That’s insanely fast for the 14th century. People could travel huge distances and share stories about other cultures. It helped connect East and West much better

China was the world leader in population and economy. 120 million people is huge! It’s crazy to think one country had 30% of the world’s population.

This is really interesting as he was known for being merciful. Contrasts with the brutal Crusader capture in 1099. Shows how back then, leadership can include honor, and not to reckless violence

Constantine is a great example of someone moving between cultures. He helped bring knowledge from the Islamic world to Europe.

Basically, it was a giant census for money and control. tells us a lot about medieval England. Historians still use it today to understand economy and society.

It’s wild that we don’t know how to fully make it anymore. Even with modern technology, recreating it is difficult. goes to show how skilled ancient metalworkers were.

This is huge because Cyrillic is still used today. Language helped religion spread faster. It made the Slavs feel included instead of forced to use Greek or Latin.

The boundaries help visualize how massive his empire was. how it wasn’t a small kingdom and it crossed many modern countries. Geography clearly mattered a lot for power.

It’s interesting that they didn’t settle right away and kept moving. Building a brand-new city shows confidence and ambition. They wanted a capital designed for their empire, not inherited.

It’s interesting that trade stayed important even after they were forced to leave Mecca. Trading helped them survive in Medina and meet new people. This also made it easier for Islam to spread because merchants traveled a lot.

Trade routes that were connected Mecca to Africa, Asia, and Europe. Pilgrimage brought diverse religious practices into the city. Really helps explain Muhammad’s strong opposition to idolatry.

The term “barbarian” was definitely Roman bias. Many of these groups had long interacted with Rome through trade, military service, and settlement. This is more to the fact that they were outsiders completely opposed to Roman civilization.

This sentence is reminds me and other people have been around for only a short time compare to how long the earth has existed, and our writing history is just a small part of that long time. This help us stay humble and remember that many thing we think are important may not last forever

Add a 3rd blue box with this text:

Join the next LIVE round from January 12-31, 2026!

Tackle the 9 steps during the LIVE experience with my support, and guest expert feedback for your website in a LIVE session! Plus: Ask your questions and get support in the LIVE Challenge Community.

The next LIVE round is happening from January, 12-31!

OR

Join us for the next LIVE round from January 12-31, 2026!

The next LIVE round is happening: January 12-31, 2026

It is very interesting that people in South America were growing many different types of potatoes 10,000 years ago. They even learned how to freeze dry them to keep them for a long time. This was long before the Spanish brought potatoes to Europe.

This comment the writer thinks the idea is interesting and connects it to something they heard before. This makes me believe they understand how people in the past saw the taming of animals. It’s not completely clear but I can see the link.

Add this question: How does the LIVE challenge work?

Every Monday, Wednesday and Friday I’ll prompt you with a new lesson.

Every lesson includes: - A bite-sized video to watch - A prompt to work on one specific component of your website

You can also get guest expert feedback for your website in a LIVE session!

Plus: Ask your questions and get support in the LIVE Challenge Community.

Can't make it live? Catch the replay shortly after the LIVE sesisons.

update this

Tackle the 9 steps during the LIVE experience with my support, and guest expert feedback for your website in a LIVE session! Plus: Ask your questions and get support in the LIVE Challenge Community.

The next LIVE round is happening from January, 12-31!

OR

Join us for the next LIVE round from January 12-31, 2026!

I get the following warning on startup:

REDIRECT_DOMAINS is not set, Anubis will only redirect to the same domain a request is coming from, see https://anubis.techaro.lol/docs/admin/configuration/redirect-domains"}

Some personal take away's,

• This Ann lady fits perfectly into the Leftoid stereotype of someone who chases feelings and is incapable of dealing with concrete goals backed by facts and logic.
  • I bet if we do some research we can find idiots like Ann funded with lots of money that would have been better put in other peoples hands to solve the same problems.
• There exists a caste system, the people with access to cheap debt, and normal people who deal with credit cards and mortgages. The people that sit on money, leveraged investments, that grow faster than they spend it, also known as the bourgeois. Then there is the other caste of people that have to "earn" money.
  • Giving money to the "earning" caste leads to them investing in the stock market which in turns gives money to the bourgeois caste.
• "Housing is an informal part of what economists would call the money supply."
  • Anecdote, I went to a community trust housing project and talked to the lead organizer there. I explained how low interest rates make houses unaffordable to "earners". He had no idea what I was talking about. Lesson here is that not only do the people complaining about hosing prices have no idea why they are high but the people working in the low income housing industry have no idea why they are high either.
• "Basically, if you have a functioning reputation network, you can sell to a buyer at point A in exchange for a letter entitling you to some of the buyer's account held at point B."
  • Contracts are the root of society, that is an axiom of some kind for designing new social systems
• "So, now you give the Devil the benefit of law!"
  • The idea that rule of law also applies to the Devil, and therefore must be applied to every human being equally is a very strong concept that is relevant to those who want to design new social systems.
• Without the founding of the Federal Reserve System and the american Accumulation of British and French war debt it would have been very unlikely America would have entered World War 1.
  • Central banks, their bonds, and access to cheap capital are the real reason America Entered World War 1.
  • The cheap money printer known as The Federal Reserve is why the "Military Industrial Complex" keeps running and killing the way it does

I would like to see a list of these leveraged buy outs and who did them, let's see if the AI can do it?

https://grok.com/share/bGVnYWN5_b54d1b5b-4d00-4342-a7eb-d978645ab27e

Wow, that's one way to deconstruct an argument, I assign one Philosopher King point to this Ben Guy who this comment alone.

Are you saying that Autists would make for moral Politicians?

On "Geeks, MOPs, and Sociopaths" — LessWrong

Wow that's one way to describe "Blockchain"

Wow this is a loaded statement,

Who are the bourgeoisie, they are the creditors.

What happens to creditors in a deflationary economy?

I don't think they care they are getting their money back, but the debtors get really dam mad because it becomes harder and hard to pay back the creditors.

Ah so it was caste warfare

And at the end of the way Europe was off the Gold Standard

Oh selling British and French war bonds to Americas made America dependent on the British and French to win the war otherwise those Bonds would have never gotten repaid

Wait, the first world war would not have been fundable without the Federal Reserve.

In fact since it's easy for a central bank to repay money people that loan money out are incentived to start wars for the government to spend money that has to be loaned to the government.

Ah that's the "Military Industrial Complex" Eisenhower was talking about in his speach

Do any "Game B" people have any solutions to this inventive problem that creates perpetual war?

Wait I never heard about this, turns out it is true,

Bank Panic of 1907: Causes, Effects, and Importance

Ah the Central Bankers,

https://youtu.be/mII9NZ8MMVM?si=f89eO_JNjVX6YAsX&t=828

Smells just like the European Union

The Devil Beholden to the laws of man, now that's a sight to behold.

Sounds like a transition from Feudal to "State Capitalist" transition going on here

Interesting scene, but why is this relevant to debt?

Ah the caste system. This guy does not belong in the caste of leveraged low interest debt havers

If you put people in debt you can make them do amazing things. Do you know any other examples from history or fiction of someone pushed to do horrible things to pay off their debt?

Viewing your own creditors as a threat? That's one way to look at it

I wonder if the Devil had anything to do with the creation of Capitalism?

So the aristocrats with access to cheap debt in the Victorian era are like the Banking Corporate class now with access to cheap debt, whomever has access to cheap debt are the people of privilege

And voila, Contract Law was born

The Banks are the true winners of war, here's a beautiful clip that explains it.

The Collapse of The American Dream Explained in Animation - YouTube

IMDB Link for Memex Reference -> The American Dream (Short 2010) - IMDb

It would be nice to have a more comprehensive analysis of the relationship between OPU and ASV since there may be many drivers of correlation between OPU and ASV, prevalence of species being one, but also, you might have differing environmental factors diving correlation in OPU that deviates from ASV. If you could examine the correlation between OPU sets or features and environmental factors (such as organic\non-organic, or plant type) after controlling for ASV it might more directly identify aspects of biology that are driven to be similar based on growth conditions and not different species presence.

conclusion- Dowd concludes that the unchecked expansion of A.I. in media and culture is dangerous and should be approached with caution or resistance.

Slippery Slope: This claim suggests an extreme outcome without fully demonstrating that intermediate steps are inevitable, which may weaken the argument’s strength.

premise- This premise suggests that reliance on simulations and fakes leads to psychological and social harm, reinforcing the argument that A.I. poses risks beyond economic concerns.

Dowd concludes that widespread A.I.-generated media will undermine public trust in evidence and shared reality, especially in political contexts.

Dowd appeals to expert opinion to support the claim that realistic A.I.-generated media will exacerbate social conflict and misinformation.

Dowd introduces Sora as evidence that A.I. media can convincingly fabricate realistic videos, expanding the argument beyond Hollywood labor to the problem of misinformation and deception.

This introduces the concern that A.I. performers are perceived as a real threat by human actors. It functions as an illustrative premise that frames the argument emotionally and establishes that resistance to A.I. in Hollywood already exists among industry professionals.

Gives us uncertainty about Reagan’s involvement, showing how the Iran-Contra scandal raised questions about presidential accountability and oversight.

Shows the dangers and human cost of U.S. military involvement abroad, showing that even peacekeeping missions can result in devastating casualties and political consequences.

Shows Reagan’s belief that cutting taxes for individuals and businesses would increase investment, production, job creation, and overall economic growth.

Shows that scientists largely agree climate change is real, even though many politicians and members of the public continue to doubt or argue against it.

This makes us understand the length and cost of the Afghanistan war following the 9/11 attacks.

This shows the unprecedented scale and shock of the September 11 terrorist attacks.

Excellently/clearly explained

This is concerning because it shows that AI tools prioritize sounding correct over being accurate.

The irony here is striking. A report that calls for teaching ethics and responsible AI use failing to meet those standards itself weakens the message.

Wow! I had no idea and this is very good to know.

Good comparison and interesting to know.

I do agree, we all make mistakes.

Very Important! And I agree 100%.

Very important to emphasize and to keep in mind.

A very good suggestion. The value of interdependence in learning is very important.

Giving students the extra support and giving them the little push to expand more is ideal to have them share a little more.

This would be a good idea and beneficial for the students as well.

In exactly the same words as were used originally.

The action of depriving someone of membership in an organization.

Maybe we note that the parallel fibers provide good tensile (straight-line) strength, not very good shear (perpendicular) strength, leading to tears when perpendicular forces are applied as in many knee injuries.

Maybe a note that the deepest layer of the tissue is the only one that divides, and as cells get pushed closer to the surface they flatten out and die.

Maybe add that this contributes to its role as a barrier, preventing infectious material from directly entering the bloodstream from the environment.

Các thuốc chống đông đường tiêm có thể được sử dụng để ức chế quá trình đông máu huyết tương cả dự phòng lẫn điều trị, ví dụ nhằm phòng ngừa và điều trị huyết khối.

Heparin là nhóm thuốc được sử dụng phổ biến nhất; các thuốc khác thường chỉ được dùng khi có không dung nạp. Bên cạnh tăng nguy cơ biến chứng chảy máu, tác dụng phụ đáng sợ nhất là giảm tiểu cầu do heparin (HIT) — một biến chứng hiếm gặp, trong đó tự kháng thể gây kết tập tiểu cầu, dẫn đến thuyên tắc huyết khối nguy hiểm. Do biến chứng này có thể được nhận biết qua sự giảm mạnh số lượng tiểu cầu, nên với việc sử dụng heparin rất thường xuyên trong thực hành lâm sàng, cần theo dõi công thức máu.

https://www.theriaults.com/events/listing/14714/shirley-temples-personal-childhood-typewriter-with-inscription

the public expression of moral opinions or beliefs to signal one's good character and alignment with a particular group

Do we want to mention a very brief overview of meiosis and the reasons we need it here for those students who will not move on the Advanced A&P?

I like to point out that Telophase effectively undoes Prophase, so that if we know what happens in Prophase we can predict what happens in Telophase (the opposite).

We may also mention here that this is when the spindle fibers attach to the chromosomes.

I would suggest altering to say "use of cellular energy"

I like to point out here that this still fits the definition of passive transport because the substance that is moving (in this case, water) is moving down ITS concentration gradient.

I would reemphasize here that substances move until they achieve equilibrium. This is going to set us up better for the external respiration conversation and the problems with breathing at altitude, for example.

I much prefer to define passive transport as using energy from the environment (repulsion of like charges, for example) while active transport requires the cell to provide the energy needed for movement. If we say passive transport does not require energy, students wonder why they are moving at all. If we use environmental energy that more naturally leads to a conversation about equilibrium and how particles with like charges establish a maximum distance from each other. It also sets up the conversation for moving up or down concentration gradients.

It would be useful to include a bit about the criteria used during expert evaluation

Lock IN ANSWER FOR KANT ESSAY Qsn: Does reason entail freedom? consciosuness of outer things: involves the understanding and reason

Represents the digital transformation in the baking industry and its purpose and goal.

Beneficial effects from the transition of traditional baking to technological methods.

A menos que cada uno tuviera su propio servidor y los servidores centralizados fueran usados sólo para coordinar comunicaciones, como ocurre con Fossil y ocurrirá con Cardumem. Así, la centralización ofrece conveniencia pero no usa asimetría fundamental de capacidad o poder, como ocurre actualmente y servicios/protocolos de descubrimiento de servidores podrían ser implementados sobre la infraestructura cliente servidor actual, en caso de que algún servidor sea dado de baja.

Most of the West, don't generalise.

We define what genes are made of, but never discuss their role in the body. Some reference to genes being the instructions for forming proteins is probably warranted.

We haven't introduced genes or their role in the body yet, so this may be a very confusing sentence. Maybe put the nucleic acid conversation before the protein conversation in this chapter in order to help define this concept before it is applied?

A comparison of the relative energy content of fats vs carbohydrates (kcal/g) may be useful here.

What does it actually mean when a bank “puts money into a sector”?

Banks don’t usually give money. They finance things. That happens in a few main ways:

1. Loans

Banks lend money to companies. Example: An oil company wants to drill a new field → the bank gives a loan.

If the bank says no, that project often can’t happen (or becomes much more expensive).

1. Underwriting bonds and shares

Big companies raise money by issuing:

bonds (debt)

shares (equity)

Banks act as the middlemen who:

design the deal

sell it to investors

take a fee

If a bank refuses to underwrite a coal or oil expansion, that company loses easy access to capital markets.

1. Project finance

This is very direct. Banks fund specific projects like:

coal mines

LNG terminals

pipelines

No bank finance → no project.

1. General corporate finance

Even if money isn’t tied to a single oil well, banks provide:

credit lines

working capital

refinancing

This keeps fossil fuel companies alive and growing.

So… can banks really choose NOT to fund fossil fuels?

Yes. And many already do — selectively.

Banks set internal policies, for example:

“We will not finance new coal projects”

“We will stop funding Arctic drilling”

“We will only fund companies with transition plans”

These are choices, not laws of nature.

Then why do banks say “it’s complicated”?

Because of three real-world pressures:

1. Profit

Fossil fuels still make money. Oil and gas companies are:

large

politically powerful

seen as “safe” borrowers

Banks are profit-driven institutions.

1. Energy demand today

The world still runs on fossil fuels. Banks argue: “If we stop financing now, energy prices spike and economies suffer.”

There’s some truth here — but it’s also used as a convenient excuse to delay change.

1. Competition

If Bank A stops funding fossil fuels, Bank B might step in. So banks fear: “We’ll lose business, but emissions won’t go down.”

This is why collective action matters — not individual PR pledges.

So what’s the core criticism in reports like Banking on Climate Chaos?

Not that banks should:

shut off fossil fuels overnight

But that they:

publicly promise climate action

privately fund expansion of fossil fuels

Especially:

new oil and gas fields

long-life infrastructure that locks emissions in for decades

That’s the hypocrisy the report is calling out.

A sentence here about how forming compounds changes how these substances interact with the body may be useful here. An example to potentially include could be that elemental sodium explodes when exposed to water, but when combined with chloride it becomes table salt and is critical to the functioning of the nervous system. There is a lot of misinformation out there claiming that some substances are dangerous to the body in all forms when they are demonstrably perfectly safe in compounded forms.

Support aimed at restoring the crier’s independence and competence.Examples:

“Let’s talk through what you can do next.”

Teaching a child how to manage frustration rather than doing the task for them

Offering advice, reassurance, or practical strategies

Emotional message to the crier:

“You’re capable, and I can support you.

within the individual ,Effects on the person who is crying)

As previously discussed, it could be interesting to divide these taxa fractions into bacteria, fungi, metazoa, viruses, etc. Maybe there is a trend...

I find this quite interesting and I think maybe we should further try to understand why in some cases the similarity between contig-based & read-based tax annotation in samples is greater (points far away from each other of same sample) and in some cases it is lower (points close to each other in PCoA of the same sample). I there anyway to explain this?

Maybe it could be also interesting to color points according to season and use instead of red and blue forms like circle and square of the same color to understand if differences between read-based and contig-based approaches are influenced by seasonality.

According to my knowledge: If unemployment consistently rises month over month or spikes sharply beyond 4.4%, it could mean the economy is deteriorating, prompting you to take action, such as adjusting your portfolio.

Waiting for confirmation on December 2025 reading.

Acá hay transiciones en los siguientes cuadrantes:

Cardumem toma una ruta alterna y más sencilla para explorar transiciones similares.

1. Inicia por el wiki, como software documental asíncrono.
2. Se conectará con HedgeDoc como software documetal síncrono.
3. Se conectará con Hypothesis como software dialógico asíncrono.
4. Implementará progresivamente funcionalidades síncronas vía sistemas hipermedia en tiempo real.

La idea de local primero ocurrirá debido a que el servidor puede correr de manera local o remota.

Preventing learning

Upwind means on the side the wind is coming from.

If the wind is blowing West → East

Upwind = West side (where the wind starts)

Downwind = East side (where the wind goes)

So an upwind wind farm hits the wind first, creates a wake, and a downwind wind farm can receive slower/turbulent air.

