life lacks "third places" and that active effort—like the author's experiment—is now required to build a community.

I find this interesting because I have never really put much thought into any of my data being personal and never minded being identified by it. I can understand that others are more sensitive to their information being shared. Personally, I will fill out information forms and not think about it but I'm curious if others hold back certain information? if so let me know what kinds of information?

This simple statement is incredibly profound and important.

no wonder we're all running on Red Bull and love ;) This is just another reminder of why taking good care of ourselves mentally and physcally is so important if we want to be effective and regulated educators!

Il n'y a pas eu d'explication quant à ce code ici

Súmula nº 39/TST - PERICULOSIDADE - Os empregados que operam em bomba de gasolina têm direito ao adicional de <u>periculosidade</u> (Lei nº 2.573, de 15.08.1955).

• A regra é a responsabilidade subsidiária do sócio retirante, no entanto, por prazo de 2 anos após a modificação contratual.

• Logo, a respeito do sócio retirante:

  • Responsabilidade subsidiária, via de regra;
  • A responsabilidade perdura por 2 anos;
  • O termo inicial é averbação da modificação no contrato social;
  • No caso de má-fé, a reponsabilidade é solidária

1940s advertisement with Underwood Standard that uses the phrase "Rhythm Touch", but which features a short armed carriage return lever.

On This Day in Typewriter History: Underwood and the Emperor’s New Old Clothes<br /> by [[Robert Messenger]] for Oz Typewriter Blog<br /> accessed on 2026-05-06T12:33:38

https://oztypewriter.blogspot.com/2012/11/on-this-day-in-typewriter-history_25.html

via Robert Messenger at https://oztypewriter.blogspot.com/2012/11/on-this-day-in-typewriter-history_25.html

I remember all of those "explain your thinking" questions where I had to pay attention to penmanship and grammar, not math.

basically, talking heads in videos are not helpful.

https://www.facebook.com/groups/TypewriterCollectors/posts/10161712887224678/

None of the discussion here seems definitive for differentiating the "two models".

Differentiating between an Underwood SS and the Underwood Rhythm Touch:

comment to James Grooms at https://typewriterdatabase.com/show.23202.typewriter

James, perhaps it's hiding somewhere else in the comments on the database, but I'm curious if you've come across definitive differences between the Underwood SS and the Underwood Rhythm Touch models which have separate pages within the database:<br /> - SS https://typewriterdatabase.com/Underwood.SS.4.bmys - Rhythm Touch https://typewriterdatabase.com/Underwood.Rhythm+Touch.4.bmys

Most of my Google searches don't return anything definitive or with actual sourcing of any sort.

The main page has the SS starting in May 1946 and the Rhythm Touch beginning in July of that year, but doesn't seem to specify between the two in any substantive way. Neither of the two models seems to have had a name printed on it.

Your description here uses both designators, but knowing your penchant for newspaper and magazine advertisements, I would suspect you may have seen specific differentiators.

This Facebook post has some handwaving differentiators: https://www.facebook.com/groups/TypewriterCollectors/posts/10161712887224678/ but none seem definitive or sourced. It also uses the phrase carriage shift, though presumably with these models Underwood had moved to a segment/basket shift on their standards.

Other than the chrome side detailing moving from 3 strips to 5 as you've noted, one of the few differentiators I can see in this era is the shift from the shorter carriage return lever to the longer armed version around 1948 which Robert Messenger notes in https://oztypewriter.blogspot.com/2012/11/on-this-day-in-typewriter-history_25.html. However that same page also has an advertisement on it with the words Rhythm Touch featuring a short armed (older style) carriage return.

Is there really a difference between the SS and the Rhythm Touch or are they the same model with the phrase "Rhythm Touch" used as a marketing tag to compete potentially with Smith-Corona's "Floating Shift"?

Thanks!

For differentiating between the early and later model Underwood SS pre-1948 and after.

via James Grooms at https://typewriterdatabase.com/1950-underwood-ss.23202.typewriter

The end goal is a high converting funnel that sells your diversified product offerings in one place, increasing revenue through showing the advantages of upsells and increasing the average order value through bump ups etc.

• All ThriveCart users get access to Learn
• Learn+ is for premium members who pay a one time fee for lifetime access.

ThriveCart learn started as as way for us to teach our own clients how to leverage the power of the tool but has since grown to be utility for our clients. Meaning? Just like an NFT once you purchase something from the client you can get access to client built training courses.

test

Mrs turner is racist to her own kind. Which also means she hates herself or her background.

Mrs. Turner liked Janie because she had lighter skin, which made her overlook everything “bad” about her. She didn’t like Tea Cake because he was darker skinned so she wanted to make Janie get with her brother instead.

Mrs. Turner’s obsession of whiteness creates tension, which shows how racism can exist within the black community and affect relationships.

They racist

Tea Cake hit Janie because he wanted to prove to Mrs. Turner that he is in control over her and has more power even though he is darker skinned.

The hurricane reveals the power of nature over people, which forces Janie and Tea Cake to confront the limits of human control.

Teak cake and Janie get revenge by punishing Mrs turner for her colorist views and for trying to break their marriage

Tea cake is insecure and treats janie like he’s in control of her

He would rather choose violence than talk to her

This text shows a boastful description of a women’s extreme strength and ferocity

Tea cake tells Janie how Mrs. Turner isn’t a normal person but thinks bad about her own people

Tea Cake is jealous of Mrs. Turners brother and beats Janie for possession and control. They destroy Mrs. Turners restaurant on Saturday night. She returns back to Miami.

Chapter 17 shows how insecure tea cake really is.. and to make himself feel better he hits Janie to show her “he’s in control”

There deciding to move back to Miami as they think they might have a better life over there.

We see that tea cake is really just an insecure man that can’t handle himself.

Chapter 17 Tea Cake hits Janie to show control after getting jealous. Mrs. Turner tries to separate them because of colorism and causes tension. A big drunken fight breaks out in her restaurant, causing chaos and injuries. Afterward, Mrs. Turner decides to leave.

After meeting Mrs turners sibling and being flirted with by other men teacake starts hitting her and his friend approves of it

They are trying to get rid of her, because she is using black folks for her business while looking down at them.

It’s striking how in Chapter 17. the muck’s sense of community starts to curdle. Jody’s control is gone, but now Tea Cake’s possessiveness and the violent fight over Mrs. Turner show that Janie still isn’t fully free, she’s just exchanged one kind of constraint for another. The party turns into a brawl, and you realize the Everglades can be just as trapping as Eatonville.

They’re shaming turner because he is letting everyone trample over his wife and they are saying he isn’t a real man.

It seems like Tea Cake is acting like Janie’s past husbands. His insecurities are leading to him hitting Janie.

Tea Cake resorts to violence against Janie because of his insecurity and jealousy. No one bats an eye at the abuse and they even support it.

Tea cake is becoming just like Janie’s past husbands. He is hitting her physically just out of jealousy.

Tea cake hitting Janie is showing his jealousy and insecurity issues.

Tea cake starts to fight with Janie and hits her and starts a fight in Mrs turners place

It seems that if tea cake doesn’t get his way, or gets jealous he instantly think of abuse or hitting women

Janie isn’t being passive anymore

Tea Cake beats Janie out of jealousy, then a drunken fight breaks out at Mrs. Turner’s place and wrecks everything, showing how tense and chaotic things have gotten

Ai iterates itself to death

https://thetyee.ca/News/2024/08/26/BC-Illegally-Collected-Personal-Info-Wetsuweten/

What are the actual consequences when corporations and the government illegally use data from Canadians?

This trickle down effect is something that goes for all organizations, teams, schools, etc.

This feels like a way to look from a utilitarianism perspective

Feel's like it is hard for a Wall Street employee for example to have a long-term perspective when their performance is graded on a short-term scale

via Erik Bruchez at https://typewriterdatabase.com/1949-underwood-rhythm-touch.10882.typewriter

lmao

given that the vast majority of these are outside of your sample, how accurate is the task construction?

.............

This is also insane! What about literally yes or no questions!

Yes, but do you establish how frequently? I want to see this data. Taking the corpus size of 345k for 150 employees over 4 years works out as ~1.6 emails/employee-day. Is the claim here genuinely that people's work is summarized by two emails a day?

This cannot possibly have evidence

what's the fifth layer!!

very important

"dependia"

Feels like this can create conflict when two people of contradicting training growing up meet and disagree, while both having good character in their own ways.

I would argue that there are situations where you can be behaving ethically but not meeting the standards of the law.

I've seen most companies have a code of ethics or code of conduct that goes beyond legal compliance

Stakeholders definition. Learned this in Business Ethics

Why we should care about ethics in our lives = Main idea

comment comment comment

Test: is this what we want the title to be?

Can you "unbold" the # of respondents and make sure this is all appearing as one continuous sentence? Right now it is "wrapping" around for me at odd places.

Do you need the links? Here they are:

Background analysis: https://docs.google.com/document/d/1-0x4Qd_cvaWbFemKGGhjWAm_SqqVTc6xDYgzcQ82NSs/edit?usp=sharing

Survey design: https://docs.google.com/document/d/18QhPkN8K64RplmitD6GPOkLTGBurjOMaf3P0aeN5QLk/edit?usp=sharing

This is running too long - can you make the space inbetween the two columns LARGER so this will wrap earlier?

Summarization of what is going on in this chapter

Rowland Manthrope, in this article, explains an incident where President Donald Trump's retweet of a quote from Benito Mussolini was gaining attention and going viral on social media. He connects this to a broader picture where he explains that gaining people's attention and going viral on the internet is important to be successful in public life in this modern attention-driven digital world. One piece of detail that Manthrope shared that most stood out to me was that it was actually a bot that caused that incident of making that post.

The Wikipedia article on “meme” explains that the term was first introduced by Richard Dawkins to describe how ideas and cultural behaviors spread through imitation, kind of like how genes evolve over time. It also goes into the word’s origins from the Greek “mimema,” meaning “something imitated,” and shows how the concept has expanded today to include internet memes as a major way culture spreads online.

This article discusses how a 27 year old composer made a viral trend on TikTok. It highlights how individual contributions turned a simple video into a crowd-created performance, which suggest potential for TikTok's potential for creative collaboration.

The source on chain letters explains how chain messages have existed for a long time, even before the internet, often spreading through physical mail and later through email and social media. Many chain letters relied on emotional pressure by promising rewards for forwarding the message or threatening bad luck if the chain was broken. Reading about this made me realize how similar modern internet culture still is. Even though chain letters sound outdated, social media trends today often use the same idea of encouraging participation and rapid sharing. One detail I found memorable was how chain letters became especially widespread through email in the early internet era because forwarding messages suddenly became instant and effortless.

This source talks about the pyramid scheme being a business model where you enroll others to enroll more people, giving it a snowballing effect. It also goes into depth on different models of the scheme because apparently there are more than one. The source also covers notable cases of pyramid schemes being deployed.

This meme is interesting because this kid reached fame unknowingly and many different versions were made. It seemed to have negative effects on the boy and lawsuits were made to the families of the boys how made the video originally.

This source details the story of the "Star Wars Kid" meme, including its creation and the aftermath. The video came from a boy in his high school who was playing with a golf ball retriever; unknowingly to him, there was a camera in the room recording him. Some of his classmates found the recording and published it. The meme took off from there, and people edited the original video to include a realistic lightsaber and sound effects. After the meme became viral, it is reported that the boy from the video finished his schooling in a psychiatric ward, and his parents filed a lawsuit against the families of the boys who originally posted the video.

Monica is a shocking example of virality; overnight, her life changed forever. I think it's because the affair was so public. But just like internet memes, they have consequences as well, but these things come in cycles. There is always going to be new drama, a new meme, or something freshly viral. That is just the nature of the internet.