Wind theft = “upwind farm steals your wind” (not literally). It’s the nickname for when one wind farm sits upwind and reduces the wind energy available to another farm downwind, cutting its power output.

Wake effect = the physics behind it. A turbine pulls energy out of the air, so behind it there’s a wake: wind is slower and more turbulent. With big offshore arrays, those wakes can merge and stretch far enough to reach other projects.

Why you should care: power (and money) drops fast. Wind power is very sensitive to wind speed, so even “small” wake slowdowns can mean meaningful generation losses, which becomes a financing + revenue + ROI problem.

Why it’s getting worse now. Offshore wind is scaling up and clusters are getting denser, so the chance that one project’s wake overlaps another project is rising—especially in busy seas.

Countries/examples mentioned. The article points to UK disputes, and a cross-border example where a planned farm in Norway could impact a farm in Denmark; it also flags potential future disputes involving UK vs Netherlands/Belgium/France.

What the “fix” looks like (not one magic lever). Better planning/spacing, better wake modelling in approvals, and clearer rules/agreements on how to handle cross-farm impacts—so projects don’t end up in endless developer vs developer fights.

This should be change according to the date of the report issue

this is where you will be going to school for the duration of the hovedforløb 2

eLife Assessment

This fundamental study provides new evidence of a change in how microglia survey neurons during the chronic phase of neurodegeneration, which researchers studying neuroinflammation and its role in neurodegenerative disease should find interesting. In this research, using time-lapse imaging of acute brain slices from prion-affected mice, the researchers show that, unlike in healthy brains, microglia become reactive, lose their territorial boundaries, and become highly mobile, exhibiting "kiss-and-ride" behavior, migrating into brain tissue and forming reversible, transient body-to-body contact with neurons. The evidence is compelling, with well-executed time-lapse imaging, good quantitative analysis across several disease stages, pharmacological validation of P2Y6 involvement, and the very surprising finding that this mobile behavior persists after microglia are removed from the brain.

Reviewer #1 (Public review):

Summary:

In this manuscript, Subhramanian et al. carefully examined how microglia adapt their surveillance strategies during chronic neurodegeneration, specifically in prion-infected mice. The authors used ex vivo time-lapse imaging and in vitro strategies and found that reactive microglia adopt a highly mobile, "kiss-and-ride" behavior, contrasting the more static surveillance typical of homeostatic microglia. The manuscript provides fundamental mechanistic insights into the dynamics of microglia-neuron interactions, implicates P2Y6 signaling in regulating mobility, and suggests that intrinsic reprogramming of microglia might underlie this behavior, the conclusions are therefore compelling.

Strengths:

(1) The novelty of the study is high, particularly the demonstration that microglia lose territorial confinement and dynamically migrate from neuron to neuron under chronic neurodegeneration.

(2) The possible implications of a stimulus-independent high mobility in reactive microglia are particularly striking. Although this is not fully explored.

(3) The use of time-lapse imaging in organotypic slices rather than overexpression models provided a more physiological approach.

(4) Microglia-neuron interactions in neurodegeneration have broad implications for understanding the progression of diseases, such as Alzheimer's and Parkinson's, that are associated with chronic inflammation.

Weaknesses:

Previous weaknesses were addressed.

Reviewer #2 (Public review):

This is a nice paper focused microglial responses to different clinical stages of prion infection in acute brain slices. The key here is the use of time-lapse imaging that captures the dynamics of microglial surveillance, including morphology, migration, and intracellular neuron/microglial contacts. The authors use a myeloid GFP-labeled transgenic mouse to track microglia in SSLOW-infected brain slices, quantifying differences in motility and microglial-neuronal interactions via live fluorescence imaging. Interesting findings include the elaborate patterns of motility among microglia, the distinct types and durations of intracellular contacts, the potential role of calcium signaling in facilitating hypermobility, and the fact that this motion-promoting status is intrinsic to the microglia, persisting even after the cells have been isolated from infected brains. Although largely a descriptive paper, it offers mechanistic insights, including the role of calcium in supporting microglial movement, with bursts of signaling identified even within the time lapse format, and inhibition studies implicating the purinergic receptor and calcium transient regulator P2Y6 in migratory capacity.

Strengths:

(1) The focus on microglia activation and activity in the context of prion disease is interesting

(2) Two different prions produce largely the same response

(3) Use of time-lapse provides insight into the dynamics of microglia, distinguishing between types of contact - mobility vs motility - and providing insight on the duration/transience and reversibility of extensive somatic contacts that include brief and focused connections in addition to soma envelopment.

(4) Imaging window selection (3 hours) guided by prior publications documenting preserved morphology, activity, and gene expression regulation up to 4 hours.

(5) The distinction between high- and low-mobility microglia is interesting, especially given that hypermobility seems to be an innate property of the cells.

(6) The live-imaging approach is validated by fixed tissue confocal imaging.

(7) The variance in duration of neuron/microglia contacts is interesting, although there is no insight into what might dictate which status of interaction predominates

(8) The reversibility of the enveloping action, which is not apparently a commitment to engulfment, is interesting, as is the fact that only neurons are selected for this activity.

(9) The calcium studies use the fluorescent dye calbryte-590, which picks up neuronal and microglial bursts -prolonged bursts are detected in enveloped neurons and in the hyper-mobile microglia - the microglial lead is followed up using MRS-2578 P2Y6 inhibitor that blunts the mobility of the microglia

Comments on revisions:

The authors have addressed my concerns in full - I think this is a very nice addition to the literature.

Author response:

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

Public Reviews:

Reviewer #1 (Public review)

The Cx3cr1/EGFP line labels all myeloid cells, which makes it difficult to conclude that all observed behaviors are attributable to microglia rather than infiltrating macrophages. The authors refer to this and include it as a limitation. Nonetheless, complementary confirmation by additional microglia markers would strengthen their claims. 

We appreciate the reviewer’s insightful comment regarding the cellular identity of the enveloping myeloid cells. As suggested, we performed triple co-immunostaining of SSLOW-infected Cx3cr1/EGFP mice using markers for neurons (NeuN), myeloid cells (IBA1), and resident microglia (TMEM119 or P2Y12). Because formic acid treatment used to deactivate prions abolishes the EGFP signal, we relied on IBA1 staining to identify the myeloid population. Our results confirmed that IBA1⁺ cells exhibiting the envelopment behavior are also TMEM119⁺ and P2Y12⁺, consistent with a resident microglial phenotype. These new data are presented in Figures S3 and S4 and described in the final section of the Results.

Although the authors elegantly describe dynamic surveillance and envelopment hypothesis, it is unclear what the role of this phenotype is for disease progression, i.e., functional consequences. For example, are the neurons that undergo sustained envelopment more likely to degenerate? 

We appreciate this important question regarding the functional implications of neuronal envelopment. At present, technical limitations prevent us from continuously tracking the fate of individual enveloped neurons in prion-infected mice. Nevertheless, our recent study demonstrated that P2Y12 knockout increases the prevalence of neuronal envelopment and accelerates disease progression (Makarava et al., 2025, J. Neuroinflammation). These findings suggest that while microglial envelopment may represent an adaptive response to increased neuronal surveillance demands, excessive envelopment, as observed in the absence of P2Y12, appears to be maladaptive. A new paragraph has been added to the Discussion to address this point.

Moreover, although the increase in mobility is a relevant finding, it would be interesting for the authors to further comment on what the molecular trigger(s) is/are that might promote this increase. These adaptations, which are at least long-lasting, confer apparent mobility in the absence of external stimuli. 

We thank the reviewer for this thoughtful suggestion. The molecular mechanisms underlying the increased mobility of microglia in prion-infected brains remain to be identified, and we plan to pursue this question in future studies. One possibility we briefly discuss in the revised manuscript is that proinflammatory signaling, mediated by secreted cytokines or interleukins, may drive this phenotype. Supporting this hypothesis, recent work has shown that IFNγ enhances microglial migration in the adult mouse cortex (doi:10.1073/pnas.2302892120). This work has been cited in the revised manuscript.

The authors performed, as far as I could understand, the experiments in cortical brain regions. There is no clear rationale for this in the manuscript, nor is it clear whether the mobility is specific to a particular brain region. This is particularly important, as microglia reactivity varies greatly depending on the brain region. 

We appreciate this insightful comment highlighting the importance of regional determinants of microglial reactivity, which indeed aligns with our ongoing research interests. In our previous studies, neuronal envelopment by microglia was observed consistently across all prion-affected brain regions exhibiting neuroinflammation. Assuming that envelopment requires microglial mobility, it is reasonable to speculate that microglia are mobile in all brain regions affected by prions and displaying neuroinflammatory responses. In the current study, we focused exclusively on the cortex because this region was used for quantifying the prevalence of neuronal envelopment as a function of disease progression in our prior work (DOI: 10.1172/JCI181169), which guided the present study design. Our ongoing investigations indicate that the prevalence of envelopment is region-dependent and correlates with microglial reactivity/the degree of neuroinflammation. In prion diseases, the degree of microglial reactivity is dictated by the tropism of specific prion strains to distinct brain regions. Notably, our prior studies have shown that strain-specific sialylation patterns of PrP^Sc glycans play a key role in determining both regional strain tropism and the extent of neuroinflammatory activation (DOI: 10.3390/ijms21030828, DOI: 10.1172/JCI138677). In response to this comment, we have added a brief rationale for using the cortex in the Results section.

It would be relevant information to have an analysis of the percentage of cells in normal, sub-clinical, early clinical, and advanced stages that became mobile. Without this information, the speed/distance alone can have different interpretations.

We thank the reviewer for this valuable suggestion. The percentage of mobile cells across normal, sub-clinical, early clinical, and advanced disease stages is presented in Figure 3b and described in the final paragraph of the section “Enveloping behavior of reactive myeloid cells.”

Reviewer #2 (Public review)

The number of individual cells tracked has been provided, but not the number of individual mice. The sex of the mice is not provided. 

We used N = 3 animals per group throughout the study; this information has now been added to the figure legends. Animals of both sexes were included in random proportions. The sex information is now listed for each experiment in the Animals subsection of the Methods.

The statistical approach is not clear; was each cell treated as a single observation? 

Yes, with the exception of the heat map in Figure 2d, all mobility parameters are analyzed and presented at the level of individual cells, with each cell treated as an independent observation. The primary aim of this study is to characterize behavioral patterns of single reactive myeloid cells. Analyzing data at the cell level allows us to capture the full distribution of cell behaviors and to preserve biologically meaningful heterogeneity within and across animals. By contrast, averaging values per animal would largely mask this variability. In the heat map in Figure 2d, data are averaged per animal, specifically to illustrate inter-animal variability within each group and to visualize changes across disease progression.

The potential for heterogeneity among animals has not been addressed. 

To address this concern, we now include a new Supplemental Figure (Figure S4)  presenting the data using Superplots, in which individual cells are shown as dots, animal-level average as circles, and group means calculated based on animals as black horizontal lines. These plots demonstrate that cell mobility measures are highly consistent across animals within each group, indicating limited inter-animal heterogeneity.

Validation of prion accumulation at each clinical stage of the disease is not provided. 

We now provide validation of PrP^Sc accumulation across disease stages by Western blot, along with quantitative analysis, in a new Supplemental Figure (Figure S2). This confirms progressive PrP^Sc accumulation with advancing disease.

How were the numerous captures of cells handled to derive morphological quantitative values? Based on the videos, there is a lot of movement and shape-shifting.

The following description has been added to Methods to clarify morphology analysis: For microglial morphology analysis, we quantified morphological parameters (radius, area, perimeter, and shape index) for individual EGFP⁺ cells in each time frame of the time-lapse recordings using the TrackMate 7.13.2 plugin in FIJI. Parameter values for each cell were then averaged across the entire three-hour imaging period to obtain a single mean value per cell.

While it is recognized that there are limits to what can be measured simultaneously with live imaging, the authors appear to have fixed tissues from each time point too - it would be very interesting to know if the extent or prion accumulation influences the microglial surveillance - i.e., do the enveloped ones have greater pathology. 

This is very interesting question which is difficult to answer due to technical challenges in monitoring the pathology or faith of individual neuronal cells as a function of their envelopment in live prion-infected animals. Our previous work revealed that both accumulation of total PrP^Sc in a brain and  accumulation of PrP^Sc specifically in lysosomal compartments of microglia due to phagocytosis precedes the onset of neuronal envelopment (DOI: 10.1172/JCI181169).  Moreover, the onset of neuronal envelopment occurred after a noticeable decline in neuronal levels of Grin1, a subunit of the NMDA receptor essential for synaptic plasticity. Reactive microglia were observed to envelop Grin1-deficient neurons, suggesting that microglia respond to neuronal dysfunction. However, considering that envelopment is very dynamic and - in most cases - reversible, correlating the degree of envelopment with dysfunction of individual neurons is technically challenging.

Recommendations for the authors

Reviewer #1 (Recommendations for the authors): 

(1) I recommend performing additional immunostaining using microglial markers to address specificity. 

These new data showing immunostaining for markers of resident microglia TMEM119 and P2Y12 are presented in Figures S6 and S7 and described in the final section of the Results.

(2) The authors can at least further discuss the functional consequences of their findings in further detail. 

A new paragraph has been added to the Discussion to address this point.

(3) Quantify the % of cells that become mobile in the different conditions. 

The percentage of mobile cells across normal, sub-clinical, early clinical, and advanced disease stages is presented in Figure 3b and described in the final paragraph of the section “Enveloping behavior of reactive myeloid cells.”

(4) Improve method details on the brain regions used and further expand the statistical section. 

We have expanded the Statistical Analysis section to indicate whether statistical comparisons and mean values were calculated at the single-cell level or the animal level for each analysis. The specific statistical tests used and the number of animals (N) are now reported in the corresponding figure legends. The sex of animals is provided in Table 1 (Methods). Only the cortical region was examined in this study; this information is stated in the Methods and is now also noted in the figure legends for clarity.

Reviewer #2 (Recommendations for the authors): 

(1) More details on members of the PY2 receptor family expressed in microglia would be helpful. The study highlights a previously published prion-induced decline in the expression of P2Y12, a microglial marker that is required for intracellular neuron-microglial contacts, and P2Y6, involved in calcium transients, which is required for hypermotility. How are members of this family of receptors regulated at the gene and/or protein level in microglial and given their responsiveness to nucleotide ligands, are other members implicated in the properties being quantified here? 

We appreciate the reviewer’s insightful comment. To address this point, we examined the expression of multiple P2Y receptors and ATP-gated P2X channels known to contribute to microglial surveillance, activation, motility, and phagocytosis, alongside the activation markers Tlr2, Cd68, and Trem2. Bulk brain transcript analyses indicated that all examined genes were upregulated in SSLOW-infected mice relative to controls (new Figure S5a). However, because microglial proliferation substantially increases microglial numbers during prion disease progression, bulk tissue measurements do not necessarily reflect per-cell expression levels. Therefore, we normalized gene expression values to the microglia-specific marker Tmem119, whose per-cell expression remains stable across disease stages (Makarava et al., 2025, J. Neuroinflammation). After normalization, Tlr2, Cd68, and Trem2 were increased approximately 10-, 6-, and 4-fold, respectively. In contrast, P2 receptor genes showed more modest changes: P2ry6 increased ~3-fold, P2ry13 ~2-fold, and P2rx7 ~1.3-fold, while P2rx4 remained unchanged (Figure S5a). Within the scope of the present study, we focused on P2Y6 due to (i) its role in regulating calcium transients, (ii) the magnitude of its upregulation relative to other P2 receptors, and (iii) its highly microglia-specific expression in the CNS. We note that currently available commercial P2Y6 antibodies lack sufficient specificity, making reliable assessment of protein-level expression challenging.

(2) Is P2Y6 expressed in any other cell type that might account for the blunted mobility of the microglia? The authors mention P2Y12 also identifies the GFP cells; however, it would be beneficial to highlight the specificity of the target in the ex vivo treatment of the infected slices.

In the brain, both P2Y12 and P2Y6 are considered highly specific to resident microglia under physiological and neuroinflammatory conditions. P2Y12 is, in fact, widely used as a canonical marker of homeostatic and resident microglia. While P2Y6 is also expressed in peripheral myeloid cells such as macrophages, our phenotypic characterization indicates that the cells exhibiting neuronal envelopment are TMEM119⁺ and P2Y12⁺, consistent with a resident microglial identity. These data, including new analyses added to the revised manuscript, support that the cells responding to P2Y6 signaling in our ex vivo slice experiments are resident microglia.

(3) The fluorescent mouse lacks Cx3cr1 - have the authors investigated why there were no apparent consequences, at least in the context of prion infection? Are there functional redundancies that might be harnessed? Does this impact the generalizability of the findings here?

The role of Cx3cr1 in prion disease has been directly examined in two independent studies (doi: 10.1099/jgv.0.000442; doi: 10.1186/1471-2202-15-44). One study reported no effect of Cx3cr1 deficiency on disease incubation time, whereas the other observed only a minor difference. Importantly, both studies found no detectable alterations in microglial activation patterns, cytokine expression, or PrP^Sc deposition in Cx3cr1-deficient mice compared to wild-type controls. Our own data (Figure S1) are consistent with these findings: disease course and PrP^Sc deposition were comparable between Cx3cr1/EGFP and wild-type mice. Moreover, we observed reactive microglial envelopment of neurons in both genotypes. Microglia isolated from SSLOW-infected Cx3cr1/EGFP mice also displayed similarly elevated mobility in vitro, in agreement with our previous observations of high mobility of microglia isolated from SSLOW-infected wild-type mice (Makarava et al., 2025, J. Neuroinflammation). Taken together, these results indicate that Cx3cr1 is not a key determinant of reactive microglial mobility or envelopment behavior in prion disease. Thus, the use of the Cx3cr1/EGFP reporter line does not compromise the generalizability of our conclusions.

(4) The distinction between high mobility and low mobility microglia is interesting - is there any evidence to suggest that the slow-moving microglia are actually a separate class - do enveloping microglia exhibit both mobility states - can the authors comment on plasticity here? 

We appreciate this insightful comment, which closely aligns with our ongoing interests. At present, we do not have evidence to support that high- versus low-mobility microglia represent distinct molecular phenotypes. Given that our time-lapse imaging spans only a three-hour window, it remains unclear whether these mobility states reflect stable cell-intrinsic properties or transient phases within a dynamic surveillance process. Notably, we observed that individual cells can transition between more stationary, neuron-associated states and highly mobile states within the same imaging session. In future work, we intend to investigate whether prolonged interactions with neuronal somas or other microenvironmental cues may drive diversification of reactive myeloid cell phenotypes.

(5) In the discussion, the authors speculate about "collective coordinated decision making" - that seems a stretch unless greater context is provided. The fact that several microglia can be found in contact with an individual neuron and that each microglia can connect with multiple neurons simultaneously is certainly interesting; however, evidence for hive behavior is entirely lacking.

We agree with the reviewer that our previous wording overstated the interpretation. The statement regarding collective decision-making has been removed.

eLife assessment

This important work is the first to suggest a model that the nematode C. elegans prefers specific bacteria (its major food source) that release high amounts of the known attractant isoamyl alcohol when supplemented with exogenous leucine and has also identified a likely receptor for the odorant isoamyl alcohol. The evidence supporting the claims of the authors is solid, and the manuscript would be improved by changes to the text that clarify and address the distinction between "supplemented" versus "enriched". The renaming of srd-12 to snif-1 should also be addressed.

Reviewer #1 (Public review):

Summary:

Siddiqui et al., investigate the question of how bacterial metabolism contributes to the attraction of C. elegans to specific bacteria. They show that C. elegans prefers three bacterial species when cultured in a leucine-enriched environment. These bacterial species release more isoamyl alcohol, a known C. elegans attractant, when cultured with leucine supplement than without leucine supplement. The study shows correlative evidence that isoamyl alcohol is produced from leucine by the Ehrlich pathway. In addition, they show that SNIF-1 is a receptor for isoamyl alcohol because a null mutant of this receptor exhibits lower chemotaxis to isoamyl alcohol and that chemotaxis to isoamyl alcohol is rescued by expression of snif-1 in AWC.

Strengths:

(1) This study takes a creative approach to examine the question of what specific volatile chemicals released by bacteria may signify to C. elegans by examining both bacterial metabolism and C. elegans preference behavior. Although C. elegans has long been known to be attracted to bacterial metabolites, this study may be one of the first to examine the possible role of a specific bacterial metabolic pathway in mediating attraction.

(2) A strength of the paper is the identification of SNIF-1 as a receptor for isoamyl alcohol. The ligands for very few olfactory receptors have been identified in C. elegans and so this is a significant addition to the field. The SNIF-1 null mutant strain will likely be a useful reagent for many labs examining olfactory and foraging behaviors.

Weaknesses:

(1) The authors write that the leucine metabolism via the Ehrlich pathway is required for production of isoamyl alcohol by three bacteria (CEent1, JUb66, BIGb0170), but their evidence for this is correlation and not causation. They show that the gene, ilvE (which is part of the Ehrlich pathway) is upregulated in CEent1 bacteria upon exposure to leucine. Although this indicates that the ilvE gene may be involved in leucine metabolism, it does not show causation. To show causation, they need to knockout ilvE from one of these strains, show that the bacteria does not have increased isoamyl alcohol production when cultured on leucine, and that the bacteria is no longer attractive to C. elegans.

(2) Although the authors do show that the three bacterial strains they focus on (CEent1, JUb66, and BIGb0170) are more attractive to C. elegans when supplemented with leucine. Some other strains such as BIGb0393 are also more attractive with leucine supplementation and produce isoamyl alcohol (Fig 1B and Supp Table 2). It is unclear why these other strains are not included with the selected three strains.

(3) The behavioral evidence that snif-1 gene encodes a receptor for isoamyl alcohol is compelling because of the mutant phenotype and rescue experiments. The evidence would be stronger with calcium imaging of AWC neurons in response to isoamyl alcohol in the receptor mutant with the expectation that the response would be reduced or abolished in the mutant compared to wildtype.

Reviewer #2 (Public review):

Summary:

Siddiqui et al. show that C. elegans prefers certain bacterial strains that have been supplemented with the essential amino acid (EEA) leucine. They convincingly show that some leucine enriched bacteria stimulate the production of isoamyl alcohol (IAA). IAA is an attractive odorant that is sensed by the AWC. The authors an identify a receptor, SRD-12, that is expressed in the AWC chemosensory neurons and is required for chemotaxis to IAA. The authors propose that IAA is a predominant olfactory cue that determines diet preference in C. elegans. Since leucine is an EAA, the authors propose that worm IAA sensing allows the animal provides a proxy mechanism to identify EAA rich diets.

Strengths:

The authors propose IAA as a predominant olfactory cue that determines diet preference in C. elegans providing molecular mechanism underlying diet selection. They show that wild isolates of C. elegans have strong chemotactic response to IAA indicating that IAA is an ecologically relevant odor for the worm. The paper is well written, and the presented data are convincing and well organized. This is an interesting paper that connects chemotactic response with bacterially produced odors and thus provides an understanding how animals adapt their foraging behavior through the perception of molecules that may indicate the nutritional value.

Weaknesses:

Major: While I do like the way the authors frame C. elegans IAA sensing as mechanisms to identify leucine (EAA) rich diets, it is not fully clear whether bacterial IAA production is a proxy for bacterial leucine levels.

(1) Can the authors measure leucine (or other EAA) content of the different CeMbio strains? This would substantiate the premise in the way they frame this in the introduction. While the authors convincingly show that leucine supplementation induces IAA production in some strains, it is not clear if there are lower leucine levels in the different in the non-preferred strains.

(2) It is not clear whether the non-preferred bacteria in Figure 1A and 1B have the ability to produce IAA. To substantiate the claim that C. elegans prefers CEent1, JUb66, and BIGb0170 due to their ability to generate IAA from leucine, it would be measure IAA levels in non-preferred bacteria (+ and - leucine supplementation). If the authors have these data it would be good to include this.

(3) The authors would strengthen their claim if they could show that deletion or silencing ilvE enzyme reduces IAA levels and eliminates the increased preference upon leucine supplementation.

(4) While the three preferred bacteria possess the ilvE gene, it is not clear whether this enzyme is present in the other non-preferred bacterial strains. As far as I know, the CeMbio strains have been sequenced, so it should be easy to determine if the non-preferred bacteria possess the capacity to make IAA. Does expression of ilvE in e.g. E. coli increase its preference index or are the other genes in the biosynthesis pathway missing?

(5) It is strongly implied that leucine rich diets are beneficial to the worm. Do the authors have data to show the effect on leucine supplementation on C. elegans healthspan, life-span or broodsize?

Comments on revisions:

(1) The authors have addressed most of the earlier questions. The main unresolved issue is the link between iaa production is a reflection of bacterial leucine levels. It is not clear if there are lower leucine levels in the different in non-preferred strains.

The main conclusions that: 1. some bacterial strains can convert exogenous leucine into IAA which is an attractant to C. elegans. 2. The identification of a GPCR required for IAA responses are solid. These are important results that carry the paper. My outstanding concern remains with the overinterpretation of the framing that C. elegans IAA sensing is used as a mechanism to identify leucine (EAA) rich diets. It is fine to leave this a favorite hypothesis in the discussion but statements throughout the paper need to be nuanced without leucine measurement of the different bacterial strains. (Also since for the bacterial chemotaxis assays there were only done with a single concentration of leucine makes it difficult to infer bacterial leucine concentrations). I recommend softening claims related to leucine-rich diet detection unless quantitative measurements are provided.

Part of the issue in the text lies in the difference between "supplemented" and "chemotaxis" (lab based constructs) and enriched and foraging (natural environment based). This is also the way it is set up in the introduction "Do animals use specific sensing mechanisms to find an EAA-enriched diet?". If enriched is used strictly the same as supplemented then it would be fine but in the text this distinction gets blurred and enriched drifts to the more ethological explanation.

Then it is more than just semantics since leucine-supplemented diets are not something that occurs in the natural environment. IAA production by bacteria could be a signal for a leucine rich environment and it is fine to speculate about this in the discussion.

Examples where the wording needs to be more precise to reflect the experimental results rather than the possible impact in its natural environment:

The title:' The olfactory receptor SNIF-1 mediates foraging for leucine-rich diets in C. elegans"

The intro:"Taken together, SNIF-1 regulates the dietary preference of worms to IAA-producing bacteria and thereby mediates the foraging behavior of C. elegans to leucine-enriched diets. Thus, IAA produced by bacteria is a dietary quality code for leucine-enriched bacteria."

Results "Figure 1. C. elegans relies on odors to select leucine-enriched bacteria"

Supplementation is used more in the text and the figure legends whereas headings and abstract use enriched. The experiments in the paper only describe leucine-supplemented experiments. So use I would supplemented instead of enriched when describing experiments for clarity.

For instance:

Page 4:"Microbial odors drive the preference of C. elegans for leucine-enriched diet"

Page 5: "Altogether, these findings suggested that worms rely on odors to distinguish various bacteria and find leucine-enriched bacteria"

Page 7: "Isoamyl alcohol odor is a signature for a leucine-enriched diet"

Page 9: AWC odor sensory neurons facilitate the diet preference of C. elegans for leucine-enriched diets"

page 20 "Leucine-enriched diets produce significantly higher levels of IAA odor, making up to 90% of their headspace"

(2) As suggested in the first round of review the authors now add data IAA levels in non-preferred bacteria (+ and - leucine supplementation) in table S2. While it is good to have this data, the table is not very clear. Not clear what ND stands for in the table S2. Not determined or not detected? I assume not determined since some strains Jub44, BiGb0393 Jub134 produce IAA even in the absence of LEU. The authors mention that "the abundance of IAA in these strains is significantly less". However, the table just reflects yes or no. Can the authors give an indication of the concentration to understand what significantly less means? Fig. 2c at least gives a heat map.

(3) On wormbase the gene is still called srd-12. The authors should seek permission to rename srd-12 to snif-1.

Author response:

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

eLife Assessment:

This is an important study, supported by solid to convincing data, that suggests a model for diet selection in C. elegans. The significance is that while C. elegans has long been known to be attracted to bacterial volatiles, what specific bacterial volatiles may signify to C. elegans is largely unknown. This study also provides evidence for a possible odorant/GPCR pairing.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Siddiqui et al., investigate the question of how bacterial metabolism contributes to the attraction of C. elegans to specific bacteria. They show that C. elegans prefers three bacterial species when cultured in a leucine-enriched environment. These bacterial species release more isoamyl alcohol, a known C. elegans attractant, when cultured with leucine supplement than without leucine supplement. The study shows correlative evidence that isoamyl alcohol is produced from leucine by the Ehrlich pathway. In addition, they show that SRD-12 (SNIF-1) is likely a receptor for isoamyl alcohol because a null mutant of this receptor exhibits lower chemotaxis to isoamyl alcohol and lower preference for leucine-enriched bacteria.

Strengths:

(1) This study takes a creative approach to examine the question of what specific volatile chemicals released by bacteria may signify to C. elegans by examining both bacterial metabolism and C. elegans preference behavior. Although C. elegans has long been known to be attracted to bacterial metabolites, this study may be one of the first to examine the role of a specific bacterial metabolic pathway in mediating attraction.

(2)  A strength of the paper is the identification of SRD-12 (SNIF-1) as a likely receptor for isoamyl alcohol. The ligands for very few olfactory receptors have been identified in C. elegans and so this is a significant addition to the field. The srd-12 (snif-1) null mutant strain will likely be a useful reagent for many labs examining olfactory and foraging behaviors.

Weaknesses:

(1) The authors write that the leucine metabolism via the Ehrlich pathway is required for the production of isoamyl alcohol by three bacteria (CEent1, JUb66, BIGb0170), but their evidence for this is correlation and not causation. They write that the gene ilvE is a bacterial homolog of the first gene in the yeast Ehrlich pathway (it would be good to include a citation for this) and that the gene is present in these three bacterial strains. In addition, they show that this gene, ilvE, is upregulated in CEent1 bacteria upon exposure to leucine. To show causation, they need to knockout ilvE from one of these strains, show that the bacteria does not have increased isoamyl alcohol production when cultured on leucine, and that the bacteria is no longer attractive to C. elegans.

Thank you for the comment. We have added the appropriate citation [1,2]. We agree that worms’ diet preference for the preferred strains upon ilvE knockout will further strengthen the claim for IAA being used as a proxy for leucine-enriched diet. Currently, protocols and tools for genetic manipulations for CeMbio strains are not available, making this experiment not feasible at this time.  

(2) The authors examine three bacterial strains that C. elegans showed increased preference when grown with leucine supplementation vs. without leucine supplementation. However, there also appears to be a strong preference for another strain, JUb0393, when grown on plus leucine (Figure 1B). It would be good to include statistics and criteria for selecting the three strains.

Thanks for your comment. We agree that for Pantoea nemavictus, JUb393, worms seem to prefer the leucine supplemented (+ LEU) bacteria over unsupplemented (-LEU). However, when given a choice between the individual CeMbio bacteria and E. coli OP50, worms showed preference for only CEent1, JUb66, and BIGb0170 (Figure 1F). Consequently, CEent1, JUb66, and BIGb0170 were selected for further analyses. We have included statistics for Figure 1B-C and Figure S1A-G with details mentioned in the figure legend. 

(3) Although the behavioral evidence that srd-12 (snif-1) gene encodes a receptor for isoamyl alcohol is compelling, it does not meet the standard for showing that it is an olfactory receptor in C. elegans. To show it is indeed a likely receptor one or more of the following should be done:

(a) Calcium imaging of AWC neurons in response to isoamyl alcohol in the receptor mutant with the expectation that the response would be reduced or abolished in the mutant compared to wildtype.

(b)"A receptor swap" experiment where the SRD-12 (SNIF-1) receptor is expressed in AWB repulsive neuron in SRD-12 (SNIF-1) receptor mutant background with the expectation that with receptor swap C. elegans will now be repulsed from isoamyl alcohol in chemotaxis assays (experiment from Sengupta et al., 1996 odr-10 paper).

Thanks for all your comments and suggestions. While the lab currently does not have the necessary expertise to conduct calcium imaging of neurons, we have performed additional experiments to confirm the requirements of AWC neurons for SNIF-1 function. We generated transgenic worms with extrachromosomal array expressing snif-1 under (a) AWC-specific promoter, odr-1, and (b) AWB-specific promoter, str-1. As shown in new panel 6H in the revised manuscript and Author response image 1, we found that overexpression of snif-1 in AWC neurons completely rescues the chemotaxis defect of snif-1 mutant (referred at VSL2401), whereas upon the “receptor swap" in AWB neurons IAA is sensed as a repellent.  

Author response image 1.

(A) Chemotaxis index (CI) of WT, VSL2401, VSL2401 [AWCp::snif-1] and VSL2401 [AWBp::snif-1] worms to IAA at 1:1000 dilution. Significant differences are indicated as **** P ≤ 0.0001 determined by one-way ANOVA followed by post hoc Dunnett’s multiple comparison test. Error bars indicate SEM (n≥15).

(4) The authors conclude that C. elegans cannot detect leucine in chemotaxis assays. It is important to add the method for how leucine chemotaxis assay was done in order to interpret these results. Because leucine is not volatile if leucine is put on the plates immediately before the worms are added (as in a traditional odor chemotaxis assay), there is no leucine gradient for the worm to detect. It would be good to put leucine on the plate several hours before worms are introduced so worms have the possibility to be able to detect the gradient of leucine (for example, see Wakabayashi et al., 2009).

Previously, the chemotaxis assays with leucine were performed like traditional odor chemotaxis assays. We also performed chemotaxis assay as detailed in Shingai et al 2005[3]. Leucine was spotted on the assay plates 5 hours prior to the introduction of worms on the plates. As shown in new panel S1H in the revised manuscript, wild-type worms do not show response to leucine in the modified chemotaxis assay.

We have included the experimental details for leucine chemotaxis assays in the revised manuscript.  

(5) The bacterial preference assay entitled "odor-only assay" is a misleading name. In the assay, C. elegans is exposed to both volatile chemicals (odors) and non-volatile chemicals because the bacteria are grown on the assay plate for 12 hours before the worms are introduced to the assay plate. In that time, the bacteria is likely releasing non-volatile metabolites into the plate which may affect the worm's preference. A true odor-only assay would have the bacteria on the lid and the worms on the plate.

The ‘odor-only’ diet preference assay does not allow for non-volatile chemicals to reach worms. We achieved this by using tripartite dishes where the compartments containing worms and bacterial odors are separated by polystyrene barriers. At the time of the assay, worms were spotted in a separate compartment from that of bacteria (as shown in schematic 1A). The soluble metabolites released by the bacteria during their growth will accumulate in the agar within the bacterial compartment alone such that worms only encounter the volatile metabolites produced by bacteria wafting past the polystyrene barrier.

(6) The findings of the study should be discussed more in the context of prior literature. For example, AWC neurons have been previously shown to be involved in bacterial preference (Harris et al., 2014; Worthy et al., 2018). In addition, CeMbio bacterial strains (the strains examined in this study) have been previously shown to release isoamyl alcohol (Chai et al. 2024).

Thanks for the suggestion. We have modified the Discussion section to discuss the study in the light of relevant prior literature.  

Reviewer #2 (Public review):

Summary:

Siddiqui et al. show that C. elegans prefers certain bacterial strains that have been supplemented with the essential amino acid (EEA) leucine. They convincingly show that some leucine enriched bacteria stimulate the production of isoamyl alcohol (IAA). IAA is an attractive odorant that is sensed by the AWC. The authors an identify a receptor, SRD-12 (SNIF-1), that is expressed in the AWC chemosensory neurons and is required for chemotaxis to IAA. The authors propose that IAA is a predominant olfactory cue that determines diet preference in C. elegans. Since leucine is an EAA, the authors propose that worm IAA sensing allows the animal provides a proxy mechanism to identify EAA rich diets.