The blog Spinops by Nobu Tamura mainly shares his artwork about dinosaurs and other prehistoric animals. What stands out is how he doesn’t just draw for fun but his illustrations are based on scientific research, so they feel both creative and educational at the same time. It helps us imagine what these extinct creatures might have actually looked like in real life. This feels like a mix of art and science. It shows how one person can use the internet to share their passion and knowledge with others in a simple but effective way.

I think people should generally give credit when reposting or reusing someone else’s work, especially if it’s art, photography, writing, or originally created videos. For memes and gifs, I can see how attribution is harder because they spread so quickly, but if the original creator is known, tagging or linking them would be the respectful thing to do.

I think Wesch makes a really interesting point here about how internet culture is built around remixing and reinterpreting media, and the critical vulnerability being that most copyright laws haven’t caught up to that reality. It’s kind of strange that so much normal online creativity can technically exist in a legal gray area, with most people not even realizing it.

For me personally, I think this depends on the kind of content being copied. For example, if this meme is massively widespread and well known, it feels more unstable. However, if this is a meticulous dedicated work that displays artistry, another creator is trying to use it to amass money or attention, or it is less well known yet blatantly copied, I believe the original post should be credited.

I think taking someone's content and reposting it as your own or to gain monetary value is probably immoral. I would say it's okay to use others content on social media without giving them credit directly when you are in some way altering it. To clarify, I don't mean adding a video of someone playing a video game next to make someone else's content into short-form videos.

I believe this is a difficult question to answer, simply because with the use of social media, memes and certain phrases and thrown and passed around so easily it seems almost impossible to cite them. As of late, memes and key phrases are mostly found in comment sections or throughout certain video ideas.

This history and background of the origins of memes is both really interesting and surprising to me. I never thought something as widely used today, could trace its roots back to a biological process of DNA and genes. I completely understand how the behavior of an interesting meme is so similar to that of genes in the way it spreads information. For example, the recent 6 7 meme is what started as a normal, interesting gesture caught on camera, and soon started getting reproduced just like a gene to a point where it became viral.

This part was interesting since it suggested definition of the meme. As a social media user who watches meme every day, I thought meme was a popular image or video that goes viral. This part broaden my aspect of dealing with memes.

This passage is interesting because it connects biological evolution with cultural evolution in a simple and understandable way. The explanation of how genes survive through shared traits in a bee colony makes the idea of evolution feel more practical and less abstract. I also like how it introduces Dawkins’ idea of the “meme” as cultural information that spreads between people, similar to how genes spread biologically. Overall, the paragraph effectively combines science and culture while encouraging readers to think about how ideas, beliefs, and behaviors evolve in society.

Correction requested: Table 2 compares the paper's miniscope to other commercial options. the nVue from Inscopix has 2 LEDs, and default 60FPS, with up to 100FPS in fast frame mode.

ff

Usando la cota conocida (citar prelimminares)

I realized how social media has slowly changed creativity from being individual into something almost communal. A lot of the funniest or most memorable content online now is not made by one person alone, but by dozens of strangers building on top of each other’s ideas. The grocery store musical example reminded me of how internet culture can feel chaotic, but also strangely collaborative at the same time. Someone makes a random joke, another person adds onto it, and suddenly people across the world are participating in the same “inside joke” without ever meeting each other. I also think this changes the way people seek attention online. Instead of always trying to create something completely original, many users now try to become part of an existing trend because that gives them a higher chance of being seen by the algorithm. In a way, creativity online has become less about ownership and more about timing, participation, and adaptation. Sometimes the person who improves or remixes the original idea even becomes more popular than the person who started it. I honestly think that says a lot about internet culture today. People value interaction and relatability more than polished originality.

Youtube is kinda strange man.. The user becomes both observer and observed, locked in a recursive loop of feedback between desire and recommendation.

We may want to focus on this approach - potentially more appealing to faculty

should be moved to forefront, before technical proficiency

I have participated in helping content go viral by engaging with it. For instance, I help content go viral by liking it, commenting, and/or reposting it. In addition to this, I have used hashtags that other users have created helping them go viral, and participated in trends that others have started.

Yes, I've talked about this before in comments, but I think this is why the magnitude of virality is so much higher. It's because of how many people clip and make memes of the original post. This all adds up and becomes something like a cultural phenomenon.

I think the idea of replication on social media is really fascinating, because how these content can spread in so many ways beyond just reposting. I realized some people interacts with posts like replying, screenshotting, add some emotinal into those comment, they can helping it changing its meaning or going so far. When people add their own captions or context to a post, it can completely shift how others interpret the original message. Before reading about this, I didn’t really think about how these small changes could “carry forward” future versions.

wat

"you will know us by the trail of dead"...

17:20 glyphosate may be cheaper in the short term, but it is 100% more expensive in the long term. these people hate future generations, these people hate children, these people are the real childfuckers

I looked up this platform and it seems to be incredibly useful for a wide variety of things. Namely, it is used for analytics to configure a web page based on how many users encountered problems with a system. Glad we are using this as I am seeing only good things being said about it online.

some of the tooltips are not coming up -- like here!!

Note, it's doing the sampling in straight javascript

Could look up.

Autoantibodies (AABs) have been identified in advanced Covid, laboratory models of Covid-19 utilizing S-protein fragments. The concept that addressing the AABs is a therapeutic target is reasonable, but falls short of addressing the host-targets, the ubiquitous and critical neuroreceptors that have been rendered dysfunctional by these AABs. The a7 nicotinic acetylcholine receptors (a7Rs) rendered dysfunctional are no longer capable of stabilizing the cholinergic anti-inflammatory pathway. In our clinical experience, supported with laboratory collaborations, re-establishing a7R function plays a significant role in both acute COVID-19 as well as Post-Acute Sequelae of Covid. Focusing on therapeutics for Abs 1 or Abs2 appears to be just as short-sighted for PASC as it was for using Monoclonal Abs, IL-6 inhibitors, antivirals for acute COVID-19. The focus should be entirely on the host - targets, the a7Rs and enabling the host's CAP. Ref: doi: 10.1016/biocel.2024.106519

Chapter 18: Animals begin moving east, warning of a coming hurricane, but most people ignore it. The storm hits hard, flooding the Everglades and causing chaos and destruction. Janie, Tea Cake, and others struggle to survive as they try to escape the rising water and dangerous conditions.

Chapter 20 Janie returns home after Tea Cake’s death and tells Pheoby her story. She explains what happened and reflects on love and life. Even though she is sad, she finds peace in her memories of Tea Cake and feels at home with herself again.

Chapter 19 After the hurricane, Tea Cake gets rabies from a dog bite and becomes violent. Janie is forced to shoot him to save her own life. She is put on trial but found not guilty. In the end, she buries Tea Cake and mourns him deeply.

Jainie feels free and equal in her relationship.

They build a simple but happy life together.

Jaine and tea cake move to the Everglades.

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

Evidence, reproducibility and clarity

1) Summary

This study investigates the mechanochemistry of Arp2/3-mediated branched actin networks at the level of individual branch junctions under load. Using microfluidic single-filament/branch force assays (including constant-force flow and open-chamber imaging) the authors quantify debranching, re‑nucleation, and mother- vs daughter‑interface stability across nucleotide states of Arp2/3 (ADP-Pi, ADP, and an ADP-BeFx proxy for ADP-Pi). They further test effects by two branch regulators (GMF and cortactin). Key findings include: (i) ADP-Pi and ADP complexes share similar force dependence but differ markedly (~20×) in intrinsic dissociation rate; (ii) phosphate turnover on the Arp2/3 complex is rapid ii) affinity for Pi drops when Arp2/3 loses its daughter filament; (iii) quantification from model fits uncovers large stability differences between daughter and mother interfaces of the Arp2/3 complex; (iv) extraordinary high stability of ADP-Pi-like Arp2/3 on the mother filament; and (v) distinct effects of GMF and cortactin on force‑dependent stability. Overall, the work combines technically demanding measurements with mechanistic modeling to probe how nucleotide state and regulatory factors tune branch mechanics.

2) Major comments:

1. Low force kinetics and completeness of survival curves (Figure 1). "For all forces, the surviving curves exhibited a clear single exponential behavior...." While the data can be fitted to monoexponential decay curves, data at low forces is clearly incomplete. >90% of branches have not dissociated by the end of the experiment. For the particular data shown in 1C (F00nN, n=60 total branches) it means that the time information is coming from

Essential; experiment might already be performed. Otherwise straightforward to do (weeks time).

In figure 1B, we indeed show a Survival curve for ADP-Arp2/3 complex branch dissociation at 0 pN up to 900 seconds. As now shown in updated supp figure S2, the data was in fact acquired for at least 5000 seconds for ADP-Arp2/3 and ADP-Pi states (N=2 repeats for each condition, with n = 60 and 90 branches for ADP-Arp2/3 branches, and 90 and 132 branches for ADP-Pi-Arp2/3 branches). The debranching rates reported in the initial submission were already obtained by fitting the surviving curves over the whole duration of the experiments.

1. Stability Analysis (Figure 4). I can follow much of the arguments presented in the stability analysis of the daughter vs mother interfaces, which is in principle extremely interesting! However, there are some concerns here:

i) The authors emphasize the zero force ratio derived from fits (which is linked to the stability difference of the two interfaces in the absence of force) despite this being only weakly constrained by data. Intuitively in the model, the stability difference should grow to very large values as the re-nucleation ratio approaches 1 at low force. This combined with the noise in the data poses an issue in my opinion. Looking at the data and the error margin, I think that the authors cannot state with high confidence that there is a real difference between the relative stability of the daughter and mother interfaces between the two nucleotide states of the complex.

Essential; analysis and textual revision only

We thank the reviewer for this comment. The difference in stability between the two interfaces is strongly constrained by the shape of the branch renucleation ratio versus force curve, and its value at 0 pN. This is illustrated in the figure shown below (new Supp Fig. S8), showing the dissociation rates of the two interfaces (in 'dashed' and 'point-dashed' style) that contribute to the overall debranching rate in each nucleotide condition. Despite the limited force range at which we probed the debranching rate, the branch renucleation ratio curve informs us on which interface is the weakest, and how this evolves with force.

We have assessed the confidence intervals of the parameters obtained from the fits, taking into account the error bars on our experimental datapoints. It seems to indicate that the simultaneous fits of the debranching rate and the branch renucleation ratio curves indeed constrain the parameters quite strongly. These confidence intervals are now reported in the main text and in the summarizing table.

We have repeated branch renucleation experiments for ADP-BeFx- and ADP-Pi-Arp2/3 complex branches (see new figure 4C&D, and our response to the next point). We believe these new measurements allow a better assessment of the relative stability between the two interfaces for Arp2/3 complex branch junctions in the ADP-BeFx state.

Still, we agree with the reviewer that the dispersion of the experimental data does not allow us to have a strong confidence on the crossover force and relative stability difference of the interfaces. Therefore, we have slightly toned down the way we present and discuss the differences in stability when comparing the two nucleotide states.

ii) For ADP-Pi, the renucleation ratio essentially remains flat over the measured force range. Hence, the data can only provide little leverage to estimate both the zero force ratio and, more importantly, the differential distance to the transition state in the slip-bond model in my opinion, which will show in the crossover force. Consequently, the quoted ">100×" stability difference at F=0 and the crossover force >20pN are driven largely by extrapolation rather than direct constraint by data. Given the high number of free parameters in the model, I would anticipate that several crossover forces and differential distances might explain the data nearly equally well. Instead of loosely reporting exact number from fits, I would have hoped for some sort of sensitivity analysis, for instance relying on profile likelihoods. Also parameter values could be reported as bounds (e.g crossover force≫measured range) rather than precise point estimates. This issue re-occurs (albeit not as drastically) for the cortactin experiments (Figure 6).

Essential; analysis and textual revision only

As mentioned in our response to the previous point, we have repeated renucleation experiments for ADP-BeFx- (and also for Arp2/3 complex branches in the presence of 50 mM Pi) (see new figure 4C&D) to better characterize the differential distance between to the transition force. The crossover force for the ADP-BeFx state is now 13.5 pN and the ratio of the stability between the two interfaces is roughly 100 times.

We agree with the reviewer that the dispersion of the experimental data does not allow us to have a strong confidence on the crossover force and relative stability difference of the interfaces. We have thus toned down the way we report these values. We do believe though that the difference we report between the ADP and ADP-BeFx state appears to be significant and needs to be acknowledged.

As a side note, it has proven to be challenging to pull on branches at forces higher than 7 pN. To apply a large force on the branch junction, we need to have a high flow rate. In this case, it appeared that the height of the filaments (both mother and daughter filaments) above the surface seem to deviate from what we have established in our previous studies (Jegou et al, Nat. Comm. 2013 & Wioland et al, PNAS 2019). This may originate from the fact branched filaments have a more complex shape than an individual filament. Characterizing accurately the evolution of the branch height as a function of the flow rate and applied force would require quite extensive additional characterization, which, we believe, is beyond the current focus of this study on the stability of Arp2/3 complexes.

iii) One important expectation from the "two slip bond" model is that branch dissociation rates should not necessarily scale mono-exponentially as they mostly do over the accessible force range of the paper. However, once the "minor" pathway of dissociation from the mother starts to dominate at high forces, rates become more force sensitive. This is nicely recaptured by the model fits in Figure S6 but deserves some explanation in the text. Otherwise, people will simply remember the "ADP-Pi is 20-fold more stable than ADP at all forces" message.

Essential; textual revision only

We now have rephrased the key sentences (in the Abstract and Results sections) to more clearly state that the debranching rate is not increasing mono-exponentially with force.

In the Abstract: "Remarkably, we find that branch junctions are over 30-fold more stable when the Arp2/3 complex is in the ADP-Pi rather than ADP state, and that force accelerates debranching with similar exponential factors in both states."

In the Results section: "The debranching rate seems to increase exponentially with the applied pulling force, in the range of 0 to 6 pN (Fig. 1F; see more refined analysis below). This behaviour is predicted by the Bell-Evans model for a slip bond."

iv) One important prerequisite for the model is that isolated Arp2/3 complexes (without a daughter filament) should dissociate with equal rates from mother filaments at all flow rates. Since the Arp2/3 complex prefers mother filament curvature, forces experienced by the mother might change its off-rate. It would be good to refer to this assumption in the text and experimentally verify it. I could not find it in the paper nor in Ghasemi et al 2024.

Essential; simple experiment (a weeks time).

We thank the reviewer for this important comment.

First, we investigated whether the viscous drag force, applied on the ADP-Arp2/3 complexes which remain bound to mother filaments could affect their stability. We have performed branch renucleation experiments at different flow rates but with the same pulling force on branch junctions (average force 3.9 pN) by adapting the length of the daughter filament. As shown in new supp. figure S11 (shown below), we did not observe any significant differences between 'low' and 'high' flow rates. If the off-rate of the surviving Arp2/3 was significantly affected by the flow, this would have led to a variation of the renucleation ratio with the flow rate.

Second, we have investigated the impact of the tension experienced by the mother filament at the location of the branch junction for ADP-Arp2/3 complex branches, with the same pulling force on the branches (average 4.1 pN pulling force on branches). We have quantified the debranching rate from three groups of branches depending on their position along mother filaments. As shown in new supp. figure S12 (shown below), we can observe a small trend, where the debranching rate decreases with the tension on the mother filament at the branching point.

Doubling the tension on the mother filament from 15 to 30 pN decreases the debranching rate by a third. Though, pairwise logrank tests performed between the survival fractions of the three binned groups do not report any statistical significant difference (all p values > 0.05). One possible explanation for this is the height of the mother filament in the microfluidics flow that increases linearly from the anchoring point to the free barbed end. As a consequence the pulling force on the branches will be higher, as branches experience faster flows.

For these same groups, upon branch dissociation, all remaining-bound Arp2/3 complexes are exposed to the same flow rate; the branch renucleation ratios were similar. Thus branch renucleation ratio seems to not significantly depend on the tension experienced by the mother filament at the branching point.

Similarly, Pandit et al PNAS 2020, Extended figure S1, also reported no detectable impact of the mother filament tension on the debranching rate in their assay.

v) The force dependence of the branch re-nucleation rate (Fig 3D) has been measured previously by the same group (Ghasemi et al). While the data in the older paper has not been fitted by a model, the trend of the data in the previous paper looks conspicuously different. Are there any explanations for this? I speculate that it might be related to actin and ATP not being saturated (low-force re-nucleation rate rarely exceeds 80%) in Ghasemi et al., but it would be good to know what the authors think about this. Essential; textual revision only

This is a good point. We have plotted the data of the renucleation ratio from ADP-Arp2/3 complex from figure 1F of Ghasemi et al, Sc. Adv. 2024 (performed at 0.3 and 1 µM actin), together with the data of the current study from figure 4D (performed at 1.5 µM actin). We feel this comparison could be of interest to the readers, and have thus integrated it in the manuscript as new supp. figure S13 (shown below).

As expected, the branch renucleation ratio is lower with lower concentrations of actin. The experimental data points from Ghasemi et al are similarly well fitted by the branch renucleation function obtained for 1.5 µM multiplied by a scaling parameter, which reflects the fact that the branch renucleation ratio is actin concentration dependent (Fig. 6A in Ghasemi et al). This scaling parameter was the only free parameter of those fits.

Since the branch renucleation ratio depends on the actin concentration as follows, 0.97.kon.([actin] - Cc)kon.([actin] - Cc)+koffATP-Arp2/3 , with kon = 3.4 µM-1.s-1 and koff ATP-Arp2/3 = 0.66 s-1 from (Ghasemi et al. 2024), the scaling parameter obtained by the fits give estimates of the actin concentration in these experiments, of 0.6({plus minus}0.05) and 0.9({plus minus}0.2) µM for the experiments performed at 0.3 and 1 µM respectively in (Ghasemi et al. 2024).

1. Stability of the authentic ADP-Pi-Arp2/3 complex on the mother filament. The extraordinary stability of the isolated ADP-BeFx-Arp2/3 complex on mother filaments is surprising, especially considering that both ATP and ADP states are much more labile (Ghasemi et al 2024). I would recommend repeating this experiment in the authentic ADP-Pi state with labelled Arp2/3 complexes as a more direct readout, even if this would require working with very high phosphate concentrations.

Essential; simple experiment (a weeks time).

We have followed the recommendation of the reviewer and have performed new experiments using fluorescent Arp2/3 complexes for ADP, ADP-BeFx and ADP-Pi states, now displayed in new figure 5C (also shown below).

For fluorescent Arp2/3 complexes remaining bound to the mother filament, the Arp2/3 complex - mother filament interface is ~ 100 times more stable in the ADP-BeFx state (0.0046 s-1) compared to the ADP state (0.56 s-1). We also assessed the dissociation of surviving ADP-BeFx-Arp2/3 complexes using unlabelled Arp2/3 complexes (previously in figure 4B, repeated experiment shown in new supp. figure S10), which also indicates a remarkable stability.

The dissociation curve of surviving Arp2/3 complexes in the presence of 50 mM Pi and 200 µM ATP in solution reflects the mixture of Arp2/3 dissociating in the ADP/ATP state and ADP-Pi-Arp2/3 that can either dissociate in the ADP-Pi state or lose their Pi and dissociate in the ATP state. Despite the presence of 50 mM Pi, the rate at which ADP dissociates and ATP reloads rate is much faster than Pi binding. Fitting this survival curve with a function that accounts for the initial double populations and the evolution of the ADP-Pi population (see Methods) gives a good estimate of the Pi release rate.

OPTIONAL: Further, but beyond the scope of the present paper, would be titrating phosphate in these experiments, which would even allow the authors to independently verify the reduced Pi affinity for Arp2/3 in the mother filament. Of note, this affinity difference is needed to satisfy detailed balance in the reaction scheme (Fig 4 D)!

We thank the reviewer for this suggestion. High concentrations of phosphate in the buffer renders glass surfaces quite sticky in our assays. We've tried several different passivation strategies (BSA, PLL-PEG, K-casein, ...) but none gave satisfactory results. So titrating phosphate, by going beyond 50 mM phosphate, proved to be quite challenging.

Detailed balance, considering the two possible routes connecting the ADP-Pi-Arp2/3 complex branch junction state and the surviving ADP-Arp2/3 complex state, can be written as KPi rel.branch junction . Kdebranching ADP-Arp2/3 = KdebranchingADP-Pi-Arp2/3 . KPi rel.surviving Arp2/3.. Some of these affinity constants are not known, because of the inability to determine reverse reactions rates such as the rebinding of a daughter filament to a surviving Arp2/3. It is thus hard to determine how the affinity of Pi for Arp2/3 complex changes between Arp2/3 complexes at branch junctions and surviving Arp2/3 complexes on mother filaments.

While we cannot determine the affinity constant of Pi for a surviving Arp2.3 complex, our data indicates that the dissociation rate of Pi is higher from Arp2/3 complexes at branch junction (koff = 0.21 s-1) than from surviving Arp2/3 complexes (koff = 0.05 s-1). This unexpected finding indicates that surviving Arp2/3 complexes adopt a conformation where the nucleotides are readily exchanged, but where the 'back door' for Pi release is less open. We now discuss this point in our revised manuscript.

1. Importance of "surviving" ADP-Pi-Arp2/3 complexes. The authors show a) rapid turnover of Pi on the ADP-Arp2/3 complex in both branch- or mother filament-bound state and b) the lowered Pi affinity of the latter. Nonetheless, they emphasize the importance of long-lived "surviving" ADP-Pi bound complexes on the mother (even stated in the abstract). I understand that this fraction shows under some experimental conditions (BeFx), but unless I am missing something, most complexes should rapidly lose their phosphate and either exchange nucleotide or dissociate from the mother under physiological conditions. Please clarify or tone done.

Essential; textual revision only

We thank the reviewer for their remark. We have tried to clarify this aspect in the manuscript.

As shown now with the departure rate of fluorescent surviving Arp2/3 complexes together with branch renucleation data, we show that surviving ADP-Pi-Arp2/3 complexes are quite stable on mother filaments, because they detach and release their Pi slowly, such that branch regrowth will occur provided there is actin in solution. In the absence of actin monomers, as the reviewer correctly points out, the surviving ADP-Pi-Arp2/3 will predominantly release its Pi and thus become a surviving ADP-Arp2/3 complex. We have modified the text to avoid any confusion.

1. GMF mechanism. The authors claim that GMF "...accelerates the departure of the surviving Arp2/3 complex from the mother...". I assume that they infer this from decrease in the re-nucleation ratio. However, alternatively GMF could simply dwell on the complex, inhibiting re-nucleation without promoting dissociation from the mother. The authors should either monitor Arp2/3 dwell times directly to discriminate between these possibilities or be more cautious in their conclusions.

Essential; simple experiment (a weeks time) or textual revision.