Strengths:

The authors propose IAA as a predominant olfactory cue that determines diet preference in C. elegans providing molecular mechanism underlying diet selection. They show that wild isolates of C. elegans have a strong chemotactic response to IAA indicating that IAA is an ecologically relevant odor for the worm. The paper is well written, and the presented data are convincing and well organized. This is an interesting paper that connects chemotactic response with bacterially produced odors and thus provides an understanding of how animals adapt their foraging behavior through the perception of molecules that may indicate the nutritional value.

Weaknesses:

Major:

While I do like the way the authors frame C. elegans IAA sensing as mechanisms to identify leucine (EAA) rich diets it is not fully clear whether bacterial IAA production is a proxy for bacterial leucine levels.

(1) Can the authors measure leucine (or other EAA) content of the different CeMbio strains? This would substantiate the premise in the way they frame this in the introduction. While the authors convincingly show that leucine supplementation induces IAA production in some strains, it is not clear if there are lower leucine levels in the different in non-preferred strains.

Thanks for your suggestion. Estimating leucine levels in various bacteria will provide useful information, and we hope to do so in future studies.

(2) It is not clear whether the non-preferred bacteria in Figure 1A and 1B have the ability to produce IAA. To substantiate the claim that C. elegans prefers CEent1, JUb66, and BIGb0170 due to their ability to generate IAA from leucine, it would measure IAA levels in non-preferred bacteria (+ and - leucine supplementation). If the authors have these data it would be good to include this.

Thanks for the suggestion. We have included the table indicating the presence or absence of IAA production by all the bacteria under + LEU and – LEU conditions (Table S2). Some of the nonpreferred bacteria indeed produce isoamyl alcohol. However, the abundance of IAA in these strains is significantly less than in the preferred bacteria.  

Using the available genomic sequence data, we found that all CeMbio strains encode IlvE-like transaminase enzymes[4]. This suggests that presumably all the bacteria have the metabolic capacity to make alpha-ketoisocaproate (an intermediate in IAA biosynthetic pathway) from leucine. However, the regulation of metabolic flux is likely to be quite complex in various bacteria.  

(3) The authors would strengthen their claim if they could show that deletion or silencing ilvE enzyme reduces IAA levels and eliminates the increased preference upon leucine supplementation.

We agree that testing worms’ diet preference for the preferred strains upon ilvE knockout will further strengthen the claim for IAA being crucial for finding leucine-enriched diet. Currently the lab does not have the necessary expertise and standardize protocols to do genetic manipulations for the CeMbio strains.

(4) While the three preferred bacteria possess the ilvE gene, it is not clear whether this enzyme is present in the other non-preferred bacterial strains. As far as I know, the CeMbio strains have been sequenced so it should be easy to determine if the non-preferred bacteria possess the capacity to make IAA. Does the expression of ilvE in e.g. E. coli increase its preference index or are the other genes in the biosynthesis pathway missing?

Thanks for the suggestion. Using the available genomic sequence data, we find that all the bacteria in the CeMbio collection possess IlvE-like transaminase necessary for synthesis of alphaketoisocaproate, key metabolite in leucine turn over as well as precursor for IAA [4]. E. coli has an IlvE encoding gene in its genome [2]. However, we do not find IAA in the headspace of E. coli either with or without leucine supplementation. This indicates either (i) E. coli lacks enzymes for subsequent steps in IAA biosynthesis or (ii) leucine provided under the experimental regime is not sufficient to shift the metabolic flux to IAA production.  

Previous studies have suggested that in yeast, the final two steps leading to IAA production are catalyzed by decarboxylase and dehydrogenase enzymes1. The genomic and metabolic flux data available for CeMbio do not describe specific enzymes leading up to IAA synthesis [4].  

(5) It is strongly implied that leucine-rich diets are beneficial to the worm. Do the authors have data to show the effect on leucine supplementation on C. elegans healthspan, life-span or broodsize?

Edwards et al. 2015 reported a 15% increase in the lifespan of worms upon 1 mM leucine supplementation [5]. Wang et al 2018 also showed lifespan extension upon 1 mM and 10 mM leucine supplementation. They also reported that while leucine supplementation did not have any effect on brood size, it did make worms more resistant to heat, paraquat, and UV-stress [6]. These studies have been included in the discussion section.

Other comments:

Page 6. Figure 2c. While the authors' conclusions are correct based on AWC expts. it would be good at this stage to include the possibility that odors that enriched in the absence of leucine may be aversive.

Thanks for the comment. We have tested the chemotaxis response of the worms for most of the odors produced by CeMbio strains without leucine supplementation. We did not find any odor that is aversive to worms. However, we cannot completely rule out the possibility that a low abundance of aversive odor in the headspace of the bacteria was missed.

Interestingly, we did identify 2-nonanone, a known repellent, in the headspace of the preferred bacteria upon leucine supplementation. However, the abundance of 2-nonanone in headspace of bacteria is relatively low (less than 1% for CEent1, and JUb66, and ~10% for BIGb0170). This suggests that the relative abundance of odors in an odor bouquet may be a relevant factor in determining worms’ reference.  

Page 6. IAA increases 1.2-4 folds upon leucine supplementation. If the authors perform a chemotaxis assay with just IAA with 1-2-4 fold differences do you get the shift in preference index as seen with the bacteria? i.e. is the difference in IAA concentration sufficient to explain the shift in bacterial PI upon leucine supplementation? Other attractants such as Acetoin and isobutanol go up in -Leu conditions.

Thanks for the suggestion. As shown in Figure S2H and S2I, when given a choice between a concentration of IAA (1:1000 dilution) attractive to worms and a 4-fold higher amount of IAA, worms chose the latter. This result suggests that worms can distinguish between relatively small difference in concentrations of IAA.

We agree that the relative abundance of Acetoin and Isobutanol is high in -LEU conditions. The presence of other attractants in - LEU conditions should skew the preference of worms for – LEU bacteria. However, we found that worms prefer + LEU bacteria (Figure 1B), suggesting that the abundance of IAA mainly influences the diet preference of the worms.  

Page 14-15. The authors identify a putative IAA receptor based on expression studies. I compliment the authors for isolating two CRISPR deletion alleles. They show that the srd-12 (snif-1) mutants have obvious defects in IAA chemotaxis. Very few ligand-odorant receptors combinations have been identified so this is an important discovery. CenGen data indicate that srd-12 (snif-1) is expressed in a limited set of neurons. Did the authors generate a reporter to show the expression of srd-12 (snif-1)? This is a simple experiment that would add to the characterization of the SRD-12 (SNIF-1) receptor. Rescue experiments would be nice even though the authors have independent alleles. To truly claim that SRD-12 (SNIF-1) is the ligand for IAA and activates the AWC neurons would require GCamp experiments in the AWC neuron or heterologous expression system. I understand that GCamp imaging might not be part of the regular arsenal of the lab but it would be a great addition (even in collaboration with one of the many labs that do this regularly). Comparing AWC activity using GCaMP in response IAA-producing bacteria with high leucine levels in both wild-type and SRD-12 (SNIF-1) deficient backgrounds, would further support their narrative. I leave that to the authors.

Thanks for your comments and suggestions. To address this comment, we rescued snif-1 mutant (referred as VSL2401) with extrachromosomal array expressing snif-1 under AWC-specific promoter as well as its native promoter. As shown in Figure 6H and Author response image 2, we find that both transgenic lines show a complete rescue of chemotaxis response to isoamyl alcohol. To find where snif-1 is expressed, we generated a transgenic line of worms expressing GFP under snif-1 promoter, and mCherry under odr-1 promoter (to mark AWC neurons). As shown in Figure 6I, we found that snif-1 is expressed faintly in many neurons, with strong expression in one of the two AWC neurons marked by odr-1::mCherry. This result suggests that SNIF-1 is expressed in AWC neuron.

We hope to perform GCaMP assay and further characterization of SNIF-1 in the future.

Author response image 2.

Chemotaxis index (CI) of WT, VSL2401, VSL2401 [AWCp:: snif-1] and VSL2401 [snif-1p::snif-1] worms to IAA at 1:1000 dilution. Significant differences are indicated as **** P ≤ 0.0001 determined by one-way ANOVA followed by post hoc Dunnett’s multiple comparison test. Error bars indicate SEM (n≥15).

Minor:

Page 4 "These results suggested that worms can forage for diets enriched in specific EAA, leucine...." More precise at this stage would be to state " These results indicated that worms can forage for diets supplemented with specific EAA...".

We have changed the statement in the revised manuscript.

Page 5."these findings suggested that worms not only rely on odors to choose between two bacteria but also to find leucine enriched bacteria" This statement is not clear to me and doesn't follow the data in Fig. S2. Preferred diets in odorant assays are the IAA producing strains.

Thanks for your comment. We have revised the manuscript to make it clear. “Altogether, these findings suggested that worms rely on odors to distinguish different bacteria and find leucineenriched bacteria”. This statement concludes all the data shown in Figure 1 and Figure S1.  

Page 5. Figure S2A provides nice and useful data that can be part of the main Figure 1.

Thanks for the comment. We have incorporated the data from Figure S2A to main Figure 1.

Reviewer #3 (Public review):

Summary:

The authors first tested whether EAA supplementation increases olfactory preference for bacterial food for a variety of bacterial strains. Of the EAAs, they found only leucine supplementation increased olfactory preference (within a bacterial strain), and only for 3 of the bacterial strains tested. Leucine itself was not found to be intrinsically attractive.

They determined that leucine supplementation increases isoamyl alcohol (IAA) production in the 3 preferred bacterial strains. They identify the biochemical pathway that catabolizes leucine to IAA, showing that a required enzyme for this pathway is upregulated upon supplementation.

Consistent with earlier studies, they find that AWC olfactory neuron is primarily responsible for increased preference for IAA-producing bacteria.

Testing volatile compounds produced by bacteria and identified by GC/MS, and identified several as attractive, most of them require AWC for the full effect. Adaptation assays were used to show that odorant levels produced by bacterial lawns were sufficient to induce olfactory adaptation, and adaptation to IAA reduced chemotaxis to leucine-supplemented lawns. They then showed that IAA attractiveness is conserved across wild strains, while other compounds are more variable, suggesting IAA is a principal foraging cue.

Finally, using the CeNGEN database, they developed a list of candidate IAA receptors. Using behavioral tests, they show that mutation of srd-12 (snif-1) greatly impairs IAA chemotaxis without affecting locomotion or attraction to another AWC-sensed odor, PEA.

Comments

This study will be of great interest in the field of C. elegans behavior, chemical senses and chemical ecology, and understanding of the sensory biology of foraging.

Strengths:

The identification of a receptor for IAA is an excellent finding. The combination of microbial metabolic chemistry and the use of natural bacteria and nematode strains makes an extremely compelling case for the ecological and adaptive relevance of the findings.

Weaknesses:

AWC receives synaptic input from other chemosensory neurons, and thus could potentially mediate navigation behaviors to compounds detected in whole or in part by those neurons. Language concluding detection by AWC should be moderated (e.g. p9 "worms sense an extensive repertoire...predominantly using AWC") unless it has been demonstrated.

Thanks for your comment. We have modified the manuscript to incorporate the suggestion.

srd-12 (snif-1) is not exclusively expressed in AWC. Normally, cell-specific rescue or knockdown would be used to demonstrate function in a specific cell. The authors should provide such a demonstration or explain why they are confident srd-12 (snif-1) acts in AWC.

Thanks for the comment. We have performed AWC-specific rescue of snif-1 in mutant worms. As shown in Figure 6H, we found that AWC neurons specific rescue completely recovered the chemotaxis defect of the snif-1 mutant (referred as VSL2401) for IAA. In addition, snif-1 is expressed in one of the AWC neurons.

A comparison of AWC's physiological responses between WT and srd-12 (snif-1), preferably in an unc13 background, would be nice. Even further, the expression of srd-12 (snif-1) in a different neuron type and showing that it confers responsiveness to IAA (in this case, inhibition) would be very convincing.

Thanks for the suggestion. We have performed a receptor swap experiment, where snif-1 is misexpressed in AWB neurons. We find that these worms show slight but significant repulsion to IAA compared to WT and snif-1 mutant worms (Author response image 1).

Recommendations for the authors:

Reviewing Editor:

Please consider all of the reviewer comments. In particular, as noted in the individual reviews, the strength of the evidence would be bolstered by additional experiments to demonstrate that the iLvE enzyme affects IAA levels in the preferred bacteria. The reviewers note that the authors haven't shown that IAA production is a reflection of leucine content. Are the non-preferred bacteria low on leucine or lack iLvE or IAA synthesis pathways? Further, more direct evidence that SRD-12 (SNIF-1) is in fact the primary IAA receptor would further strengthen the study. The authors should also be aware that geographic distance for wild isolate C. elegans may not directly correlate with phylogenetic distance. This should be assessed/discussed for the strains used.

Thanks for the suggestions. Some of these have been addressed in response to reviewers. Thanks for your comments about possible disconnect between geographical and phylogenetic distances amongst natural isolates used here.

By analyzing the phylogenetic tree generated using neighbor-joining algorithm available at CaeNDR database, we found that QX1211 and JU3226 are phylogenetically close, but the remaining isolates fall under different clades separated by long phylogenetic distances [7,8].  

Reviewer #1 (Recommendations for the authors):

(1) In the first sentence of the third paragraph of the introduction, C. elegans are described as "soildwelling." Although C. elegans has been described as soil-dwelling in the past, current research indicates they are most often found on rotten fruit, compost heaps and other bacterial-rich environments, not soil. "All Caenorhabditis species are colonizers of nutrient- and bacteria-rich substrates and none of them is a true soil nematode." from Kiontke, K. and Sudhaus, W. Ecology of Caenorhabditis species (WormBook).

Your specific comment about C. elegans’ habitat is well received. However, in that sentence we are referring to the chemosensory system of soil-dwelling animals in general, and not particularly C. elegans.

(2) Figure 3K, the model would be clearer if leucine-rich diet -> volatile chemicals ->AWC (instead of leucine-rich diet -> AWC <- volatile chemicals). The leucine-rich diet results in the production of volatile chemicals which are detected by AWC.

We have modified the figure to make it clearer.

(3) Figure 4 - it would help to include a table summarizing the volatile chemicals that each bacteria releases. Then the reader could more easily evaluate whether the adaptation to each specific odor is consistent with the change in preference for the specific bacteria based on what it releases in its headspace. In addition, Figure 4 would help to clarify whether bacteria in these experiments were cultured with or without leucine supplementation.

Table S2 summarizes the odors released by all the bacteria under + LEU and – LEU conditions.

In Figure 4, adaptation was performed by odors of bacteria when cultured under leucineunsupplemented conditions.

Reviewer #2 (Recommendations for the authors):

Page 9. Previous studies e.g. Bargmann Hartwieg and Horvitz have shown IAA is sensed by the AWC. Would be good to cite appropriately.

Thanks for the comment. The reference has been cited at p9 and p16.

References:

(1) Yuan, J., Mishra, P., and Ching, C.B. (2017). Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae. Journal of Industrial Microbiology and Biotechnology 44, 107-117. 10.1007/s10295-016-1855-2 %J Journal of Industrial Microbiology and Biotechnology.

(2) Kanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y., and Ishiguro-Watanabe, M. (2025). KEGG: biological systems database as a model of the real world. Nucleic Acids Res 53, D672-d677. 10.1093/nar/gkae909.

(3) Shingai, R., Wakabayashi, T., Sakata, K., and Matsuura, T. (2005). Chemotaxis of Caenorhabditis elegans during simultaneous presentation of two water-soluble attractants, llysine and chloride ions. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 142, 308-317. 10.1016/j.cbpa.2005.07.010.

(4) Dirksen, P., Assié, A., Zimmermann, J., Zhang, F., Tietje, A.M., Marsh, S.A., Félix, M.A., Shapira, M., Kaleta, C., Schulenburg, H., and Samuel, B.S. (2020). CeMbio - The Caenorhabditis elegans Microbiome Resource. G3 (Bethesda, Md.) 10, 3025-3039. 10.1534/g3.120.401309.

(5) Edwards, C., Canfield, J., Copes, N., Brito, A., Rehan, M., Lipps, D., Brunquell, J., Westerheide, S.D., and Bradshaw, P.C. (2015). Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans. BMC genetics 16, 8. 10.1186/s12863-015-0167-2.

(6) Wang, H., Wang, J., Zhang, Z.J.J.o.F., and Research, N. (2018). Leucine Exerts Lifespan Extension and Improvement in Three Types of Stress Resistance (Thermotolerance, AntiOxidation and Anti-UV Irradiation) in C. elegans. 6, 665-673.

(7) Crombie, T.A., McKeown, R., Moya, N.D., Evans, Kathryn S., Widmayer, Samuel J., LaGrassa, V., Roman, N., Tursunova, O., Zhang, G., Gibson, Sophia B., et al. (2023). CaeNDR, the Caenorhabditis Natural Diversity Resource. Nucleic Acids Research 52, D850-D858. 10.1093/nar/gkad887 %J Nucleic Acids Research.

(8) Cook, D.E., Zdraljevic, S., Roberts, J.P., and Andersen, E.C. (2017). CeNDR, the Caenorhabditis elegans natural diversity resource. Nucleic Acids Res 45, D650-d657. 10.1093/nar/gkw893.

How is it possible that it is virtually impossible to sneak an invalid proof ?