In Ghasemi et al. Sci. Adv. 2024, we examined the departure of Arp2/3 from the mother filament after GMF-induced debranching using fluorescent Arp2/3. Most of the fluorescent Arp2/3 dissociated from mother filaments within the same frame as the branch, i.e. within 0.5 seconds after the debranching event, and none were visible after another second . This could be due to Arp2/3 departing with the branch or an accelerated departure after branch dissociation. In any case, this rules out the possibility that GMF would dwell on the surviving complex for a substantial amount of time without promoting dissociation from the mother.

In the present manuscript, we now show that increasing the ATP concentration 10-fold (from 0.2 to 2 mM) is sufficient to restore the branch renucleation ratio to its level without GMF. This shows that GMF does not cause Arp2/3 to leave with the branch, but rather that it (also) acts on the surviving Arp2/3 complex, in a way that is countered by high concentrations of ATP. More specifically, it suggests that GMF accelerates the departure of the surviving ADP-Arp2/3 complex, either directly and by hindering the reloading of ATP, and that GMF does not affect the surviving Arp2/3 complex once it has reloaded ATP.

We now discuss these two non-mutually exclusive possibilities for the accelerated dissociation of the surviving ADP-Arp2/3 complex in the manuscript.

6.Cortactin mechanism and the "leash model". I must say that the cortactin data are the most puzzling part of the paper and hard to reconcile with what we know from structure. I was hoping to find some of this resolved in the discussion. However, I do not understand the "leash model" in the discussion section for cortactin-mediated branch stabilization: "This would explain the observed increase in branch survival compared to the absence of cortactin. As the pulling force is increased, this rebinding mechanism becomes less efficient." According to my understanding of the data, this is opposite to what happens. Cortactin only stabilizes the labile interface at elevated forces! Some re-writing might help here.

Essential; textual revision.

We thank the reviewer for having us think more thoroughly about the model we initially proposed. We now believe that our 'leash' mechanism is not able to fully recapitulate our observations in a simple and satisfactory manner.

We now propose a much simpler model, where the binding of cortactin to the Arp2/3 complex at the branch junction simply changes the energy landscape of the Arp2/3-daughter interface without the need to invoke a rebinding of the daughter filament upon branch departure. We have updated our interpretation of the data in the Discussion section accordingly.

Overall, our results on the impact of cortactin on branch renucleation highlights a surprising behaviour that would require further investigation to fully decipher the underlying molecular mechanism.

3) Minor comments

Organization: - I do not want to impose on how to best tell the story, but I felt that Fig1 A-D and Fig 2 A-B belong to one logical unit (nucleotide dependence), whereas Fig 1 E-F and Fig 2 C belong to the other (Pi binding and exchange). Perhaps consider re-organizing to streamline presentation?

We thank the reviewer for their suggestion. We agree that it flows more naturally as suggested, and have made the changes! Thank you.

Semantics/Typos: - Abstract: „... ADP-Pi and ADP-Arp2/3 detach with the same exponential increase as a function of force...". Increase should refer to the dissociation rate, which should be added to the sentence.

We have corrected this.

Results page 8: "...and the majority of Arp2/3 complexes detach from the mother filament while remaining bound to the branch at the debranching time." "Branch" should likely be daughter here, as there is no branch after dissociation of either interface.

We have corrected this, thank you.

Results page 13: "Exposing ADP-BeFx-Arp2/3 complex branch junctions to a saturating amount of GMF...". It is strange to imply saturation, because GMF likely simply does not bind to the complex in this nucleotide state with appreciable affinity. Suggest to change to "high".

We have made the changes accordingly.

Discussion page 18: "Moreover, in mammalian Arp2/3, His80 in Arp3 (corresponding to His73 in mammalian actin) is not methylated, and corresponds to residue N77 in Arp3, which is also not modified." N77 likely belongs to Arp2?

We have made the changes accordingly.

Discussion page 19: "We showed that Pi affinity for Arp2/3 complexes at branch junctions is around 3.7 mM (Fig. 1), a value which lies within the reported 1-10 mM Pi concentration measured in the cytosol in different mammalian cell types". Notably, this is not too different from F-actin, which should be mentioned. By this measure alone, free inorganic phosphate could also directly regulate actin filament stability!

We now mention this and discuss that intracellular Pi can also impact actin filament nucleotide state.

Future interest (non essential): - It would be utterly exciting (but beyond current scope) to quantify how instantaneous debranching rates evolve for naturally aging branches starting from ATP-Arp2/3 complexes!

We thank the reviewer for this remark. It is indeed quite beyond the scope of the current study, as this would require a way to probe ATP-Arp2/3 complex branches while daughter filaments are still quite short (so pulling on them is difficult). An interesting alternative could be to use ATP analogs, such as App-NHp (aka AMP-PNP), to stabilize this state. However, some studies have mentioned that App-NHp is not very stable.

Significance

General assessment:

This is a compelling and carefully executed study that delivers a clear mechanistic framework for how Arp2/3 branch junctions fail and re‑form under load. The central strength is the tight integration of state‑of‑the‑art reconstitutions with careful and original kinetic analysis. The experimental design is elegant and experiments have been carried out to a masterful standard. The figures are clear, the statistics are appropriate with some exceptions as detailed above. There are very few labs in the world that could have achieved this feat!

A few aspects could be further strengthened, most notably the explanation and application of the "two slip bond" model as well as slightly more restraint in speculating around specific regulatory mechanisms. However, these are minor refinements that do not detract from the important contributions of the paper.

Overall, the clearly work merits publication with high priority after revision; most requested changes are textual/analytical with very few targeted experiments, which would substantially strengthen core claims.

We thank the reviewer for their positive evaluation of our manuscript. We hope that our responses to the detailed points above, along with the corresponding revisions of the manuscript, will alleviate their concerns.

Advance relative to prior literature: The major novel findings of the paper are already summarized above. There is some recent work done on the subject of branch mechanics by the authors (Ghasemi et al 2024, PMID: 38277459) and others (Pandit et al 2020 PMID: 32461373), but the focus of the present work is clearly unique and the there is plenty of novel insight.

Audience and impact: Primary audience: specialists in cytoskeleton dynamics, in vitro reconstitution single molecule biophysics, and mechanobiochemistry. Secondary: researchers in cell motility, morphogenesis and mechanobiology, physicists working on active matter and modelers studying force producing and load-bearing biopolymer networks. The results and analysis framework should inform quantitative models of branched network turnover under load and the interpretation of regulatory factor action in vivo and in cells.

Reviewer expertise: Actin dynamics; biochemical reconstitution; single molecule approaches; biophysics.

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

Xiao et al examine the molecular events occurring when Arp2/3 complex-mediated actin filament branches are removed from mother actin filaments. They do this using microfluidics assay with purified proteins combined with single filament TIRF imaging of branched actin filaments with distinct fluorescent labels. The contribution of different nucleotide states of Arp2/3 complex are tested in conjunction with the relationship force exerted on the branches and regulatory protein involvement from GMF and cortactin. The data seem comprehensive and highly quantified in response to concentration, force, fraction of branches and survival times and branching rates. They find that ADP-BeFx and high phosphate concentrations (leading to the ADP-Pi state) leads to a slower debranching rate at a given level of force applied. The ability to rapidly switch the buffer gives powerful information about response times of debranching compared with other actin remodelling events. They use renucleation experiments to determine that the previous debranching event most often occurs at the Arp2/3 complex/daughter interface, showing that filaments will be ready to re-branch in the stable ADP-Pi bound state. GMF addition allows debranching of the ADP state to occur at a lower force. Cortactin acts similarly to the ADP-Pi state to increase branch stability.

Specific comments

The pulling force on the branches seems to arise from different flow rates in the microfluidics. Viscous drag is mentioned and I can see there is methylcellulose in the buffer. It would be helpful to have the explanation of the conversion between flow and force, even if it has been standard in previous work.

We apologize if this was unclear: in microfluidics experiments, the buffer does not contain methylcellulose. Methylcellulose is only used for 'open chamber' experiments, where no force is applied to Arp2/3 branches, to maintain them in the TIRF field of excitation (Figure S2).

To better clarify the conversion between flow and force, we have rephrased and extended the Methods section to explain how the force on the branch junction is computed based on the local flow velocity and the length of the daughter filament.

Pg 5 - what was the motivation to titrate phosphate? It seems a stretch that intracellular Pi levels are tuning branching inside cells more than protein-mediated control (GMF or cortactin) - can the authors evidence this at all?

We are not claiming that the level of Pi plays a stronger regulatory role than proteins. We show that inorganic phosphate tunes the state of the Arp2/3 complex, which in turn modulates the action of regulatory proteins, such as GMF and cortactin.

Nonetheless, we do show that the contribution of inorganic phosphate is quite central as it can (1) strongly stabilize branch junctions (~30-fold decrease in the dissociation rate), and (2) tune the activity of GMF and cortactin on Arp2/3 complexes at branch junctions as well as on the 'surviving' Arp2/3 complexes that remain bound to mother filaments.

We thus titrated phosphate and found that its impact on Arp2/3 complex stability is significant in the range of Pi concentration that is explored in cells. For the sake of completeness, and following a comment from reviewer #1, we now also mention the affinity of Pi for actin subunits in filaments in the Discussion, and discuss the impact of intracellular Pi on actin itself.

Minor comments

• In the introduction, while the structural and mutagenesis evidence is clearly stated, in other cases a bit more detail would be helpful e.g. 'biochemical studies', which referred measurement of hydrolysis rates using radiolabelling

We have made changes to more precisely define which biochemical assays were used in previous studies.

• Page 3 Figures shouldn't be referenced in the introduction

We have removed the references to the figures from the introduction.

• Page 3 slip bond behaviour needs explanation

We now explain the concept when first using this concept in the manuscript, as follows: "The debranching rate seems to increase exponentially with the applied pulling force, in the range of 0 to 6 pN (Fig. 1F; see more refined analysis below). This behaviour of accelerated debranching with the increase of the applied force is similar to the 'slip bond' concept, as predicted by the Bell-Evans model of the force-dependent lifetime of the interaction between two proteins".

• Figure 1B seems to be a theoretical schematic which is superfluous

We suppose that the reviewer is actually referring to figure 3B of the initial manuscript, describing the energy potential of a molecular interaction as a function of the reaction coordinate. We agree with the reviewer that it is not absolutely required and we have removed it.

• Figure 4D is helpful, different weight lines might help even more to explain the dominant pathways

We have made modifications to the biochemical reaction scheme in this figure (now figure 5F in the revised version). We hope we succeeded in improving its readability. Since the different paths depend on mechano-chemical parameters, there is no real dominant pathway per se.

**Referee cross-commenting**

Rev1 sounds like the specialist here. I can't comment on their requests. Some similar points arise between the reviewers which need addressing.

Reviewer #2 (Significance (Required)):

Significance

Taking a look at references 16 and 19, I do not find it clear what is achieved differently in the current work compared to these papers and what agrees and what disagrees. If it's a species difference I might expect the two species would be analysed side-by-side in this paper.

We thank the reviewer for this important comment. The goal of our study was not to compare the behaviour of mammalian and yeast Arp2/3 complexes.

We now try to better explain that the motivation of the present work is to address how the nucleotide state of the Arp2/3 complex tunes actin branch mechanosensitive stability, and regulates interactions with well known Arp2/3 complex binding proteins. Most of the reactions are quantified here for the first time. Moreover, the experiments with branch junctions in different nucleotide states are done under controlled mechanical conditions, providing the first direct measurements of the force-dependence of the debranching reactions. Our detailed kinetic analysis of the full reaction scheme allows us to model the different binding interfaces of the Arp2/3 complex.

In addition, it is worth noting that:

1. Species matter and this is why ref 16 and 19 can give the impression to disagree on the ability to renucleate branches thanks to the stability of surviving Arp2/3 complexes on mother filaments.
2. In ref 16 (Pandit et al, PNAS 2020) species are mixed (yeast Arp2/3 and mammalian alpha actin from skeletal muscle), likely leading to a different behaviour compared to the only mammalian protein situation we examine in our current work. In particular, with mixed species one misses the ability to renucleate, as shown in our previous study Ghasemi et al (ref 19). However, since mixing species does not correspond to anything physiological, we do not think it is worth repeating these conditions alongside our experiments.
3. Further, the analysis carried out in ref 16 suffers from important limitations: the force was unknown (not calibrated) and the data was fitted by a model that compounded several reactions, providing only an indirect estimation of the rates, in particular at zero force. In contrast, we have worked with calibrated forces (including dedicated experiments at zero force) and we have carried out specific experiments to directly measure several rates.
4. In ref 19 (our earlier work) we did not investigate the impact of the nucleotide state of the branch junction at all, and we did not systematically measure the dissociation rates as a function of force. Contrary to Pandit et al, we directly measure the difference in branch stability at zero force between ADP and ADP-Pi states and show that the ~ 30 fold difference holds true at all probed forces. Last, the force dependence of the branch renucleation success rate gives us crucial information on which of the two Arp2/3 complex interfaces ruptures first.

I'm not understanding how the authors can distinguish effects of adding phosphate and BeFx on Arp 2 and 3 compared to effects on actin. Importantly, are possible accompanying changes in the actin filament a confounding factor?

We have checked that the nucleotide state (ADP-BeFx and ADP-Pi versus ADP) of the mother and daughter filaments have no impact on branch stability:

• In the experiments shown in figure 2F, where the buffer condition to which branches are exposed is quickly changed from phosphate buffer to buffer without phosphate, we observe a rapid change of branch stability. Actin subunits at the branch junction are in F-actin conformation according to recent cyroEM observations (ref. Chavani et al, Nat Comm. 2024; Liu et al, NSMB 2024). These actin subunits, initially in the ADP-Pi state, are expected to age and become ADP with a rate of ~ 0.007 s-1 (ie half-time of 100 s; ref. Jegou et al, PLoS Biology 2011, Ooosterhert et al, NSMB 2023), a much lower rate than the observed change of the debranching rate (0.21 s-1). This means that the debranching rate is independent of the nucleotide state of daughter and mother filaments.

• In new supp. Figure S4, we show that the debranching rate is similar for ADP-Arp2/3 complex branch junctions initiated from ADP- or ADP-BeFx-actin mother filaments.

• In new supp. Figure S9, we initially exposed branch junctions to a BeFx solution then monitored debranching and branch renucleation in our standard buffer (ie without BeFX or Pi). We observed multiple rounds of branch renucleation, the first with ADP-BeFx-actin daughter filaments, and the following with daughter filaments never exposed to BeFx. They all had the same debranching rates and renucleation success rates.

The paper is quite specialist to read and the advance appears to be incremental. My expertise is in molecular pathways to actin regulation outside the main area of the paper.

The results we present in this study are often unexpected, and some go counter long-standing assumptions. The regulation of Arp2/3-nucleated branches is of importance for the stability and the force-generating capabilities of many actin networks in cells. Last, most of the measurements that we present had never been done, mainly because experiments are difficult to achieve, and require specific tools to monitor several events while controlling the applied force.

We believe our results are of broad interest as they go counter long-standing assumptions. We have rewritten the text in several instances to convey our message more clearly.

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

Please find enclosed the review of the manuscript "Inorganic phosphate in Arp2/3 complex acts as a rapid switch for the stability of actin filament branches" by Xiao et al.

The authors provide a detailed investigation of how the nucleotide bound to the Arp2/3 complex affects branch stability under flow force. From a kinetic perspective, this is an elegant study with generally high-quality data, although some conclusions rest on assumptions rather than direct experimental evidence.

We thank the reviewer for their positive feedback. We have improved our manuscript and performed important additional experiments to provide more direct experimental evidence of our conclusions.

A key question concerns the physiological relevance of these findings. For instance, the concept of branch regrowth may not be applicable in cellular contexts, since forces by actin polymerization would displace existing branches away from sites where they generate this active forces. The authors should clarify the relevance of regrowth during active force generation by branched networks.

We thank the reviewer for this comment. Our in vitro results indeed point to a previously unreported property of branched actin networks, i.e. the ability of Arp2/3 complexes to readily renucleate branches in the ADP-Pi state and that it does require reloading ATP within Arp2/3.

Branched actin networks, especially the lamellipodia or endocytotic patches, do exert active force thanks to actin polymerization of the individual branches at the forefront. Though, the whole actin network is exposed to stress, and the architecture of the network (inter-branch distance, crosslink between branches, ...) presumably strongly impact its mechanical properties.

In the case of other types of branched actin networks, such as the actin cortex, myosin motor put the whole network under tension. Such pulling forces on actin branches, depending on the amplitude of the pulling force, can lead to branch regrowth, and network self-repair.

We have modified the text to make the physiological relevance clearer.

Additionally, all experiments employ flow conditions that branches would probably not experience in cells-notably, the flow direction in the cellular context would be reversed. Altering the flow direction relative to the branches could affect not only the relationship between flow rate and branch stability, but potentially other system properties as well.

We agree with the reviewer that in cells branches will not experience flow conditions similar to the ones we use in our in vitro assay. Nonetheless, in cells we expect mechanical stress on the branch junction to be applied in all directions. In lamellipodia, the compressive force applied at the leading edge is expected to result in diverse local orientations of the force on individual branch junctions within the network (as explained in Lappalainen et al. Nat Rev MBC 2022). Also, branch junctions are found in the cell cortex, where they are exposed to pulling forces resulting from the action of myosin motors and crosslinkers on mother and daughter filaments.

This impact of the direction of the flow was addressed in our previous publication (Ghasemi et al, Sc. Adv. 2024, figure 2) and, to a lesser extent, by the lab of Enrique de la Cruz in Pandit et al, PNAS 2020 (ref. 16). We reported that flow direction has a minimal effect, if any, on branch dissociation rate and renucleation ratio.

Reviewer #3 (Significance (Required)):

Furthermore, the study appears not to account for the mother filament (particularly its nucleotide state) or the actin subunit bound to the Arp2/3 complex. The authors should discuss why their interpretation focuses exclusively on the Arp2/3 complex rather than on the actin filaments or Arp2/3-bound actin subunit.

We have checked that the nucleotide state (ADP-BeFx and ADP-Pi versus ADP) of the mother and daughter filaments has no impact on branch stability :

• In the experiments shown in figure 2F, where the buffer condition to which branches are exposed is quickly changed from phosphate buffer to buffer without phosphate, we observe a rapid change of branch stability. Actin subunits at the branch junction are in F-actin conformation according to recent cyroEM observations (ref. Chavani et al, Nat Comm. 2024; Liu et al, NSMB 2024). These actin subunits, initially in the ADP-Pi state, are expected to age and become ADP with a rate of ~ 0.007 s-1 (ie half-time of 100 s; ref. Jegou et al, PLoS Biology 2011, Ooosterhert et al, NSMB 2023), a rate much lower than the observed change of the debranching rate (0.21 s-1). This means that the debranching rate is independent of the nucleotide state of daughter and mother filaments.

• In new supp. Figure S4, we show that the debranching rate is similar for ADP-Arp2/3 complex branch junctions initiated from ADP- or ADP-BeFx-actin mother filaments.

• In new supp. Figure S9, we initially exposed branch junctions to a BeFx solution then monitored debranching and branch renucleation in a regular buffer. We observed multiple rounds of branch renucleation, the first with ADP-BeFx-actin daughter filaments, and the following with daughter filaments never exposed to BeFx. They all had the same debranching rates and renucleation success rates.

An important concern involves the use of KPi (inorganic phosphate). Based our experience, KPi appears to have effects beyond simply impacting nucleotide state-actin filaments seem to assemble differently in the presence of KPi. The authors should exercise caution in their interpretation of KPi-based experiments.

Concentration of KPi (up to 50 mM Pi) did not slow down barbed end elongation rate in our experiments.

Overall, while the technical quality and kinetic analyses are state-of-the-art, relating this work to physiological contexts remains challenging, and some conclusions appear overstated.

We have made changes in the discussion to try to more clearly relate our in vitro observations and conclusions with the cellular context where branch renucleation could have a strong impact on the architecture and mechanics of actin networks.

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Referee #3

Evidence, reproducibility and clarity

Please find enclosed the review of the manuscript "Inorganic phosphate in Arp2/3 complex acts as a rapid switch for the stability of actin filament branches" by Xiao et al.

The authors provide a detailed investigation of how the nucleotide bound to the Arp2/3 complex affects branch stability under flow force. From a kinetic perspective, this is an elegant study with generally high-quality data, although some conclusions rest on assumptions rather than direct experimental evidence.

A key question concerns the physiological relevance of these findings. For instance, the concept of branch regrowth may not be applicable in cellular contexts, since forces by actin polymerization would displace existing branches away from sites where they generate this active forces. The authors should clarify the relevance of regrowth during active force generation by branched networks.

Additionally, all experiments employ flow conditions that branches would probably not experience in cells-notably, the flow direction in the cellular context would be reversed. Altering the flow direction relative to the branches could affect not only the relationship between flow rate and branch stability, but potentially other system properties as well.

Significance

Furthermore, the study appears not to account for the mother filament (particularly its nucleotide state) or the actin subunit bound to the Arp2/3 complex. The authors should discuss why their interpretation focuses exclusively on the Arp2/3 complex rather than on the actin filaments or Arp2/3-bound actin subunit.

An important concern involves the use of KPi (inorganic phosphate). Based our experience, KPi appears to have effects beyond simply impacting nucleotide state-actin filaments seem to assemble differently in the presence of KPi. The authors should exercise caution in their interpretation of KPi-based experiments.

Overall, while the technical quality and kinetic analyses are state-of-the-art, relating this work to physiological contexts remains challenging, and some conclusions appear overstated.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Xiao et al examine the molecular events occurring when Arp2/3 complex-mediated actin filament branches are removed from mother actin filaments. They do this using microfluidics assay with purified proteins combined with single filament TIRF imaging of branched actin filaments with distinct fluorescent labels. The contribution of different nucleotide states of Arp2/3 complex are tested in conjunction with the relationship force exerted on the branches and regulatory protein involvement from GMF and cortactin. The data seem comprehensive and highly quantified in response to concentration, force, fraction of branches and survival times and branching rates. They find that ADP-BeFx and high phosphate concentrations (leading to the ADP-Pi state) leads to a slower debranching rate at a given level of force applied. The ability to rapidly switch the buffer gives powerful information about response times of debranching compared with other actin remodelling events. They use renucleation experiments to determine that the previous debranching event most often occurs at the Arp2/3 complex/daughter interface, showing that filaments will be ready to re-branch in the stable ADP-Pi bound state. GMF addition allows debranching of the ADP state to occur at a lower force. Cortactin acts similarly to the ADP-Pi state to increase branch stability.

Specific comments

The pulling force on the branches seems to arise from different flow rates in the microfluidics. Viscous drag is mentioned and I can see there is methylcellulose in the buffer. It would be helpful to have the explanation of the conversion between flow and force, even if it has been standard in previous work.

Pg 5 - what was the motivation to titrate phosphate? It seems a stretch that intracellular Pi levels are tuning branching inside cells more than protein-mediated control (GMF or cortactin) - can the authors evidence this at all?