R.A Is the study of the interrelationships of each system, and how each nerve, muscle, blood vessel interact in order to

And just like in the Western zodiac, all 12 signs in the Chinese zodiac have an emoji representation as well, all of which were standardized by Unicode in 2010 to fill in gaps in some of the earliest Japanese emoji sets. These emojis are:

🐀 Rat (or 🐭 Mouse Face or 🐁 Mouse) 🐂 Ox (or 🐃 Water Buffalo) 🐅 Tiger (or 🐯 Tiger Face) 🐇 Rabbit (or 🐰 Rabbit Face) 🐉 Dragon (or 🐲 Dragon Face) 🐍 Snake 🐎 Horse (or 🐴Horse Face) 🐐 Goat (or 🐑 Ewe or 🐏 Ram) 🐒 Monkey (or 🐵 Monkey Face) 🐓 Rooster (or 🐔 Chicken) 🐕 Dog (or 🐶 Dog Face) 🐖 Pig (or 🐷 Pig Face or 🐗 Boar) No matter what zodiac you turn to for gaining insights about the world, yourself, and the unfolding of your life, emojis are excellent tools for adding some personality, flair, and meaning to the experiences of every season, sign, and symbol.

eLife Assessment

This important study characterized and identified clonal MSC populations from human synovium. The authors provide convincing evidence that clonal MSC populations can be isolated and expanded from both normal and osteoarthritic synovium and that CD47 represents a potential marker for improved chondrogenic potential of MSC sub-populations. These findings could provide new avenues for osteoarthritis treatment in the future and deeper mechanistic understanding of the factors involved in the repair.

Reviewer #1 (Public review):

Summary:

This work by Al-Jezani et al. focused on characterizing clonally derived MSC populations from the synovium of normal and osteoarthritis (OA) patients. This included characterizing the cell surface marker expression in situ (at time of isolation), as well as after in vitro expansion. The group also tried to correlate marker expression with trilineage differential potential. They also tested the ability of the different sub-populations for their efficacy in repairing cartilage in a rat model of OA. The main finding of the study is that CD47hi MSCs may have a greater capacity to repair cartilage than CD47lo MSCs, suggesting that CD47 may be a novel marker of human MSCs that have enhanced chondrogenic potential.

Strengths:

Studies on cell characterization of the different clonal populations isolated indicate that the MSC are heterogenous and traditional cell surface markers for MSCs do not accurately predict the differentiation potential of MSCs. While this has been previously established in the field of MSC therapy, the authors did attempt to characterize clones derived from single cells, as well as evaluate the marker profile at the time of isolation. While the outcome of heterogeneity is not surprising, the methods used to isolate and characterze the cells were well developed. The interesting finding of the study is the identification of CD47 as a potential MSC marker that could be related to chondrogenic potential. The authors suggest that MSCs with high CD47 repaired cartilage more effectively than MSC with low CD47 in a rat OA model.

Comments on revisions:

Thank you for addressing the comments from the first review. No additional revisions.

Reviewer #2 (Public review):

Summary:

This is a compelling study that systematically characterized and identified clonal MSC populations derived from normal and osteoarthritis human synovium. There is immense growth in the focus on synovial-derived progenitors in the context of both disease mechanisms and potential treatment approaches, and the authors sought to understand the regenerative potential of synovial-derived MSCs.

Strengths:

This study has multiple strengths. MSC cultures were established from an impressive number of human subjects, and rigorous cell surface protein analyses were conducted, at both pre-culture and post-culture timepoints. In vivo experiments using a rat DMM model showed beneficial therapeutic effects of MSCs vs non-MSCs, with compelling data demonstrating that only "real" MSC clones incorporate into cartilage repair tissue and express Prg4. Proteomics analysis was performed to characterize non-MSC vs MSC cultures, and high CD47 expression was identified as a marker for MSC. Injection of CD47-Hi vs CD47-Low cells in the same rat DMM model also demonstrated beneficial effects, albeit only based on histology. A major strength of these studies is the direct translational opportunity for novel MSC-based therapeutic interventions, with high potential for a "personalized medicine" approach.

Weaknesses:

Weaknesses of this study include the rather cursory assessment of the OA phenotype in the rat model, confined entirely to histology (i.e. no microCT, no pain/behavioral assessments, no molecular readouts). This is relevant given the mixed results in therapeutic experiments demonstrating lower OA scores, but not lower inflammation scores, in CD47-Hi-treated rats. Thus, future work should focus on characterizing the therapeutic mechanism further given the clinical relevant of inflammation and pain in OA. It is somewhat unclear how the authors converged on CD47 vs other factors, but despite its somewhat broad profile, it was shown to be a useful marker to differentiate functional effects of MSCs. Additional work is needed to understand whether MSCs also engraft in ectopic cartilage (in the context of osteophyte/chondrophyte formation) or whether their effects are limited to articular cartilage. Despite these areas for improvement, this is a strong paper with a high degree of rigor, and the results are compelling, timely, and important.

Overall, the authors achieved their aims, and the results support not just the therapeutic value of clonally-isolated synovial MSCs but also the immense heterogeneity in stromal cell populations (containing true MSCs and non-MSCs) that must be investigated further. Of note, the authors employed the ISCT criteria to characterize MSCs, with mixed results in pre-culture and post-culture assessments. This work is likely to have a long-term impact on methodologies used to culture and study MSCs, in addition to advancing the field's knowledge about how synovial-derived progenitors contribute to cartilage repair in vivo.

Comments on revisions:

I commend the authors for a good revision. While the revision primarily entailed re-analysis or additional analysis of existing data, as well as text-based changes, it improved the clarity and completeness of the manuscript.

I do encourage the authors to expand their phenotyping assessments in future studies given that the interaction between structural disease, inflammation, and pain is complex, and our understanding of how the two interact and affect each other is evolving. There are multiple recent publications that show that a therapeutic or knock-out is protective against cartilage damage but doesn't alleviate pain, or vice versa. Thus, as a field, understanding which therapies target which pathological manifestations is an important next step to advance treatments. I also look forward to the follow-up studies on the MSC's role in ectopic cartilage.

Author response:

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

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

This work by Al-Jezani et al. focused on characterizing clonally derived MSC populations from the synovium of normal and osteoarthritis (OA) patients. This included characterizing the cell surface marker expression in situ (at time of isolation), as well as after in vitro expansion. The group also tried to correlate marker expression with trilineage differential potential. They also tested the ability of the different subpopulations for their efficacy in repairing cartilage in a rat model of OA. The main finding of the study is that CD47hi MSCs may have a greater capacity to repair cartilage than CD47lo MSCs, suggesting that CD47 may be a novel marker of human MSCs that have enhanced chondrogenic potential. 

Strengths: 

Studies on cell characterization of the different clonal populations isolated indicate that the MSC are heterogenous and traditional cell surface markers for MSCs do not accurately predict the differentiation potential of MSCs. While this has been previously established in the field of MSC therapy, the authors did attempt to characterize clones derived from single cells, as well as evaluate the marker profile at the time of isolation. While the outcome of heterogeneity is not surprising, the methods used to isolate and characterize the cells were well developed. The interesting finding of the study is the identification of CD47 as a potential MSC marker that could be related to chondrogenic potential. The authors suggest that MSCs with high CD47 repaired cartilage more effectively than MSC with low CD47 in a rat OA model. 

Weaknesses: 

While the identification of CD47 as a novel MSC marker could be important to the field of cell therapy and cartilage regeneration, there was a lack of robust data to support the correlation of CD47 expression to chondrogenesis. The authors indicated that the proteomics suggested that the MSC subtype expressed significantly more CD47 than the non-MSC subtype. However, it was difficult to appreciate where this was shown. It would be helpful to clearly identify where in the figure this is shown, especially since it is the key result of the study. The authors were able to isolate CD47hi and CD47 low cells. While this is exciting, it was unclear how many cells could be isolated and whether they needed to be expanded before being used in vivo. Additional details for the CD47 studies would have strengthened the paper. Furthermore, the CD47hi cells were not thoroughly characterized in vitro, particularly for in vitro chondrogenesis. More importantly, the in vivo study where the CD47hi and CD47lo MSCs were injected into a rat model of OA lacked experimental details regarding how many cells were injected and how they were labeled. No representative histology was presented and there did not seem to be a statistically significant difference between the OARSI score of the saline injected and MSC injected groups. The repair tissue was stained for Sox9 expression, which is an important marker of chondrogenesis but does not show production of cartilage. Expression of Collagen Type II would be needed to more robustly claim that CD47 is a marker of MSCs with enhanced repair potential. 

Reviewer #2 (Public review): 

Summary: 

This is a compelling study that systematically characterized and identified clonal MSC populations derived from normal and osteoarthritis human synovium. There is immense growth in the focus on synovial-derived progenitors in the context of both disease mechanisms and potential treatment approaches, and the authors sought to understand the regenerative potential of synovial-derived MSCs. 

Strengths: 

This study has multiple strengths. MSC cultures were established from an impressive number of human subjects, and rigorous cell surface protein analyses were conducted, at both pre-culture and post-culture timepoints. In vivo experiments using a rat DMM model showed beneficial therapeutic effects of MSCs vs non-MSCs, with compelling data demonstrating that only "real" MSC clones incorporate into cartilage repair tissue and express Prg4. Proteomics analysis was performed to characterize non-MSC vs MSC cultures, and high CD47 expression was identified as a marker for MSC. Injection of CD47-Hi vs CD47-Low cells in the same rat DMM model also demonstrated beneficial effects, albeit only based on histology. A major strength of these studies is the direct translational opportunity for novel MSC-based therapeutic interventions, with high potential for a "personalized medicine" approach. 

Weaknesses: 

Weaknesses of this study include the rather cursory assessment of the OA phenotype in the rat model, confined entirely to histology (i.e. no microCT, no pain/behavioral assessments, no molecular readouts). It is somewhat unclear how the authors converged on CD47 vs the other factors identified in the proteomics screen, and additional information is needed to understand whether true MSCs only engraft in articular cartilage or also in ectopic cartilage (in the context of osteophyte/chondrophyte formation). Some additional discussion and potential follow-up analyses focused on other cell surface markers recently described to identify synovial progenitors is also warranted. A conceptual weakness is the lack of discussion or consideration of the multiple recent studies demonstrating that DPP4+ PI16+ CD34+ stromal cells (i.e. the "universal fibroblasts") act as progenitors in all mesenchymal tissues, and their involvement in the joint is actively being investigated. Thus, it seems important to understand how the MSCs of the present study are related to these DPP4+ progenitors. Despite these areas for improvement, this is a strong paper with a high degree of rigor, and the results are compelling, timely, and important. 

Overall, the authors achieved their aims, and the results support not just the therapeutic value of clonally-isolated synovial MSCs but also the immense heterogeneity in stromal cell populations (containing true MSCs and non-MSCs) that must be investigated further. Of note, the authors employed the ISCT criteria to characterize MSCs, with mixed results in pre-culture and post-culture assessments. This work is likely to have a longterm impact on methodologies used to culture and study MSCs, in addition to advancing the field's knowledge about how synovial-derived progenitors contribute to cartilage repair in vivo.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors): 

In all figures, it would be beneficial to report the sample number used for the data reported. It is difficult to appreciate the statistical analysis without that information.

Understood, the sample number and replicates have been added to each figure legend.

Please check that Table S7 is part of the manuscript. It could not be found.

It was added as an additional excel file since it was too large to fit in the word document.

Lines 377-379 (Figure 2E): the authors write that rats receiving MSCs had a significantly lower OARSI and Krenn score vs. rats injected with non-MSCs. However, none of the bars indicating statistical significance run between these two groups. Please verify the text and figure.

This has been corrected

The details surrounding the labeling of the cells with tdTomato were not presented in the methods. 

This has been added to the methods

The fluorescent antibodies used should be listed and more details provided in the methods rather than a general statement that fluorescent antibodies were used.

Our apologies, the clones and companies have been added.

Additional information on the CD47 experiments (# cells, # animals) would have strengthened the study.

This has been added to the methods and figure legend.

Reviewer #2 (Recommendations for the authors): 

My comments span minor corrections, requests for additional analyses, some suggestions for additional experiments, and requests for additional discussion of recent important studies. 

Introduction: 

The introduction is thorough and well-written. I recommend a brief discussion about the emerging evidence demonstrating that DPP4+ PI16+ CD34+ synovial cells, i.e. the "universal fibroblasts", act as stromal progenitors in development, homeostasis, and disease. Relevant osteoarthritis-related papers encompass human and mouse studies (PMIDs: 39375009, 38266107, 38477740, 36175067, 36414376).

This has been added.

Relatedly, as DPP4 is CD26 and therefore useful as a cell-surface antigen for flow cytometry, sorting, etc, it would be interesting to understand the relationship and similarities between the CD47-High cells identified in this study and the DPP4/PI16+ cells previously described. Do they overlap in phenotype/identity?

We have added a new flow cytometry figure for address this question. 

Results: 

Note type-o on Line 311: "preformed" instead of "performed". Line 313 "prolife" instead of "profile"

Thank you for catching these.

The identified convergence of the cell surface marker profile of all normal and OA clones in culture is a highly intriguing result. Do the authors have stored aliquots of these cells to demonstrate whether this would also occur in soft substrate, i.e. low stiffness culture conditions? This could be done with standard dishes coated with bulk collagen or with commercially available low-stiffness dishes (1 kPa). This is relevant to multiple studies demonstrating the induction of a myofibroblast-like phenotype by stromal cells cultured on high-stiffness plastic or glass. This is also the experiment where assessment of DPP4/CD26 could be added, if possible.

While we agree it would be interesting to determine the mechanism by which the cells phenotypes converge, we would argue that it is outside of the scope of the current manuscript. We have instead added a sentence to the discussion. 

Line 353 regarding the use of CD68 as a negative gate: can the authors pleasecomment on why they employed CD68 here and not CD45? While monocytes/macs/myeloid cells are the most abundant immune cells in synovium, CD45 would more comprehensively exclude all immune cells. 

That is a fair point, and we really don’t have any reason to have picked CD68 over CD45. In our opinion either would be a fair negative marker to use based on the literature. 

Fig 2, minor suggestion: consider adding "MSC vs non-MSC" on the experimental schematic to more comprehensively summarize the experiment. 

This has been modified 

Fig 2E should show all individual datapoints, not just bar graphs. 

This has been modified

Fig 2: Given the significant reduction in Krenn score in DMM-MSC injected knees compared to DMM-saline knees, Fig 2 should also show representative images of the synovial phenotype to demonstrate which aspects of synovial pathology were mitigated. Was the effect related to lining hyperplasia, subsynovial infiltrate, fibrosis, etc? Similarly, can the authors narrate which aspects of the OARSI score drove the treatment effect (proteoglycans vs structure vs osteophytes, etc). 

We have added a new sup figure breaking down the Krenn score as well as higher magnification images of representative synovium.

Fig 2: In the absence of microCT imaging, can the authors quantify subchondral bone morphometrics using multiple histological sections? The tibial subchondral bone in Fig 2D appears protected from sclerosis/thickening.

Unfortunately, this is beyond what are able to add to the manuscript. 

The Fig 3 results are highly compelling and interesting. Congratulations.

Thank you very much.

Fig 4A: the cell highlighted in the high-mag zoom box in Fig 4A appears to be localized within the joint capsule or patellar tendon (it is unclear which anatomic region this image represents). The highly aligned nature of the tissue and cells along a fibrillar geometry indicates that this is not synovium. The interface between synovium and the tissue in question can be clearly observed in this image. Please choose an image more representative of synovium.

We completely agree with the reviewers assessment. However, it is the synovium that overlays this tissue (Fig 4A arrow). We are simply showing that there were very few MSCs that took up residence in the synovium or the adjacent tissues. 

Fig 4C and F: please show individual data points.

This has been added

Fig 5D: I see DPP4 and ITGA5 were also hits in the proteomics analysis, which is intriguing. Besides my comments/suggestions regarding DPP4 above, please note this recent paper identifying a ITGA5+ synovial fibroblast subset that orchestrates pathological crosstalk with lymphocytes in RA, PMID: 39486872

Thank you for the information. We have added the reference in the results section. 

Fig 5B-D: How did the authors converge on CD47 as the target for follow-up study? It does not appear to be a differentially-expressed protein based on the Volcano plot in Fig 5B, and it's unclear why it is a more important factor than any of the other proteins shown in the network diagram in Fig 5D, e.g. CTSL, ITGA5, DPP4. Can the authors add a quantitative plot supporting their statement "the MSC sub-type expressed significantly more CD47 than the non-MSCs" on Line 458? 

We have re-written this line. It was incorrect to discuss amount of CD47. That was shown later with the flow analysis.  

Fig 6D: Please show individual data points and also representative histology images to demonstrate the nature of the phenotypic effect.

This has been added. 

Fig 6E-F: In what anatomic region are these images? Please add anatomic markers to clarify the location and allow the reader to interpret whether this is articular cartilage or ectopic cartilage

We have redone the figure to show the area as requested.

Relevant to this, do the authors observe this type of cellular engraftment in ectopic cartilage/osteophytes or only in articular cartilage? Understanding the contribution of these cells to the formation/remodeling of various cartilage types in the context of OA is a critical aspect of this line of investigation.

We didn’t see any contribution of these cells to ectopic cartilage formation and are actively working on a follow up study discussing this point specifically. 

Discussion: 

Besides my comments regarding DPP4 and ITGA5 above, the authors may also consider discussing PMID: 37681409 (JCI Insight 2023), which demonstrates that adult Prg4+ progenitors derived from synovium contribute to articular cartilage repair in vivo. 

We agree that there are numerous markers we could look at in future studies and that other people in the field are actively studying.

Pathos appeals to emotions such as sadness, fear, or happiness. Writers use pathos to help the audience emotionally connect to the message and feel motivated to respond.

Logos focuses on logic, facts, and reasoning. Using evidence and clear explanations helps make an argument more convincing and reasonable to the audience.

Ethos is about credibility and trust. If the audience believes the writer is knowledgeable and trustworthy, they are more likely to accept the argument being made.

A rhetorical situation includes the writer, the audience, and the purpose. All three must work together for communication to be effective, and changing one affects how the message is understood.

Rhetoric is about persuading an audience through writing or speaking. This shows that communication is not just about sharing information, but about influencing how people think or act.

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

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

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary

In this paper, Wang and Shu et al. investigate the extent to which the negative binomial (NB) distribution captures the statistical properties of single-cell like count data and the effects of using this model to interpret biophysical parameters. Assuming an underlying telegraph model of transcription, they demonstrate how the NB can produce similar if not equivalent fits to simulated data from various parameter regimes, regimes which can, notably, fall outside of the bursty transcription limit in which the telegraph model is known to have a NB form. The authors then assess how model selection favors the NB or Poisson models over the underlying telegraph model, and how technical noise can lead to greater selection/representation of the NB over the parameter regime. Finally, they demonstrate how the broader applicability of the NB impacts inference of burst size and frequency (commonly inferred from NB fits on single-cell data), preserving relative rather than absolute information.

The authors use both method of moments and MLE-based approaches to obtain and compare model fits over the same parameter regimes. They also develop the aeBIC metric which balances parametric complexity and distributional similarity to the desired, ground truth distribution, to more quickly approximate the BIC (used for model selection).

Major comments:

The likelihood of model fits is used as a main criteria for model selection and comparison (e.g., in the BIC/aeBIC metrics), however it is possible that analysis of the curvature of the likelihood may suggest greater uncertainty/less information about parameter estimates for the different statistical models across the transcriptional regimes tested. Since a major component of this study is to demonstrate to readers that nuanced model selection is important for interpreting single-cell data, it would support these efforts to see if the telegraph versus NB model fits, for example, demonstrate differences in their respective Hessian matrices for the MLE estimates. This would help determine, for those interested in comparing these fits on their data, if there is potential here to distinguish the more optimal/true model or not (i.e., what the extent of the limitations are). The authors describe how in the infinite limit of N_sigma the NB and telegraph models converge to the same distribution, which provides another biological scenario outside transcriptional bursting where the NB can be interpreted as a good statistical model. However, though many parameter regimes are possible not all are observed in real data. Thus for readers to understand how likely these regimes are to be present in the data it would be helpful to discuss in what biological scenarios such a limit may appear and if it is likely to be a common instance, etc (perhaps given the ranges of on/off times observed in the literature https://pmc.ncbi.nlm.nih.gov/articles/PMC10860890/). This would parallel the discussion in the study on the bursty transcription model, often described in the literature as a widespread phenomenon. The p_cap parameter is described as representing technical capture and affords the conclusion in the Discussion that the NB can improve capture of technical noise beyond the biological noise in the system. However, as mentioned later in the Discussion, this effect could also arise from cell to cell differences in transcription rate (extrinsic, biological noise), which cannot be distinguished in this model. This point should be made clearer earlier on, as without use of control genes/spike-ins/etc we cannot distinguish the biological and technical components encompassed by the p_cap term (i.e., whether or not a spread in total UMIs observed over droplets is due to biological or technical capture differences). Since the aeBIC is being presented as a new, faster method in this study, the timing and memory usage in performing these calculations, for each model, should be presented somewhere. The Methods should also have a more explicit description of the steps/tools used to calculate the aeBIC.

Minor comments:

Figure S4 mentioned comparison of scRNA-seq with smFISH data to approximate p_cap, however given that smFISH data would have its own technical biases it does not seem exactly clear how a map from smFISH to scRNA-seq would work such as to illuminate the gap incurred by technical bias/capture. Perhaps previous literature/methods doing this can be cited here, or this idea can be fleshed out in the Discussion text for readers interested in better estimating p_cap. In Figure 4 the pink color of the Poisson in c is hard to see, and it may be easier to write the names of the different models in the respective regions that they cover (similarly in Figure 5 c) For Figure 8, it may be easier for the reader to interpret the several plots in a row by repeating the x-axis labels under each set of plots and collating all the legend labels into one box somewhere near the first plots.

Significance

General assessment: Overall, the paper is a clear and concise view on the use of the NB in analysis of sparse, transcriptomic count data, the potential effects of technical and biological noise on the pertinence of the NB as the statistical representation, and the impacts on user interpretation of biophysical parameters from these model fits. This study is useful for both biologists and computational scientists looking to gain mechanistic insight from single-cell data.

The strength of the paper is that the methodology is straightforward and uses simple numerical experiments to demonstrate how and when several common distributions can describe the type of data we encounter in single-cell genomics. They additionally connect these results to common biological interpretations from single-cell measurements and outline regimes in which inferences are likely to be incorrect.

The paper could benefit from more discussion on the biological interpretations of the findings and regimes analyzed, particularly to help readers interested in how this impacts their data analysis. Supplemental analysis on whether other criteria could potentially distinguish the models in question would also help support the conclusions of model selection/identifiability and if other properties of these model fits can be used for selection or not.

Advance: The study builds on others in the field by not just fitting several common models to this type of sparse, transcriptomic count data but also describing why these overlapping fits arise and how that affects biological interpretation. Often the focus is more on choosing a sufficient statistical representation without the underlying, mechanistic connections between the models. The results here are thus more technical and mechanistic in nature, describing both the theoretical connections between common single-cell count models and their biophysical interpretations.

Audience: This result is likely to be of interest to scientists performing data analysis and method development in single-cell genomics, particularly with mechanistic insight in mind. This would be more of interest within the domain of transcriptomics, but it also presents a methodology for studying limitations of identifiability in noisy systems which could be of interest to other biological domains.

My expertise is in developing representation learning methods and stochastic models of transcription for single-cell biology, which covers the classical models described in this study.

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

The study is generally well reasoned and thorough, and should be of interest to the community. My only critique relates to the treatment of extrinsic noise and the related discussion: Many studies have concluded that extrinsic noise (e.g., cell-to-cell variability in the transcription rate) is a larger contribution to noise in gene expression than intrinsic noise. (For example, see the seminal review by Raj and van Oudenaarden (PMID: 18957198) and early examples such as Raser and O'Shea: Raser JM, O'Shea EK. Science. 2004;304:1811. doi: 10.1126/science.1098641). For this reason, one must be careful in assuming that the telegraph model by itself fully captures biological variability. I believe this point could be more clearly made in the paper. Did the authors treat a case in which the gene undergoes state switching, but where there is also a significant contribution of extrinsic noise, for example, through variability in the transcription rate and/or other papers? I could not tell for sure if this was explicitly studied. This would be an important scenario to study, because it may be the most likely. I would have thought that this is the most biologically realistic scenario (i.e., strong contributions of both intrinsic and extrinsic noise, along with state switching). My prior assumption has been that the NB model is often empirically indicated because it somehow well captures this combination of intrinsic (including state switching) + extrinsic noise. Could the authors comment on whether this assumption is consistent with their findings? (Neither Case I or Case II in the manuscript captures this scenario). Related to the treatment of extrinsic noise, I was confused by this sentence: "Any variation in the effective transcription rate due to variability in the transcription rate (extrinsic noise on the transcription rate) between cells is indistinguishable from variability in the transcript capture probability and hence is automatically accounted for in our present method. " But doesn't the distribution of transcription rates vary significantly, depending on whether the variation comes from technical noise versus extrinsic biological variability? For example, one source of extrinsic biological variability is differences in RNA polymerase concentrations in different cells. Wouldn't one need to know what kind of distribution to use to capture these effects? In this case, I believe one would need to study various types of compound distributions, depending on the assumptions underlying the biological extrinsic variability.

Significance

This paper presents a thorough study of the conditions under which the negative binomial model of transcript distributions can map onto other widely used models, namely the telegraph model of stochastic gene expression. The study is generally well reasoned and thorough, and should be of interest to the community (namely: single cell transcriptomics community, bio mathematicians, biological noise community).

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

The authors present an investigation into the surprising effectiveness of the negative-binomial distribution in modelling transcript counts in single cell RNA sequencing experiments. With experimentally motivated ground-truth models that incorporate transcriptional bursting, they show that when transcription activity is large compared to degradation these distributions coincide. With a novel model selection metric, they indicate the regions of parameter space in which the negative-binomial model is a good approximation to the underlying true model. With this procedure, they also indicate that transcriptional burst parameters are unlikely to be reconstructed by an effective negative-binomial function, but that nevertheless, relative rankings between genes can be identified robustly.

I would like to commend the authors on an interesting and fairly comprehensive investigation on a topic of considerable importance in the interpretation of single cell RNA sequencing experiments, and on a well written paper. I have no major comments on issues that affect the conclusions of the paper, although I have a few minor suggestions that might aid reader's understanding of the results and their applicability.

General

It would be nice to have a comparison with some real data for the burst frequency and size, just to indicate to the reader how important these regions are compared to what might be measured. For example, if most genes are outside of the region that does not accommodate the NB distribution, then the conclusion is quite different than if most real counts are unlikely to accommodated by the NB.

Inter-cellular variability of transcription dynamics is quite a significant point of interest, so it would be good to have stated earlier that this is not considered, with the mitigation that is noted later. This is particularly important given that in the introduction, the cases mentioned seem to imply that an NB distribution would be more likely with higher inter-cellular variability.

Introduction

It would be nice to have a bit more detail here, for example on what UMIs are, and what the parameters of the NB distribution represent in general.

For smFISH, I would have thought that the more simple explanation is that the NB is often the simplest distribution with some overdispersion that fits the data, and the parameters don't necessarily need to be biologically interpretable?

It's noted later that the capture probability of modern RNASeq protocols can be ~0.3, which doesn't seem very different compared to 0.7-0.9 of smFISH, so some context here would be good.

Results

Eq 1: I don't think you lose anything by giving the Pochammer symbol and Kummer confluent geometric function explicitly here, and it would make it it a lot easier to read. That said, this equation also seems to come out of nowhere, so a reference would be nice.

I think the moment matching is reasonably convincing, but it might require a little more explicit motivation for a more general audience.

Thm. 1: Do these converge at similar rates, and if not, does that have any implications for the interpretation of the comparisons (as these are evaluated with specific values)? This might be worth a short comment.

Fig 3. In the description for this in the text, it would be nice to have an expression of the KL divergence (and what order the arguments are in), for anyone unfamiliar.

The discussion of the aeBIC seems a bit circuitous. A reasonable prior intention might be to average (or apply a voting function) to individual BIC values, rather than the aeBIC constructed here. And in fact the text goes on to note after the description that this is a good estimate of the expectation of the BIC after all, with some computational advantages. So it might be better to have a more straightforward presentation where this is proposed as an approximation to the expectation of the BIC in the first place.

Section 2.4: The intro to this section could do with a bit more background of the capture, PCR, sequencing, etc, stages, and what exactly the data generated here represents. Otherwise the discussion of zero inflation and UMIs is a little confusing.

It would also be nice to have a comment here on the effect of sequencing depth, or similar (compared to capture probability), even if this wouldn't change the interpretation.

Significance

The paper provides novel arguments towards the support of the negative-binomial distribution in describing single cell RNA sequencing data, with particular relevance to transcriptional bursting observed in numerous datasets. The paper follows from some notable prior work in the field, and integrates these into a more consistent description, particularly in relation to newer techniques such as UMIs.

The ubiquity of the negative-binomial distribution means that these arguments will be of relevance to those that perform theoretical or statistical modelling of single cell RNA sequencing data, and theoretically justifies many widely held assumptions. However, the paper does not make any reference to specific reference datasets or commonly observed values, so where in the parameter space data likely lies would still need to be evaluated on a case-by-case basis.

My expertise is in mathematical modelling and statistics, with some experience of the analysis of single cell RNA sequencing data.

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

The PDF version of point-by-point response includes figures (I, II, III,... IX) that are not included in the manuscript nor in this post but serve to illustrate and clarify our replies to the reviewers' comments.

Dear Editor,

Many thanks for forwarding the comments from reviewers #1-#4 regarding our manuscript (Preprint #RC-2025-03087144), entitled "HIV-1 Envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane", by Quijada-Freire A. et al.

We have carefully reviewed all reviewer comments and prepared our specific, detailed responses. Alongside this, we have created a revised version of the manuscript to post them on BioRxiv, and we are pleased to announce that we will transfer this new version to an affiliate journal for consideration.

Reviewer #1

Thank you very much for considering that our manuscript evaluates an important question and that the reagents used are well prepared and characterized. We also much appreciate that you consider the information generated as potentially useful for those studying HIV infection processes and strategies to prevent infection.

• While a single particle tracking routine was applied to the data, it's not clear how the signal from a single GFP was defined and if movement during the 100 ms acquisition time impacts this. My concern would be that the routine is tracking fluctuations, and these are related to single particle dynamics, it appears from the movies that the density or the GFP tagged receptors in the cells is too high to allow clear tracking of single molecules. SPT with GFP is very difficult due to bleaching and relatively low quantum yield. Current efforts in this direction that are more successful include using SNAP tags with very photostable organic fluorophores. The data likely does mean something is happening with the receptor, but they need to be more conservative about the interpretation. *

Some of the paradoxical effects might be better understood through deeper analysis of the SPT data, particularly investigation of active transport and more detailed analysis of "immobile" objects. Comments on early figures illustrate how this could be approached. This would require selecting acquisitions where the GFP density is low enough for SPT and performing a more detailed analysis, but this may be difficult to do with GFP.

When the authors discuss clusters of 3, how do they calibrate the value of GFP and the impact of diffusion on the measurement. One way to approach this might be single molecules measurements of dilute samples on glass vs in a supported lipid bilayer to map the streams of true immobility to diffusion at >1 µm2/sec.

We fully understand the reviewer's apprehensions regarding the application of these high-end biophysical techniques, in particular the associated complexity of the data analysis. We provide below extensive explanations on our methodology, which we hope will satisfactorily address all of the reviewer's concerns.

We would first like to emphasize that the experimental conditions and the quantitative analysis used in our current experiments are similar to the established protocols and methodologies applied by our group previously (Martinez-Muñoz et al. Mol. Cell, 2018; García-Cuesta et al. PNAS, 2022; Gardeta et al. Frontiers in Immunol., 2022; García-Cuesta et al.eLife, 2024; Gardeta et al. Cell. Commun. Signal., 2025) and by others (Calebiro et al. PNAS, 2013; Jaqaman et al. Cell,2011; Mattila et al. Immunity, 2013; Torreno-Pina et al. PNAS, 2014; Torreno-Pina et al. PNAS, 2016).

As SPT (single-particle tracking) experiments require low-expressing conditions in order to follow individual trajectories (Manzo & García-Parajo Rep. Prog. Phys., 2015), we transiently transfected Jurkat CD4+ cells with CXCR4-AcGFP or CXCR4R334X-AcGFP. At 24 h post-transfection, cells expressing low CXCR4-AcGFP levels were selected by a MoFlo Astrios Cell Sorter (Beckman-Coulter) to ensure optimal conditions for SPT. Using Dako Qifikit (DakoCytomation), we quantified the number of CXCR4 receptors and found ∼8,500 - 22,000 CXCR4-AcGFP receptors/cell, which correspond to a particle density ∼2 - 4.5 particles/mm2 (Figure I, only for review purposes) and are similar to the expression levels found in primary human lymphocytes.

These cells were resuspended in RPMI supplemented with 2% FBS, NaPyr and L-glutamine and plated on 96-well plates for at least 2 h. Cells were centrifuged and resuspended in a buffer with HBSS, 25 mM HEPES, 2% FBS (pH 7.3) and plated on glass-bottomed microwell dishes (MatTek Corp.) coated with fibronectin (FN) (Sigma-Aldrich, 20 mg/ml, 1 h, 37{degree sign}C). To observe the effect of the ligand, we coated dishes with FN + CXCL12; FN + X4-gp120 or FN + VLPs, as described in material and methods; cells were incubated (20 min, 37{degree sign}C, 5% CO2) before image acquisition.

For SPT measurements, we use a total internal reflection fluorescence (TIRF) microscope (Leica AM TIRF inverted) equipped with an EM-CCD camera (Andor DU 885-CS0-#10-VP), a 100x oil-immersion objective (HCX PL APO 100x/1.46 NA) and a 488-nm diode laser. The microscope was equipped with incubator and temperature control units; experiments were performed at 37{degree sign}C with 5% CO2. To minimize photobleaching effects before image acquisition, cells were located and focused using the bright field, and a fine focus adjustment in TIRF mode was made at 5% laser power, an intensity insufficient for single-particle detection that ensures negligible photobleaching. Image sequences of individual particles (500 frames) were acquired at 49% laser power with a frame rate of 10 Hz (100 ms/frame). The penetration depth of the evanescent field used was 90 nm.

We performed automatic tracking of individual particles using a very well established and common algorithm first described by Jaqaman (Jaqaman et al. Nat. Methods, 2008). Nevertheless, we would stress that we implemented this algorithm in a supervised fashion, i.e., we visually inspect each individual trajectory reconstruction in a separate window. Indeed, this algorithm is not able to quantify merging or splitting events.

We follow each individual fluorescence spot frame-by-frame using a three-by-three matrix around the centroid position of the spot, as it diffuses on the cell membrane. To minimize the effect of photon fluctuations, we averaged the intensity over 20 frames. Nevertheless, to assure the reviewer that most of the single molecule traces last for at least 50 frames (i.e., 5 seconds), we provide the following data and arguments. We currently measure the photobleaching times from individual CD86-AcGFP spots exclusively having one single photobleaching step to guarantee that we are looking at individual CD86-AcGFP molecules. The distribution of the photobleaching times is shown below (Figure II, only for review purposes). Fitting of the distribution to a single exponential decay renders a t0 value of ~5 s. Thus, with 20 frames averaging, we are essentially measuring the whole population of monomers in our experiments. As the survival time of a molecule before photobleaching will strongly depend on the excitation conditions, we used low excitation conditions (2 mW laser power, which corresponds to an excitation power density of ~0.015 kW/cm2 considering the illumination region) and longer integration times (100 ms/frame) to increase the signal-to-background for single GFP detection while minimizing photobleaching.

To infer the stoichiometry of receptor complexes, we also perform single-step photobleaching analysis of the TIRF trajectories to establish the existence of different populations of monomers, dimers, trimers and nanoclusters and extract their percentage. Some representative trajectories of CXCR4-AcGFP with the number of steps detected are shown in new Supplementary Figure 1.