Minor comments

• In the introduction, while the structural and mutagenesis evidence is clearly stated, in other cases a bit more detail would be helpful e.g. 'biochemical studies', which referred measurement of hydrolysis rates using radiolabelling
• Page 3 Figures shouldn't be referenced in the introduction
• Page 3 slip bond behaviour needs explanation
• Figure 1B seems to be a theoretical schematic which is superfluous
• Figure 4D is helpful, different weight lines might help even more to explain the dominant pathways

Referee cross-commenting

Rev1 sounds like the specialist here. I can't comment on their requests. Some similar points arise between the reviewers which need addressing.

Significance

Taking a look at references 16 and 19, I do not find it clear what is achieved differently in the current work compared to these papers and what agrees and what disagrees. If it's a species difference I might expect the two species would be analysed side-by-side in this paper.

I'm not understanding how the authors can distinguish effects of adding phosphate and BeFx on Arp 2 and 3 compared to effects on actin. Importantly, are possible accompanying changes in the actin filament a confounding factor?

The paper is quite specialist to read and the advance appears to be incremental. My expertise is in molecular pathways to actin regulation outside the main area of the paper.

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

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Referee #1

Evidence, reproducibility and clarity

Summary

This study investigates the mechanochemistry of Arp2/3-mediated branched actin networks at the level of individual branch junctions under load. Using microfluidic single-filament/branch force assays (including constant-force flow and open-chamber imaging) the authors quantify debranching, re‑nucleation, and mother- vs daughter‑interface stability across nucleotide states of Arp2/3 (ADP-Pi, ADP, and an ADP-BeFx proxy for ADP-Pi). They further test effects by two branch regulators (GMF and cortactin). Key findings include: (i) ADP-Pi and ADP complexes share similar force dependence but differ markedly (~20×) in intrinsic dissociation rate; (ii) phosphate turnover on the Arp2/3 complex is rapid ii) affinity for Pi drops when Arp2/3 loses its daughter filament; (iii) quantification from model fits uncovers large stability differences between daughter and mother interfaces of the Arp2/3 complex; (iv) extraordinary high stability of ADP-Pi-like Arp2/3 on the mother filament; and (v) distinct effects of GMF and cortactin on force‑dependent stability. Overall, the work combines technically demanding measurements with mechanistic modeling to probe how nucleotide state and regulatory factors tune branch mechanics.

Major comments:

1. Low force kinetics and completeness of survival curves (Figure 1). "For all forces, the surviving curves exhibited a clear single exponential behavior...." While the data can be fitted to monoexponential decay curves, data at low forces is clearly incomplete. >90% of branches have not dissociated by the end of the experiment. For the particular data shown in 1C (F00nN, n=60 total branches) it means that the time information is coming from <6 observations, which is rather low for the single molecule field. I am slightly worried by this point, since the debranching rates under ADP-Pi conditions at zero force, are even by one magnitude slower. Yet, no raw data is shown. Given that the dissociation rate at low forces is a contentious point, the authors should show the raw data and the corresponding fits. At present, they only show an experimental scheme and images for these "open chamber" assay (Fig S2). Ideally, they would image for much longer than 900s with lower sampling time in those assays, to firmly establish that 20-fold difference also holds at 0 force.

Essential; experiment might already be performed. Otherwise straightforward to do (weeks time).

1. Stability Analysis (Figure 4). I can follow much of the arguments presented in the stability analysis of the daughter vs mother interfaces, which is in principle extremely interesting! However, there are some concerns here:

i) The authors emphasize the zero force ratio derived from fits (which is linked to the stability difference of the two interfaces in the absence of force) despite this being only weakly constrained by data. Intuitively in the model, the stability difference should grow to very large values as the re-nucleation ratio approaches 1 at low force. This combined with the noise in the data poses an issue in my opinion. Looking at the data and the error margin, I think that the authors cannot state with high confidence that there is a real difference between the relative stability of the daughter and mother interfaces between the two nucleotide states of the complex.

Essential; analysis and textual revision only

ii) For ADP-Pi, the renucleation ratio essentially remains flat over the measured force range. Hence, the data can only provide little leverage to estimate both the zero force ratio and, more importantly, the differential distance to the transition state in the slip-bond model in my opinion, which will show in the crossover force. Consequently, the quoted ">100×" stability difference at F=0 and the crossover force >20pN are driven largely by extrapolation rather than direct constraint by data. Given the high number of free parameters in the model, I would anticipate that several crossover forces and differential distances might explain the data nearly equally well. Instead of loosely reporting exact number from fits, I would have hoped for some sort of sensitivity analysis, for instance relying on profile likelihoods. Also parameter values could be reported as bounds (e.g crossover force≫measured range) rather than precise point estimates. This issue re-occurs (albeit not as drastically) for the cortactin experiments (Figure 6).

Essential; analysis and textual revision only

iii) One important expectation from the "two slip bond" model is that branch dissociation rates should not necessarily scale mono-exponentially as they mostly do over the accessible force range of the paper. However, once the "minor" pathway of dissociation from the mother starts to dominate at high forces, rates become more force sensitive. This is nicely recaptured by the model fits in Figure S6 but deserves some explanation in the text. Otherwise, people will simply remember the "ADP-Pi is 20-fold more stable than ADP at all forces" message.

Essential; textual revision only

iv) One important prerequisite for the model is that isolated Arp2/3 complexes (without a daughter filament) should dissociate with equal rates from mother filaments at all flow rates. Since the Arp2/3 complex prefers mother filament curvature, forces experienced by the mother might change its off-rate. It would be good to refer to this assumption in the text and experimentally verify it. I could not find it in the paper nor in Ghasemi et al 2024.

Essential; simple experiment (a weeks time).

v) The force dependence of the branch re-nucleation rate (Fig 3D) has been measured previously by the same group (Ghasemi et al). While the data in the older paper has not been fitted by a model, the trend of the data in the previous paper looks conspicuously different. Are there any explanations for this? I speculate that it might be related to actin and ATP not being saturated (low-force re-nucleation rate rarely exceeds 80%) in Ghasemi et al., but it would be good to know what the authors think about this.

Essential; textual revision only 3. Stability of the authentic ADP-Pi-Arp2/3 complex on the mother filament. The extraordinary stability of the isolated ADP-BeFx-Arp2/3 complex on mother filaments is surprising, especially considering that both ATP and ADP states are much more labile (Ghasemi et al 2024). I would recommend repeating this experiment in the authentic ADP-Pi state with labelled Arp2/3 complexes as a more direct readout, even if this would require working with very high phosphate concentrations.

Essential; simple experiment (a weeks time).

OPTIONAL: Further, but beyond the scope of the present paper, would be titrating phosphate in these experiments, which would even allow the authors to independently verify the reduced Pi affinity for Arp2/3 in the mother filament. Of note, this affinity difference is needed to satisfy detailed balance in the reaction scheme (Fig 4 D)! 4. Importance of "surviving" ADP-Pi-Arp2/3 complexes. The authors show a) rapid turnover of Pi on the ADP-Arp2/3 complex in both branch- or mother filament-bound state and b) the lowered Pi affinity of the latter. Nonetheless, they emphasize the importance of long-lived "surviving" ADP-Pi bound complexes on the mother (even stated in the abstract). I understand that this fraction shows under some experimental conditions (BeFx), but unless I am missing something, most complexes should rapidly lose their phosphate and either exchange nucleotide or dissociate from the mother under physiological conditions. Please clarify or tone done.

Essential; textual revision only 5. GMF mechanism. The authors claim that GMF "...accelerates the departure of the surviving Arp2/3 complex from the mother...". I assume that they infer this from decrease in the re-nucleation ratio. However, alternatively GMF could simply dwell on the complex, inhibiting re-nucleation without promoting dissociation from the mother. The authors should either monitor Arp2/3 dwell times directly to discriminate between these possibilities or be more cautious in their conclusions.

Essential; simple experiment (a weeks time) or textual revision. 6. Cortactin mechanism and the "leash model". I must say that the cortactin data are the most puzzling part of the paper and had to reconcile with what we know from structure. I was hoping to find some of this resolved in the discussion. However, I do not understand the "leash model" in the discussion section for cortactin-mediated branch stabilization: "This would explain the observed increase in branch survival compared to the absence of cortactin. As the pulling force is increased, this rebinding mechanism becomes less efficient." According to my understanding of the data, this is opposite to what happens. Cortactin only stabilizes the labile interface at elevated forces! Some re-writing might help here.

Essential; textual revision.

Minor comments

Organization:

• I do not want to impose on how to best tell the story, but I felt that Fig1 A-D and Fig 2 A-B belong to one logical unit (nucleotide dependence), whereas Fig 1 E-F and Fig 2 C belong to the other (Pi binding and exchange). Perhaps consider re-organizing to streamline presentation?

Semantics/Typos:

• Abstract: „... ADP-Pi and ADP-Arp2/3 detach with the same exponential increase as a function of force...". Increase should refer to the dissociation rate, which should be added to the sentence.
• Results page 8: "...and the majority of Arp2/3 complexes detach from the mother filament while remaining bound to the branch at the debranching time." "Branch" should likely be daughter here, as there is no branch after dissociation of either interface.
• Results page 13: "Exposing ADP-BeFx-Arp2/3 complex branch junctions to a saturating amount of GMF...". It is strange to imply saturation, because GMF likely simply does not bind to the complex in this nucleotide state with appreciable affinity. Suggest to change to "high".
• Discussion page 18: "Moreover, in mammalian Arp2/3, His80 in Arp3 (corresponding to His73 in mammalian actin) is not methylated, and corresponds to residue N77 in Arp3, which is also not modified." N77 likely belongs to Arp2?
• Discussion page 19: "We showed that Pi affinity for Arp2/3 complexes at branch junctions is around 3.7 mM (Fig. 1), a value which lies within the reported 1-10 mM Pi concentration measured in the cytosol in different mammalian cell types". Notably, this is not too different from F-actin, which should be mentioned. By this measure alone, free inorganic phosphate could also directly regulate actin filament stability!

Future interest (non essential):

• It would be utterly exciting (but beyond current scope) to quantify how instantaneous debranching rates evolve for naturally aging branches starting from ATP-Arp2/3 complexes!

Significance

General assessment:

This is a compelling and carefully executed study that delivers a clear mechanistic framework for how Arp2/3 branch junctions fail and re‑form under load. The central strength is the tight integration of state‑of‑the‑art reconstitutions with careful and original kinetic analysis. The experimental design is elegant and experiments have been carried out to a masterful standard. The figures are clear, the statistics are appropriate with some exceptions as detailed above. There are very few labs in the world that could have achieved this feat!

A few aspects could be further strengthened, most notably the explanation and application of the "two slip bond" model as well as slightly more restraint in speculating around specific regulatory mechanisms. However, these are minor refinements that do not detract from the important contributions of the paper.

Overall, the clearly work merits publication with high priority after revision; most requested changes are textual/analytical with very few targeted experiments, which would substantially strengthen core claims.

Advance relative to prior literature:

The major novel findings of the paper are already summarized above. There is some recent work done on the subject of branch mechanics by the authors (Ghasemi et al 2024, PMID: 38277459) and others (Pandit et al 2020 PMID: 32461373), but the focus of the present work is clearly unique and the there is plenty of novel insight.

Audience and impact:

Primary audience: specialists in cytoskeleton dynamics, in vitro reconstitution single molecule biophysics, and mechanobiochemistry. Secondary: researchers in cell motility, morphogenesis and mechanobiology, physicists working on active matter and modelers studying force producing and load-bearing biopolymer networks. The results and analysis framework should inform quantitative models of branched network turnover under load and the interpretation of regulatory factor action in vivo and in cells.

Reviewer expertise:

Actin dynamics; biochemical reconstitution; single molecule approaches; biophysics.