The emitted fluorescence (arbitrary units, a.u.) of each spot in the cells is quantified and normalized to the intensity emitted by monomeric CD86-AcGFP spots that strictly showed a single photobleaching step (Dorsch et al. Nat. Methods,2009). We have preferred to use CD86-AcGFP in cells rather than AcGFP on glass to exclude any potential effect on the different photodynamics exhibited by AcGFP when bound directly to glass. We have also previously shown pharmacological controls to exclude CXCL12-mediated receptor clustering due to internalization processes (Martinez-Muñoz et al. Mol. Cell, 2018) that, together with the evaluation of single photobleaching steps and intensity histograms, allow us to exclude the presence of vesicles in our data. Thus, the dimers, trimers and nanoclusters found in our data do correspond to CXCR4 molecules on the cell surface. Finally, distribution of monomeric particle intensities, obtained from the photobleaching analysis, was analyzed by Gaussian fitting, rendering a mean value of 980 {plus minus} 86 a.u. This value was then used as the monomer reference to estimate the number of receptors per particle in both cases, CXCR4-AcGFP and CXCR4R334X-AcGFP (new Supplementary Figure 1).

• I understand that the CXCL12 or gp120 are attached to the substrate with fibronectin for adhesion. I'm less clear how how that VLPs are integrated. Were these added to cells already attached to FN?*

For TIRF-M experiments, cells were adhered to glass-bottomed microwell dishes coated with fibronectin, fibronectin + CXCL12, fibronectin + X4-gp120, or fibronectin + VLPs. As for CXCL12 and X4-gp120, the VLPs were attached to fibronectin taking advantage of electrostatic interactions. To clarify the integration of the VLPs in these assays, we have stained the microwell dishes coated with fibronectin and those coated with fibronectin + VLPs with wheat germ agglutinin (WGA) coupled to Alexa647 (Figure III, only for review purposes) and evaluated the staining by confocal microscopy. These results indicate the presence of carbohydrates on the VLPs and are, therefore, indicative of the presence of VLPs on the fibronectin layer.

Moreover, it is important to remark that the effect of the VLPs on CXCR4 behavior at the cell surface observed by TIRF-M confirmed that the VLPs remained attached to the substrate during the experiment.

• Fig 1A- The classification of particle tracks into mobile and immobile is overly simplistic description that goes back to bulk FRAP measurements and it not really applicable to single molecule tracking data, where it's rare to see anything that is immobile and alive. An alternative classification strategy uses sub-diffusion, normal diffusion and active diffusion (or active transport) to descriptions and particles can transition between these classes over the tracking period. Fig 1B- this data might be better displayed as histograms showing distributions within the different movement classes.*

In agreement with the reviewer's commentary, the majority of the particles detected in our TIRF-M experiments were indeed mobile. However, we also detected a variable, and biologically appreciable, percentage of immobile particles depending on the experimental condition analyzed (Figure 1A in the main manuscript). To establish a stringent threshold for identifying these immobile particles under our specific experimental conditions, we used purified monomeric AcGFP proteins immobilized on glass coverslips. Our analysis demonstrated that 95% of these immobilized proteins showed a diffusion coefficient £0.0015 mm2/s; consequently, this value was established as the cutoff to distinguish immobile from mobile trajectories. While the observation of truly immobile entities in a dynamic, living system is rare, the presence of these particles under our conditions is biologically significant. For instance, the detection of large, immobile receptor nanoclusters at the plasma membrane is entirely consistent with facilitating key cellular processes, such as enabling the robust signaling cascade triggered by ligand binding or promoting the crucial events required for efficient viral entry into the cells.

Regarding the mobile receptors (defined as those with D1-4 values exceeding 0.0015 mm2/s), we observed distinct diffusion profiles derived from mean square displacement (MSD) plots (Figure V) (Manzo & García-Parajo Rep. Prog. Phys., 2015), which were further classified based on motion, using the moment scaling spectrum (MSS) (Ewers et al. PNAS, 2005). Under all experimental conditions, the majority of mobile particles, ∼85%, showed confined diffusion: for example under basal conditions, without ligand addition, ∼90% of mobile particles showed confined diffusion, ∼8.5% showed Brownian-free diffusion and ∼1.5% exhibited directed motion (new Supplementary Figure 5A in the main manuscript). These data have been also included in the revised manuscript to show, in detail, the dynamic parameters of CXCR4.

Due to the space constraints, it is very difficult to include all the figures generated. However, to ensure comprehensive assessment and transparency (for the purpose of this review), we have included below representative plots of the MSD values as a function of time from individual trajectories, showing different types of motion obtained in our experiments (Figure IV, only for review purposes).

• Fig 1C,D- It would be helpful to see a plot of D vs MSI at a single particle level. In comparing C and D I'm surprised there is not a larger difference between CXCL12 and X4-gp120. It would also be very important to see the behaviour of X4-gp120 on the CXCR4 deficient Jurkat that would provide a picture of CD4 diffusion. The CXCR4 nanoclustering related to the X4-gp120 could be dominated by CD4 behaviour.*

As previously described, all analyses were performed under SPT conditions (see previous response to point 1 in this reply). Figure 1C details the percentage of oligomers (>3 receptors/particle) calibrated using Jurkat CD4+ cells electroporated with monomeric CD86-AcGFP (Dorsch et al. Nat. Methods, 2009). The monomer value was determined by analyzing photobleaching steps as described in our previous response to point 1.

In our experiments, we observed a trend towards a higher number of oligomers upon activation with CXCL12 compared with X4-gp120. This trend was further supported by measurements of Mean Spot Intensity. However, the values are also influenced by the number of larger spots, which represents a minor fraction of the total spots detected.

The differences between the effect triggered by CXCL12 or X4-gp120 might also be attributed to a combination of factors related to differences in ligand concentration, their structure, and even to the technical requirements of TIRF-M. Both ligands are in contact with the substrate (fibronectin) and the specific nature of this interaction may differ between both ligands and influence their accessibility to CXCR4. Moreover, the requirement of the prior binding of gp120 to CD4 before CXCR4 engagement, in contrast to the direct binding of CXCL12 to CXCR4, might also contribute to the differences observed.

We previously reported that CXCL12-mediated CXCR4 dynamics are modulated by CD4 co-expression (Martinez-Muñoz et al. Mol. Cell, 2018). We have now detected the formation of CD4 heterodimers with both CXCR4 and CXCR4R334X, and found that these conformations are influenced by gp120-VLPs. In the present manuscript, we did not focus on CD4 clustering as it has been extensively characterized previously (Barrero-Villar et al. J. Cell Sci., 2009; Jiménez-Baranda et al. Nat. Cell. Biol., 2007; Yuan et al. Viruses, 2021). Regarding the investigation of the effects of X4-gp120 on CXCR4-deficient Jurkat cells, which would provide a picture of CD4 diffusion, we would note that a previous report has already addressed this issue using single-molecule super-resolution imaging, and revealed that CD4 molecules on the cell membrane are predominantly found as individual molecules or small clusters of up to 4 molecules, and that the size and number of these clusters increases upon virus binding or gp120 activation (Yuan et al. Viruses, 2021).

• Fig S1D- This data is really interesting. However, if both the CD4 and the gp120 have his tags they need to be careful as poly-His tags can bind weakly to cells and increasing valency could generate some background. So, they should make the control is fair here. Ideally, using non-his tagged person of sCD4 and gp120 would be needed ideal or they need a His-tagged Fab binding to gp120 that doesn't induce CXCR4 binding.*

New Supplementary Figure 2D shows that X4-gp120 does not bind Daudi cells (these cells do not express CD4) in the absence of soluble CD4. While the reviewer is correct to state that both proteins contain a Histidine Tag, cell binding is only detected if X4-gp120 binds sCD4. Nonetheless, we have included in the revised Supplementary Figure 2D a control showing the negative binding of sCD4 to Daudi cells in the absence of X4-gp120. Altogether, these results confirm that only sCD4/X4-gp120 complexes bind these cells.

• Fig S4- Panel D needs a scale bar. I can't figure out what I'm being shown without this.*

Apologies. A scale bar has been included in this panel (new Supplementary Figure 6D).

Reviewer #2

• This study is well described in both the main text and figures. Introduction provides adequate background and cites the literature appropriately. Materials and Methods are detailed. Authors are careful in their interpretations, statistical comparisons, and include necessary controls in each experiment. The Discussion presents a reasonable interpretation of the results. Overall, there are no major weaknesses with this manuscript.*

We very much appreciate the positive comments of the reviewer regarding the broad interest and strength of our work.

• NL4-3deltaIN and immature HIV virions are found to have less associated gp120 relative to wild-type particles. It is not obvious why this is the case for the deltaIN particles or genetically immature particles. Can the authors provide possible explanations? (A prior paper was cited, Chojnacki et al Science, 2012 but can the current authors provide their own interpretation.)*

Our conclusion from the data is actually exactly the opposite. As shown in Figure 2D, the gp120 staining intensity was higher for NL4-3DIN particles (1,786 a.u.) than for gp120-VLPs (1,223 a.u.), indicating lower expression of Env proteins in the latter. Furthermore, analysis of gp120 intensity per particle (Figure 2E) confirmed that gp120-VLPs contained fewer gp120 molecules per particle than NL4-3DIN virions. These levels were comparable with, or even lower than, those observed in primary HIV-1 viruses (Zhu et al. Nature, 2006). This reduction was a direct consequence of the method used to generate the VLPs, as our goal was to produce viral particles with minimal gp120 content to prevent artifacts in receptor clustering that might occur using high levels of Env proteins in the VLPs to activate the receptors.

This misunderstanding may arise from the fact that we also compared Gag condensation and Env distribution on the surface of gp120-VLPs with those observed in genetically immature particles and integrase-defective NL4-3ΔIN virions, which served as controls. STED microscopy data revealed differences in Env distribution between gp120-VLPs and NL4-3ΔIN virions, supporting the classification of gp120-VLPs as mature particles (Figure 2 A,B).

Reviewer #3

We thank the reviewer for considering that our work offers new insights into the spatial organization of receptors during HIV-1 entry and infection and that the manuscript is well written, and the findings significant.

• For mechanistic basis of gp120-CXCR4 versus CXCL12-CXCR4 differences. Provide additional structural or biochemical evidence to support the claim that gp120 stabilises a distinct CXCR4 conformation compared to CXCL12. If feasible, include molecular modelling, mutagenesis, or cross-linking experiments to corroborate the proposed conformational differences.*

We appreciate the opportunity to clarify this point. The specific claim that gp120 stabilizes a conformation of CXCR4 that is distinct from the CXCL12-bound state was not explicitly stated in our manuscript, although we agree that our data strongly support this possibility. It is important to consider that CXCL12 binds directly to CXCR4, whereas gp120 requires prior sequential binding to CD4, and its subsequent interaction is with a CXCR4 molecule that is already forming part of the CD4/CXCR4 complex, as demonstrated by our FRET experiments and supported by previous studies (Zaitseva et al. J. Leuk. Biol., 2005; Busillo & Benovic Biochim. Biophys. Acta, 2007; Martínez-Muñoz et al. PNAS, 2014). This difference makes it inherently complex to compare the conformational changes induced by gp120 and CXCL12 on CXCR4.

However, our findings show that both stimuli induce oligomerization of CXCR4, a phenomenon not observed when mutant CXCR4R334X was exposed to the chemokine CXCL12 (García-Cuesta et al. PNAS, 2022).

1. CXCL12 induced oligomerization of CXCR4 but did not affect the dynamics of CXCR4R334X (Martinez-Muñoz et al. Mol. Cell, 2018; García-Cuesta et al. PNAS, 2022). By contrast, X4-gp120 and the corresponding VLPs-which require initial binding to CD4 to engage the chemokine receptor-stabilized oligomers of both CXCR4 and CXCR4R334X.

FRET analysis revealed distinct FRET50 values for CD4/CXCR4 (2.713) and CD4/CXCR4R334X (0.399) complexes, suggesting different conformations for each complex. Consistent with previous reports (Balabanian et al. Blood, 2005; Zmajkovicova et al. Front. Immunol., 2024; García-Cuesta et al. PNAS, 2022), the molecular mechanisms activated by CXCL12 are distinct when comparing CXCR4 with CXCR4R334X. For instance, CXCL12 induces internalization of CXCR4, but not of mutant CXCR4R334X. Conversely, X4-gp120 triggers approximately 25% internalization of both receptors. Similarly, CXCL12 does not promote CD4 internalization in cells co-expressing CXCR4 or CXCR4R334X, whereas X4-gp120 does, although CD4 internalization was significantly higher in cells co-expressing CXCR4.

These findings suggest that CD4 influences the conformation and the oligomerization state of both co-receptors. To further support this hypothesis, we have conducted new in silico molecular modeling of CD4 in complex with either CXCR4 or its mutant CXCR4R334X using AlphaFold 3.0 (Abramson et al. Nature, 2024). The server was provided with both sequences, and the interaction between the two molecules for each protein was requested. It produced a number of solutions, which were then analyzed using the software ChimeraX 1.10 (Meng et al. Protein Sci., 2023). CXCR4 and its mutant, CXCR4R334X bound to CD4, were superposed using one of the CD4 molecules from each complex, with the aim of comparing the spatial positioning of CD4 molecules when interacting with CXCR4.

As illustrated in Figure V (only for review purposes), the superposition of the CD4/CXCR4 complexes was complete. However, when CD4/CXCR4 complexes were superimposed with CD4/CXCR4R334X complexes using the same CD4 molecule as a reference, indicated by an arrow in the figure, a clear structural deviation became evident. The main structural difference detected was the positioning of the CD4 transmembrane domains when interacting with either the wild-type or mutant CXCR4. While in complexes with CXCR4, the angle formed by the lines connecting residues E416 at the C-terminus end of CD4 with N196 in CXCR4 was 12{degree sign}, for the CXCR4R334X complex, this angle increased to 24{degree sign}, resulting in a distinct orientation of the CD4 extracellular domain (Figure VI, only for review purposes).

To further analyze the models obtained, we employed PDBsum software (Laskowski & Thornton Protein Sci., 2021) to predict the CD4/CXCR4 interface residues. Data indicated that at least 50% of the interaction residues differed when the CD4/CXCR4 interaction surface was compared with that of the CD4/CXCR4R334X complex (Figure VII, only for review purposes). It is important to note that while some hydrogen bonds were present in both complex models, others were exclusive to one of them. For instance, whereas Cys394(CD4)-Tyr139 and Lys299(CD4)-Glu272 were present in both CD4/CXCR4 and CD4/CXCR4R334X complexes, the pairs Asn337(CD4)-Ser27(CXCR4R334X) and Lys325(CD4)-Asp26(CXCR4R334X) were only found in CD4/CXCR4R334X complexes.

These findings, which are consistent with our FRET results, suggest distinct interaction surfaces between CD4 and the two chemokine receptors. Overall, these results are compatible with differences in the spatial conformation adopted by these complexes.

• For Empty VLP effects on CXCR4 dynamics: Explore potential causes for the observed effects of Env-deficient VLPs. It's valuable to include additional controls such as particles from non-producer cells, lipid composition analysis, or blocking experiments to assess nonspecific interactions. *

As VLPs are complex entities, we thought that the relevant results should be obtained comparing the effects of Env(-) VLPs with gp120-VLPs. Therefore, we would first remark that regardless of the effect of Env(-) VLPs on CXCR4 dynamics, the most evident finding in this study is the strong effect of gp120-VLPs compared with control Env(-) VLPs. Nevertheless, regarding the effect of the Env(-) VLPs compared with medium, we propose several hypotheses. As several virions can be tethered to the cell surface via glycosaminoglycans (GAGs), we hypothesized that VLPs-GAGs interactions might indirectly influence the dynamics of CXCR4 and CXCR4R334X at the plasma membrane. Additionally, membrane fluidity is essential for receptor dynamics, therefore VLPs interactions with proteins, lipids or any other component of the cell membrane could also alter receptor behavior. It is well known that lipid rafts participate in the interaction of different viruses with target cells (Nayak & Hu Subcell. Biochem., 2004; Manes et al. Nat. Rev. Immunol., 2003; Rioethmullwer et al. Biochim. Biophys. Acta, 2006) and both the lipid composition and the presence of co-expressed proteins modulate ligand-mediated receptor oligomerization (Gardeta et al. Frontiers in Immunol., 2022; Gardeta et al. Cell. Commun. Signal., 2025). We have thus performed Raster Image Correlation Spectroscopy (RICS) analysis to assess membrane fluidity through membrane diffusion measurements on cells treated with Env(-) VLPs.

Jurkat cells were labeled with Di-4-ANEPPDHG and seeded on FN and on FN + VLPs prior to analysis by RICS on confocal microscopy. The results indicated no significant differences in membrane diffusion under the treatment tested, thereby discarding an effect of VLPs on overall membrane fluidity (Figure VIII, only for review purposes).

Nonetheless, these results do not rule out other non-specific interactions of Env(-) VLPs with membrane proteins that could affect receptor dynamics. For instance, it has been reported that C-type lectin DC-SIGN acts as an efficient docking site for HIV-1 (Cambi et al. J. Cell. Biol., 2004; Wu & KewalRamani Nat. Rev. Immunol., 2006). However, a detailed investigation of these possible mechanisms is beyond the scope of this manuscript.

• For Direct link between clustering and infection efficiency - Test whether disruption of CXCR4 clustering (e.g., using actin cytoskeleton inhibitors, membrane lipid perturbants, or clustering-deficient mutants) alters HIV-1 fusion or infection efficiency*.

Designing experiments using tools that disrupt receptor clustering by interacting with the receptors themselves is difficult and challenging, as these tools bind the receptor and can therefore alter parameters such as its conformation and/or its distribution at the cell membrane, as well as affect some cellular processes such as HIV-1 attachment and cell entry. Moreover, effects on actin polymerization or lipids dynamics can affect not only receptor clustering but also impact on other molecular mechanisms essential for efficient infection.