She starts thinking about choosing love over reputations

People in eatonville gossip about Janie and tea cake

She feels excited but also unsure because of their age difference

Janie meets tea cake, who treats her with kindness and respect

Hat hier nichts zu suchen

56

This passage uses sourdough starter as a creative example to explain how microorganisms can continue growing and spreading over time. I think the comparison is effective because it makes the concept of evolution easier to understand through something familiar and practical. The idea that people can keep feeding, dividing, and sharing the starter also connects well to how culture and information spread between humans. Overall, the paragraph is simple but engaging, and it helps readers see how biological and cultural evolution can work in similar ways.

eLife Assessment

This important study investigates the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma using multi-omics approaches. The detailed genetic analysis of two cancer genes (BRCA1 and BRCA2) demonstrated their new roles in causing the tumor microenvironment in lung cancer. The solid findings of this study provide an essential foundation for further developing drugs targeting BRCA1/2 in lung cancer therapy.

Reviewer #1 (Public review):

Summary:

Liao et al. performed a large-scale integrative analysis to explore the function of two cancer genes (BRCA1 and BRCA2) in lung cancer, which is one of the cancers with an extremely high mortality rate. The detailed genetic analysis demonstrated new roles of BRCA1/2 in causing the tumor microenvironment in lung cancer. In particular, the discovery of different mechanisms of BRCA1 and BRCA2 provides an essential foundation for developing drugs that target BRCA1 or BRCA2 in lung cancer therapy.

Strengths:

(1) This study leveraged large-scale genomic and transcriptomic datasets to investigate the prognostic implications of BRCA1/2 mutations in LUAD patients (~2,000 samples). The datasets range from genomics to single-cell RNA-seq to scTCR-seq.

(2) In particular, the scTCR-seq offers a powerful approach for understanding T cell diversity, clonal expansion, and antigen-specific immune responses. Leveraging these data, this study found that BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations were linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity.

(3) This study also performed a comprehensive analysis of genomic variation, gene expression, and clinical data from the TCGA program, which provides an independent validation of the findings from LUAD patients newly collected in this study.

(4) This study provides an exemplary integration analysis using both computational biology and wet bench experiments. The experimental testing in the A549 cell line further supports the robustness of the computational analysis.

(5) The findings of this study offer a comprehensive view of the molecular mechanisms underlying BRCA1 and BRCA2 mutations in LUAD. BRCA1 and BRCA2 are two well-known cancer-related genes in multiple cancers. However, their role in shaping the tumor microenvironment, particularly in lung cancer, is largely unknown.

(6) By focusing on PD-L1-negative LUAD patients, this study demonstrated the molecular mechanisms underlying resistance to immune therapy. These new insights highlight new opportunities for personalized therapeutic strategies to BRCA-driven tumors. For example, they found histone deacetylase (HDAC) inhibitors consistently downregulated 4-R genes in A549 cells.

(7) The deposition of raw single-cell sequencing (including scRNA-seq and scTCR-seq) data will provide an essential data resource for further discovery in this field.

Comments on revisions:

The author has revised accordingly. I have no further comments.

Reviewer #2 (Public review):

Summary:

This study investigates the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma using multi-omics approaches. The work highlights distinct roles of BRCA1 and BRCA2 mutations in shaping immune-related processes, and is logically structured with clearly presented analyses. However, the conclusions rely primarily on descriptive computational analyses and would benefit from additional immunological validation.

Strengths:

By integrating public datasets with in-house data, this study examines the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma from multiple perspectives using multi-omics approaches. The analyses are diverse in scope, with a clear overall logic and a well-organized structure.

Weaknesses:

The study is largely descriptive and would benefit from additional immunological experiments or validation using in vivo models. The fact that the BRCA1 and BRCA2 samples were each derived from a single patient also limits the robustness of the conclusions.

Comments on revisions:

The authors have addressed my concerns satisfactorily

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment

This important study investigates the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma using multi-omics approaches. The detailed genetic analysis of two cancer genes (BRCA1 and BRCA2) demonstrated new roles for these genes in causing the tumor microenvironment in lung cancer. Further experimental explorations of the immune-related changes may still be required. The solid findings of this study provide a foundation for further developing drugs targeting BRCA1/2 in lung cancer therapy.

We would like to express our sincere gratitude for your thoughtful and constructive comments on our manuscript. We carefully considered each comment from these two reviewers and revised the manuscript accordingly. Below, we provided a point-by-point response to each comment.

Reviewer #1 (Public review):

Summary:

Liao et al. performed a large-scale integrative analysis to explore the function of two cancer genes (BRCA1 and BRCA2) in lung cancer, which is one of the cancers with an extremely high mortality rate. The detailed genetic analysis demonstrated new roles of BRCA1/2 in causing the tumor microenvironment in lung cancer. In particular, the discovery of different mechanisms of BRCA1 and BRCA2 provides an essential foundation for developing drugs that target BRCA1 or BRCA2 in lung cancer therapy.

Strengths:

(1) This study leveraged large-scale genomic and transcriptomic datasets to investigate the prognostic implications of BRCA1/2 mutations in LUAD patients (~2,000 samples). The datasets range from genomics to single-cell RNA-seq to scTCR-seq.

(2) In particular, the scTCR-seq offers a powerful approach for understanding T cell diversity, clonal expansion, and antigen-specific immune responses. Leveraging these data, this study found that BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations were linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity.

(3) This study also performed a comprehensive analysis of genomic variation, gene expression, and clinical data from the TCGA program, which provides an independent validation of the findings from LUAD patients newly collected in this study.

(4) This study provides an exemplary integration analysis using both computational biology and wet bench experiments. The experimental testing in the A549 cell line further supports the robustness of the computational analysis.

(5) The findings of this study offer a comprehensive view of the molecular mechanisms underlying BRCA1 and BRCA2 mutations in LUAD. BRCA1 and BRCA2 are two well-known cancer-related genes in multiple cancers. However, their role in shaping the tumor microenvironment, particularly in lung cancer, is largely unknown.

(6) By focusing on PD-L1-negative LUAD patients, this study demonstrated the molecular mechanisms underlying resistance to immune therapy. These new insights highlight new opportunities for personalized therapeutic strategies to BRCA-driven tumors. For example, they found histone deacetylase (HDAC) inhibitors consistently downregulated 4-R genes in A549 cells.

(7) The deposition of raw single-cell sequencing (including scRNA-seq and scTCR-seq) data will provide an essential data resource for further discovery in this field.

Weaknesses:

(1) The finding of histone deacetylase (HDAC) inhibitors suggests the potential roles of epigenetic regulation in lung cancer. It would be interesting to explore epigenetic changes in LUAD patients in the future.

Thank you for your insightful comment. We fully agree that the specific situation of epigenetic dysregulation in LUAD needs to be explored. We believe that future investigations utilizing clinical specimens and animal models to map histone acetylation patterns and DNA methylation profiles were crucial for identifying novel biomarkers and therapeutic targets unique to LUAD.

(2) For some methods, more detailed information is needed.

This is a valid point. We agree that additional details regarding are necessary for clarity and reproducibility. We have expanded these method details in the revised manuscript.

(3) There are grammar issues in the text that need to be fixed.

We apologize for our irregular use of grammar. In the revised manuscript, we carefully checked the grammar and make corrections.

(4) Some text in the figures is not labeled well.

We appreciate the reviewers' comments. We have added labels to the revised version of the figures.

Reviewer #2 (Public review):

Summary:

This study investigates the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma using multi-omics approaches. The work highlights distinct roles of BRCA1 and BRCA2 mutations in shaping immune-related processes, and is logically structured with clearly presented analyses. However, the conclusions rely primarily on descriptive computational analyses and would benefit from additional immunological validation.

Strengths:

By integrating public datasets with in-house data, this study examines the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma from multiple perspectives using multi-omics approaches. The analyses are diverse in scope, with a clear overall logic and a well-organized structure.

Weaknesses:

The study is largely descriptive and would benefit from additional immunological experiments or validation using in vivo models. The fact that the BRCA1 and BRCA2 samples were each derived from a single patient also limits the robustness of the conclusions.

Thank you for this excellent suggestion. In the revised manuscript, we supplemented the additional immunological experiments and validation based on pathological tissue sections of lung adenocarcinoma patients. In addition, we elaborated on the limitations of our study in the Discussion section and provided reasonable explanations.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The abstract includes a lot of abbreviations, which makes it difficult to follow. For example, "IFN" is not defined. And "HRR" is defined but used only once in the abstract. This issue also appears in other parts, such as "OAK" on page 5, line 114; "DFS" on page 15, line 398; and "DSBs" on page 20, line 558. Please try to avoid unnecessary abbreviations.

Thank you for highlighting this. We have revised the manuscript to minimize the use of abbreviations. Specifically, we have now defined all necessary abbreviations upon first mention (including 'IFN') and have removed or spelled out those used infrequently to ensure the text flows more smoothly for the reader.

(2) Page 5, line 129, what data type is used in this part analysis?

We apologize for our negligence. The whole exome sequencing data used here has been added in the revised manuscript.

Materials and methods, page 6, lines 131-132: “The raw reads (fastq) of whole exome sequencing were pre-processed and trimmed with fastp (Version: 0.23.4) based on default parameters.”

(3) Page 6, line 138, Add citation for ANNOVAR.

Thank you for your suggestion. We have added a citation for ANNOVAR in the revised manuscript.

(4) Page 8, line 211, what cutoff is used to define the significant makers?

Thank you for your insightful comment. We provided the cutoff used to define significant markers.

Materials and methods, page 8, lines 213-215: “Differential expression genes for specific clusters were identified using the “FindMarkers” function, with a threshold of |avg_log2FC| ≥ 0.5 and adjusted P-value ≤ 0.01.”

(5) Page 11, line 276, HEK293T is not a lung cancer cell line. It would be better to label the details of this cell line.

Thank you for your correction. We have now clarified HEK293T in the text by stating: 'human embryonic kidney cell line HEK293T'.

Materials and methods, page 11, lines 277-278: “The human lung cancer cell line A549 (#SCSP-503) and the human embryonic kidney cell line HEK293T (#SCSP-502) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, China.”

(6) Page 16, line 415, what samples and how many individuals were used for the exome sequencing?

We agree that specifying the sample set is crucial. The exome sequencing was conducted on 2 individuals (four samples). The samples used were tumor tissues (2 samples) and matched blood (2 samples). This information has been clarified in the revised manuscript.

Results section, page 16, lines 415-416: “Exome sequencing was performed on four samples from two individuals: two tumor tissues and two matched blood samples.”

(7) Page 17, line 468, Replace "Differently" with "In contrast" (more appropriate for scientific writing).

Thank you for pointing this out. We agree that "In contrast" is more appropriate for scientific writing. Accordingly, we have replaced "Differently" with "In contrast" in this sentence (Results section, page 18, line 483).

(8) Page 18, line 489, what is HMG?

Thank you for pointing this out. HMG stands for High Mobility Group. We have clarified this by writing out the full term upon first mention in the manuscript (Results section, page 19, line 503).

(9) Page 19, line 527, check the grammar for this sentence.

We appreciate your careful reading. We have carefully rephrased this sentence to ensure clarity and grammatical accuracy.

Results section, page 20, line 540: “Based on pseudotime order, we divided trajectories into 10 bins and analyze the activity changes of related features.”

(10) Page 20, line 541-546. It would be better to split this long sentence into smaller ones.

Thank you for your insightful comment. We have revised the text, splitting the long sentence into smaller ones for better clarity.

Results section, page 20, lines 554-559: “MHC class I and II molecules showed increased activity in late pseudotime in BRCA1- and BRCA2-mutant cells, respectively (Fig. 4G-I). This pattern was also reflected in the cell density analysis (Fig. 4J). Furthermore, CD8⁺ Tcm and Th1 signatures exhibited higher activity in late pseudotime in BRCA1- and BRCA2-mutant cells, respectively (Fig. S5F-G). These findings suggest a differential association with CD8⁺ versus CD4⁺ T cell engagement.”

(11) Page 20, line 550, remove "." after "of".

Thank you for catching this. We have removed it (Results section, Page 21, line 563).

(12) Page 22, line 592, what is "LME"?

Thank you for pointing this out. "LME" was indeed redundant in the original manuscript, so we have removed it in the revised version (Results section, Page 22, lines 607-609).

(13) Page 24, line 674, Replace "suggest" with "suggested"?

We apologize for our negligence. In the revised manuscript, we have replaced "suggest" with "suggested" (Results section, Page 25, lines 691-693).

(14) Page 35, Figure 1I, Use "B cells" instead of "B".

Thank you for your detailed review. We have changed to the appropriate label in Figure 1I.

(15) Page 36, Figure 2H, the statistics and p-value are needed to show.

Thank you for your suggestion. We have added the statistical analysis for Figure 2H, and the p-values were indicated in the revised Figure.

Special thanks to you for your kind comments.

Reviewer #2 (Recommendations for the authors):

Major:

(1) Line 44. In the Introduction section, a brief description of the prevalence of HRD or BRCA1/2 mutations in lung cancer patients should be included to highlight the significance of the study.

This is an excellent suggestion. We revised the Introduction section (page 3, lines 61-64) to include a brief overview of the prevalence of BRCA1/2 mutations specifically in lung cancer patients. We believe this addition will strengthen the background for readers.

Introduction section, page 3, lines 61-64: “Among the key genetic mutations that drive LUAD, BRCA1 and BRCA2 mutations (with prevalence rates of approximately 4% and 5%, respectively) have been increasingly implicated in the pathogenesis and progression of lung cancer [9, 13].”

(2) Line 302-355. There are relatively serious grammatical issues, and substantial revision of the text is recommended.

We acknowledge the grammatical issues in the original text. We have now carefully revised the Materials and methods section of the manuscript (pages 11-14, lines 277-358) to correct these issues and improve the overall readability. We believe the revised version is significantly improved.

(3) Line 375. The Results section lacks detailed information on the specific BRCA1/BRCA2 mutations and data explaining how these mutations lead to functional alterations of BRCA1/2.

Thank you for your insightful comment. In the revised manuscript, we added the amino acid changes caused by the specific BRCA1/BRCA2 mutation sites and expand the text to discuss the predicted and known pathogenic mechanisms of these variants (Results section, page 16, lines 420-433).

Results section, page 16, lines 420-433: “Exome sequencing data show that these two types of tumor tissues harbor somatic nonsynonymous single nucleotide variants (SNV) in BRCA2 (p.N372H) and BRCA1 (p.E991G, p.S1566G, p.K1136R, p.P824L, and p.Y809H), respectively (Table S1). The BRCA2 p.N372H variant lies within the BRC3 or BRC4 motifs critical for RAD51 binding. It may alter binding affinity, impair high-fidelity homologous recombination repair, and promote genomic instability [39-41]. In BRCA1, mutations are distributed across two key functional domains: the Coiled-Coil domain (e.g., p.E991G, p.Y809H, p.P824L) and the BRCT domain (e.g., p.K1136R, p.S1566G). Coiled-Coil mutations disrupt BRCA1-PALB2-BRCA2 complex assembly, impairing localization to DNA damage sites and subsequent RAD51 recruitment; BRCT domain mutations compromise phospho-protein recognition and G2/M checkpoint control, leading to defective DNA damage response and unchecked proliferation of damaged cells [42-44]. Together, these defects promote the accumulation of genomic scars and chromosomal instability.”

(4) Line 492-498. Changes in genes associated with BRCA1 and BRCA2 mutations should be validated by immunofluorescence.

Thank you for your insightful comment. Immunofluorescence would provide valuable orthogonal validation of the protein-level consequences of these mutations. To address this, we obtained pathological tissue sections from patients carrying BRCA1/2 mutations and performed immunofluorescence staining for S100A10, a risk gene associated with BRCA1 mutations. We found that S100A10 was upregulated in BRCA1-mutated tumor tissue compared to adjacent non-cancerous tissue.

Results section, page 24, lines 673-675: “Immunofluorescence experiments on patient tissue sections revealed that S100A10 was upregulated in BRCA1-mutated tumor tissue relative to adjacent non-cancerous tissue (Fig. S11D-E).”

(5) Line 538. Although both BRCA1 and BRCA2 deficiencies impair DNA damage repair, BRCA1, but not BRCA2, activates the cGAS-STING pathway. This is a particularly interesting observation and should be validated by immunofluorescence experiments.

Thank you for highlighting this observation. To address this, we conducted immunofluorescence experiments to quantify STING, the key protein of cGAS-STING pathway, in BRCA1- and BRCA2-deficient tissues to confirm this phenotype. We have included these results in the revised manuscript.

Results section, page 21, lines 578-584: “Furthermore, our results revealed that BRCA1-mutant tumors showed higher activity of cGAS-STING signaling and STING mediated induction of host immune responses compared to BRCA2-mutant tumors (Fig. 5G and Fig. S6F). Also, cGAS-STING signaling gens, including cGAS, STING1, and downstream factors STAT1 and CCL5, were upregulated in BRCA1-mutant tumor cells (Fig. 5H). This observation was validated through immunofluorescence staining experiments on patient tumor tissue sections (Fig. 5I-J).”

(6) Line 599. "CD8+ Trm cells were more abundant in BRCA1-mutant sample, whereas CD4+ Trm cells were higher in BRCA2-mutant sample". This part is also recommended to be validated using immunofluorescence or more rigorous flow cytometry analyses.

We sincerely appreciate this insightful suggestion. To address this, we performed immunofluorescence staining to quantify the abundance of CD8⁺ and CD4⁺ Trm cells in BRCA1- and BRCA2-mutant tissues. We have included these results in the revised manuscript.

Results section, page 22, lines 614-617: We identified two tissue-resident memory T cell (Trm) subsets, CD8⁺ Trm and CD4⁺ Trm, both predominantly derived from tumor tissues (Fig. 6B). “Interestingly, our analysis revealed that CD8⁺ Trm cells were more abundant in BRCA1-mutant tumor, whereas CD4⁺ Trm cells were more abundant in BRCA2-mutant tumor (Fig. 6B-D, Fig. S7D, and Fig. S8A-B).”

(7) Line 643-676. The authors identified four risk genes associated with BRCA1 mutations-S100A10, LDHA, MYL12A, and GAPDH; however, MYL12A was not validated in the subsequent in vitro experiments. The authors state that "S100A10 can promote cancer metastasis by recruiting MDSC cells, and increased LDHA activity contributes to tumor immune escape." However, because immune cells were not included in the in vitro assays, these results instead suggest that these genes may directly suppress tumor cell proliferation.

We thank the reviewer for this insightful observation. Our intention was not to suggest that the reduction in proliferation observed in our in vitro assays was caused by the disruption of immune cell recruitment or immune escape pathways. As the reviewer correctly points out, those mechanisms are irrelevant in a system lacking immune cells. Our results showing that "Knockdown of S100A10, LDHA, and GAPDH reduced LUAD cell proliferation in vitro (Fig. 7D-E)" strongly suggest a direct, cell-autonomous role for these genes in regulating LUAD cell growth. For the MYL12A gene, the existing study have shown that BRCA1 transcriptionally regulates this gene involved in breast tumorigenesis (PMID: 12032322). In view of the characteristics of MYL12A in lung cancer, we will conduct in-depth in vitro and in vivo validation experiments in future studies.

(8) Line 677. The authors should emphasize the limitations arising from the small sample size and the lack of in vivo validation models in the Discussion section.

Thank you for highlighting these important limitations. We agree that the small sample size and the lack of in vivo validation are significant limitations of the current study. We have explicitly addressed these points in the Discussion section (page 27, lines 740-750) to ensure the interpretation of our data is appropriately qualified and to provide transparency regarding the scope of our conclusions.

Discussion section, page 27, lines 740-750: “Although we included both tumor tissues and matched paracancerous and blood samples, the sample size remains modest, which may limit the statistical power and generalizability of our findings. Therefore, our results should be interpreted as preliminary, and further studies with larger, independent cohorts are required to validate these observations. Single-cell RNA-seq and TCR-seq analyses in this study provide high-resolution insights into the cellular and clonal dynamics of the TME, the functional validation of key mechanisms remains largely correlative. While our in vitro experiments provide valuable mechanistic insight, the lack of in vivo validation, which cannot fully recapitulate the complex TME. Future studies utilizing murine models or patient-derived organoids are essential to establish causal relationships and elucidate the underlying molecular pathways.”

Minor:

(1) Line 163: cell/μl should be corrected to cells/μL.

Thank you for catching this. We have corrected it in the revised manuscript (Methods section, page 7, line 165).

(2) Line 388: Please clarify how the HRD score, tumor mutation burden, and neoantigen load were calculated.

We thank the reviewer for this request for clarification. In the revised manuscript, we have expanded the Methods section (page 5, lines 117-121) to provide a detailed description of how these metrics were calculated. HRD score was calculated as the unweighted sum of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST). Tumor mutation burden (TMB) was defined as the total number of somatic nonsynonymous mutations per megabase of the exome captured by the sequencing panel. Neoantigen load was predicted by NetMHCpan using the patient's HLA typing and the identified somatic mutations. The data for these three indicators all obtained from a previous study (PMID: 29628290). We believe these additions provide the necessary transparency and reproducibility for our study.

Methods section, page 5, lines 117-121: The HRD score was determined by summing specific genomic alterations, including loss of heterozygosity (LOH), large-scale state transitions (LST), and telomeric allelic imbalances (TAI). “Tumor mutation burden (TMB) was defined as the total number of somatic nonsynonymous mutations per megabase of the exome captured by the sequencing panel. Neoantigen load was predicted by NetMHCpan using the patient's HLA typing and the identified somatic mutations.”

(3) Line 421: BRCA12 should be corrected to BRCA2.

Thank you for your detailed review. We have revised it.

(4) The order of Figures 7D and 7E should be reversed.

Thank you for your insightful comment. According to your suggestion, we reversed the order of Figures 7D and 7E in the revised manuscript.

Special thanks to you for your kind comments.

Loi khuyen

eLife Assessment

This study examines the role of the fungal pathogen Candida albicans in the progression of colorectal cancer, a relevant and urgent topic given the global incidence of colon cancer. While the findings are useful and provide solid experimental work and insight into how Candida may contribute to tumor progression, the small patient sample size, reliance on in vitro models, and absence of in vivo validation may limit its impact. This work will interest scientists studying cancer progression and the role played by pathogens.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed most of the comments raised in the previous round of review.]

Summary:

This study addresses the emerging role of fungal pathogens in colorectal cancer and provides mechanistic insights into how Candida albicans may influence tumor-promoting pathways. While the work is potentially impactful and the experiments are carefully executed, the strength of evidence is limited by reliance on in vitro models, small patient sample size, and the absence of in vivo validation, which reduces the translational significance of the findings.

Strengths:

(1) Comprehensive mechanistic dissection of intracellular signaling pathways.

(2) Broad use of pharmacological inhibitors and cell line models.

(3) Inclusion of patient-derived organoids, which increases relevance to human disease.

(4) Focus on an emerging and underexplored aspect of the tumor microenvironment, namely fungal pathogens.