Many previous reports have, nonetheless, indirectly correlated receptor clustering with cell infection efficiency. Cholesterol plays a key role in the entry of several viruses. Its depletion in primary cells and cell lines has been shown to confer strong resistance to HIV-1-mediated syncytium formation and infection by both CXCR4- and CCR5-tropic viruses (Liao et al. AIDS Res. Hum. Retrovisruses, 2021). Moderate cholesterol depletion also reduces CXCL12-induced CXCR4 oligomerization and alters receptor dynamics (Gardeta et al. Cell. Commun. Signal., 2025). By restricting the lateral diffusion of CD4, sphingomyelinase treatment inhibits HIV-1 fusion (Finnegan et al. J. Virol., 2007). Depletion of sphingomyelins also disrupts CXCL12-mediated CXCR4 oligomerization and its lateral diffusion (Gardeta et al. Front Immunol., 2022). Additional reports highlight the role of actin polymerization at the viral entry site, which facilitates clustering of HIV-1 receptors, a crucial step for membrane fusion (Serrano et al. Biol. Cell., 2023). Blockade of actin dynamics by Latrunculin A treatment, a drug that sequesters actin monomers and prevents its polymerization, blocks CXCL12-induced CXCR4 dynamics and oligomerization (Martínez-Muñoz et al. Mol. Cell, 2018).

Altogether, these findings strongly support our hypothesis of a direct link between CXCR4 clustering and the efficiency of HIV-1 infection.

• CD4/CXCR4 co-endocytosis hypothesis - Support the proposed model with direct evidence from live-cell imaging or co-localization experiments during viral entry. Clarification is needed on whether internalization is simultaneous or sequential for CD4 and CXCR4.*

When referring to endocytosis of CD4 and CXCR4, we only hypothesized that HIV-1 might promote the internalization of both receptors either sequentially or simultaneously. The hypothesis was based in several findings:

1) Previous studies have suggested that HIV-1 glycoproteins can reduce CD4 and CXCR4 levels during HIV-1 entry (Choi et al. Virol. J., 2008; Geleziunas et al. FASEB J, 1994; Hubert et al. Eur. J. Immunol., 1995).

2) Receptor endocytosis has been proposed as a mechanism for HIV-1 entry (Daecke et al. J. Virol., 2005; Aggarwal et al.Traffick, 2017; Miyauchi et al. Cell, 2009; Carter et al. Virology, 2011).

3) Our data from cells activated with X4-gp120 demonstrated internalization of CD4 and chemokine receptors, which correlated with HIV-1 infection in PBMCs from WHIM patients and healthy donors.

4) CD4 and CXCR4 have been shown to co-localize in lipid rafts during HIV-1 infection (Manes et al. EMBO Rep., 2000; Popik et al. J. Virol., 2002)

5) Our FRET data demonstrated that CD4 and CXCR4 form heterocomplexes and that FRET efficiency increased after gp120-VLPs treatment.

We agree with the reviewer that further experiments are required to test this hypothesis, however, we believe that this is beyond the scope of the current manuscript.

Minor Comments:

• The conclusions rely solely on the HXB2 X4-tropic Env. It would strengthen the study to assess whether other X4 or dual-tropic strains induce similar receptor clustering and dynamics.*

The primary goal of our current study was to investigate the dynamics of the co-receptor CXCR4 during HIV-1 infection, motivated by previous reports showing CD4 oligomerization upon HIV-1 binding and gp120 stimulation (Yuan et al.Viruses, 2021). We initially used a recombinant X4-gp120, a soluble protein that does not fully replicate the functional properties of the native HIV-1 Env. Previous studies have shown that Env consists of gp120 trimers, which redistribute and cluster on the surface of virions following proteolytic Gag cleavage during maturation (Chojnacki et al. Nat. Commun., 2017). An important consideration in receptor oligomerization studies is the concentration of recombinant gp120 used, as it does not accurately reflect the low number of Env trimers present on native HIV-1 particles (Hart et al. J. Histochem. Cytochem., 1993; Zhu et al. Nature, 2006). To address these limitations, we generated virus-like particles (VLPs) containing low levels of X4-gp120 and repeated the dynamic analysis of CXCR4. The use of primary HIV-1 isolates was limited, in this project, to confirm that PBMCs from both healthy donors and WHIM patients were equally susceptible to infection. This result using a primary HIV-1 virus supports the conclusion drawn from our in vitroapproaches. We thus believe that although the use of other X4- and dual-tropic strains may complement and reinforce the analysis, it is far beyond the scope of the current manuscript.

• Given the observed clustering effects, it would be valuable to explore whether gp120-induced rearrangements alter epitope exposure to broadly neutralizing antibodies like 17b or 3BNC117. This would help connect the mechanistic insights to therapeutic relevance.*

As 3BNC117, VRC01 and b12 are broadly neutralizing mAbs that recognize conformational epitopes on gp120 (Li et al. J. Virol., 2011; Mata-Fink et al. J. Mol. Biol., 2013), they will struggle to bind the gp120/CD4/CXCR4 complex and therefore may not be ideal for detecting changes within the CD4/CXCR4 complex. The experiment suggested by the reviewer is thus challenging but also very complex. It would require evaluating antibody binding in two experimental conditions, in the absence and in the presence of oligomers. However, our data indicate that receptor oligomerization is promoted by X4-gp120 binding, and the selected antibodies are neutralizing mAbs, so they should block or hinder the binding of gp120 and, consequently, receptor oligomerization. An alternative approach would be to study the neutralizing capacity of these mAbs on cells expressing CD4/CXCR4 or CD4/CXCR4R334X complexes. Variations in their neutralizing activity could be then extrapolated to distinct gp120 conformations, which in turn may reflect differences between CD4/CXCR4 and CD4/CXCR4R334X complexes.

We thus assessed the ability of the VRC01 and b12, anti-gp120 mAbs, which were available in our laboratory, to neutralize gp120 binding on cells expressing CD4/CXCR4 or CD4/CXCR4R334X. Specifically, increasing concentrations of each antibody were preincubated (60 min, 37ºC) with a fixed amount of X4-gp120 (0.05 mg/ml). The resulting complexes were then incubated with Jurkat cells expressing CD4/CXCR4 or CD4/CXCR4R334X (30 min, 37ºC) and, finally, their binding was analyzed by flow cytometry. Although we did not observe statistically significant differences in the neutralization capacity of b12 or VRC01 for the binding of X4-gp120 depending on the presence of CXCR4 or CXCR4334X, we observed a trend for greater concentrations of both mAbs to neutralize X4-gp120 binding in Jurkat CD4/CXCR4 cells than in Jurkat CD4/CXCR4R334X cells (Figure IX, only for review purposes).

These slight alterations in the neutralizing capacity of b12 and VRC01 mAbs may thus suggest minimal differences in the conformations of gp120 depending of the coreceptor used. We also detected that X4-gp120 and VLPs expressing gp120, which require initial binding to CD4 to engage the chemokine receptor, stabilized oligomers of both CXCR4 and CXCR4R334X, but FRET data indicated distinct FRET50 values between the partners, (2.713) for CD4/CXCR4 and (0.399) for CD4/CXCR4R334X (Figure 5A,B in the main manuscript). Moreover, we also detected significantly more CD4 internalization mediated by X4-gp120 in cells co-expressing CD4 and CXCR4 than in those co-expressing CD4 and CXCR4R334X (Figure 6 in the main manuscript). Overall these latter data and those included in Figures V, VI and VII of this reply, indicate distinct conformations within each receptor complexes.

• TIRF imaging limits analysis to the cell substrate interface. It would be useful to clarify whether CXCR4 receptor clustering occurs elsewhere, such as at immunological synapses or during cell-to-cell contact.*

In recent years, chemokine receptor oligomerization has gained significant research interest due to its role in modulating the ability of cells to sense chemoattractant gradients. This molecular organization is now recognized as a critical factor in governing directed cell migration (Martínez-Muñoz et al. Mol. Cell, 2018; García-Cuesta et al. PNAS, 2022, Hauser et al.Immunity, 2016). In addition, advanced imaging techniques such as single-molecule and super-resolution microscopy have been used to investigate the spatial distribution and dynamic behaviour of CXCR4 within the immunological synapse in T cells (Felce et al. Front. Cell Dev. Biol., 2020). Building on these findings, we are currently conducting a project focused on characterizing CXCR4 clustering specifically within this specialized cellular region.

• In LVP experiments, it would be useful to report transduction efficiency (% GFP+ cells) alongside MSI data to relate VLP infectivity with receptor clustering functionally.*

These experiments were designed to validate the functional integrity of the gp120 conformation on the LVPs, confirming their suitability for subsequent TIRF microscopy. Our objective was to establish a robust experimental tool rather than to perform a high-throughput quantification of transduction efficiency. It is for that reason that these experiments were included in new Supplementary Figure S6, which also contains the complete characterization of gp120-VLPs and LVPs. In such experimental conditions, quantifying the percentage of GFP-positive cells relative to the total number of cells plated in each well is very difficult. However, in line with the reviewer's commentary and as we used the same number of cells in each experimental condition, we have included, in the revised manuscript, a complementary graph illustrating the GFP intensity (arbitrary units) detected in all the wells analyzed (new Supplementary Fig. 6E).

• To ensure that differences in fusion events (Figure 7B) are attributable to target cell receptor properties, consider confirming that effector cells express similar levels of HIV-1 Env. Quantifying gp120 expression by flow cytometry or western blot would rule out the confounding effects of variable Env surface density.*

In these assays (Figure 7B), we used the same effector cells (cells expressing X4-gp120) in both experimental conditions, ensuring that any observed differences should be attributable solely to the target cells, either JKCD4X4 or JKCD4X4R334X. For this reason, in Figure 7A we included only the binding of X4-gp120 to the target cells which demonstrated similar levels of the receptors expressed by the cells.

• HIV-mediated receptor downregulation may occur more slowly than ligand-induced internalization. Including a 24-hour time point would help assess whether gp120 induces delayed CD4 or CXCR4 loss beyond the early effects shown and to better capture potential delayed downregulation induced by gp120.*

The reviewer suggests using a 24-hour time point to facilitate detection of receptor internalization. However, such an extended incubation time may introduce some confounding factors, including receptor degradation, recycling and even de novo synthesis, which could affect the interpretation of the results. Under our experimental conditions, we observed that CXCL12 did not trigger CD4 internalization whereas X4-gp120 did. Interestingly, CD4 internalization depended on the co-receptor expressed by the cells.

• Increase label font size in microscopy panels for improved readability.*

Of course; the font size of these panels has been increased in the revised version.

• Consider adding more references on ligand-induced co-endocytosis of CD4 and chemokine receptors during HIV-1 entry.*

We have added more references to support this hypothesis (Toyoda et al. J. Virol., 2015; Venzke et al. J. Virol., 2006; Gobeil et al J. Virol., 2013).

• For Statistical analysis. Biological replicates are adequate, and statistical tests are generally appropriate. For transparency, report n values, exact p-values, and the statistical test used in every figure legend and discussed in the results.*

Thank you for highlighting the importance of transparency in statistical reporting. We confirm that the n values for all experiments have been included in the figure legends. The statistical tests used for each analysis are also clearly indicated in the figure legends, and the interpretation of these results is discussed in detail in the Results section. Furthermore, the Methods section specifies the tests applied and the thresholds for significance, ensuring full transparency regarding our analytical approach.

In accordance with established conventions in the field, we have utilized categorical significance indicators (e.g., n.s., *, **, ***) within our figures to enhance readability and focus on biological trends. This approach is widely adopted in high-impact literature to prevent visual clutter. However, to ensure full transparency and reproducibility, we have ensured that the underlying statistical tests and thresholds are clearly defined in the respective figure legends and Methods section.

Reviewer #4

We thank the reviewer for considering that this work is presented in a clear fashion, and the main findings are properly highlighted, and for remarking that the paper is of interest to the retrovirology community and possibly to the broader virology community.

We also agree on the interest that X4-gp120 clusters CXCR4R334X suggests a different binding mechanism for X4-gp120 from that of the natural ligand CXCL12, an aspect that we are now evaluating. These data also indicate that WHIM patients can be infected by HIV-1 similarly to healthy people.

• The observation that "empty VLPs" reduce CXCR4 diffusivity is potentially interesting. However, it is not supported by the data owing to insufficient controls. The authors correctly discuss the limitations of that observation in the Discussion section (lines 702-704). However, they overinterpret the observation in the Results section (lines 509-512), suggesting non-specific interactions between empty VLPs, CD4 and CXCR4. I suggest either removing the sentence from the Results section or replacing it with a sentence similar to the one in the Discussion section.*

In accordance with the reviewer`s suggestion, the sentence in the result section has been replaced with one similar to that found in the discussion section. In addition, we have performed Raster Image Correlation Spectroscopy (RICS) analysis using the Di-4-ANEPPDHQ lipid probe to assess membrane fluidity by means of membrane diffusion, and compared the results with those of cells treated with Env(-) VLPs. The results indicated that VLPs did not modulate membrane fluidity (Figure VIII in this reply). Nonetheless, these results do not rule out other potential non-specific interactions of the Env(-) VLPs with other components of the cell membrane that might affect receptor dynamics (see our response to point 2 of reviewer #3 p. 14-15 of this reply).

• In the case of the WHIM mutant CXCR4-R334X, the addition of "empty VLPs" did not cause a significant change in the diffusivity of CXCR4-R334X (Figure 4B). This result is in contrast with the addition of empty VLPs to WT CXCR4. However, the authors neither mention nor comment on that result in the results section. Please mention the result in the paper and comment on it in relation to the addition of empty VLPs to WT CXCR4.*

We would remark that the main observation in these experiments should focus on the effect of gp120-VLPs, and the results indicates that gp120-VLPs promoted clustering of CXCR4 and of CXCR4R334X and reduced their diffusion at the cell membrane. The Env(- ) VLPs were included as a negative control in the experiments, to compare the data with those obtained using gp120-VLPs. However, once we observed some residual effect of the Env(-) VLPs, we decided to give a potential explanation, formulated as a hypothesis, that the Env(-) VLPs modulated membrane fluidity. We have now performed a RICS analysis using Di-4-ANEPPDHQ as a lipid probe (Figure IX only for review purposes). The results suggest that Env(-) VLPs do not modulate cell membrane fluidity, although we do not rule out other potential interactions with membrane proteins that might alter receptor dynamics. We appreciate the reviewer's observation and agree that this result can be noted. However, since the main purpose of Figure 4B is to show that gp120-VLPs modulate the dynamics of CXCR4R334X rather than to remark that the Env(-) VLPs also have some effects, we consider that a detailed discussion of this specific aspect would detract from the central finding and may dilute the primary narrative of the study.

Minor comments

• It would be helpful for the reader to combine thematically or experimentally linked figures, e.g., Figures 3 and 4.*

• Figures 3 and 4 are very similar. Please unify the colours in them and the order of the panels (e.g. Figure 3 panel A shows diffusivity of CXCR4, while Figure 4 panel A shows MSI of CXCR4-R334X).*

While we considered consolidating Figures 3 and 4, we believe that maintaining them as separate entities enhances conceptual clarity. Since Figure 3 establishes the baseline dynamics for wild-type CXCR4 and Figure 4 details the distinct behavior of the CXCR4R334X mutant, keeping them separate allows the reader to fully appreciate the specificities of each system before making a cross-comparison.

• Some parts of the Discussion section could be shortened, moved to the Introduction (e.g.,lines 648-651), or entirely removed (e.g.,lines 633-635 about GPCRs).*

In accordance, the Discussion section has been reorganized and shortened to improve clarity.

• I suggest renaming "empty VLPs" to "Env(−) VLPs" (or similar). The name empty VLPs can mislead the reader into thinking that these are empty vesicles.*

The term empty VLPs has been renamed to Env(−) VLPs throughout the manuscript to more accurately reflect their composition. Many thanks for this suggestion.

• Line 492 - please rephrase "...lower expression of Env..." to "...lower expression of Env or its incorporation into the VLPs...".*

The sentence has been rephrased

• Line 527 - The data on CXCL12 modulating CXCR4-R334X dynamics and clustering are not present in Figure 4 (or any other Figure). Please add them or rephrase the sentence with an appropriate reference. Make clear which results are yours.*

• Line 532 - Do the data in the paper really support a model in which CXCL12 binds to CXCR4-R334X? If not, please rephrase with an appropriate reference.*

Previous studies support the association of CXCL12 with CXCR4R334X (Balabanian et al. Blood, 2005; Hernandez et al. Nat Genet., 2003; Busillo & Benovic Biochim. Biophys. Acta, 2007). In fact, this receptor has been characterized as a gain-of-function variant for this ligand (McDermott et al. J. Cell. Mol. Med., 2011). The revised manuscript now includes these bibliographic references to support this commentary. In any case, our previous data indicate that CXCL12 binding does not affect CXCR4R334X dynamics (García-Cuesta et al. PNAS, 2022).

• Line 695 - "...lipid rafts during HIV-1 (missing word?) and their ability to..." During what?*

Many thanks for catching this mistake. The sentence now reads: "Although direct evidence for the internalization of CD4 and CXCR4 as complexes is lacking, their co-localization in lipid rafts during HIV-1 infection (97-99) and their ability to form heterocomplexes (22) strongly suggest they could be endocytosed together."

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 #4

Evidence, reproducibility and clarity

This paper provides new insights into the organisational changes of the X4-tropic HIV-1 co-receptor CXCR4 upon binding of the viral receptor-binding protein X4-gp120, either in its soluble form or when displayed on virus-like particles (VLPs) as Env. The study employs single-particle tracking total internal reflection fluorescence (SPT-TIRF) microscopy to quantify the dynamics and clustering of CXCR4 on CD4+ T cells. The data show that CXCR4 clusters in the presence of X4-gp120 and VLPs, a phenomenon also observed for the primary HIV-1 receptor CD4. The authors also show that a WHIM mutant of CXCR4 (CXCR4-R334X) that does not cluster in the presence of its natural ligand, CXCL12, clusters in the presence of X4-gp120 and VLPs.

The following points should be clarified or improved prior to publication:

Major comments:

1. The observation that "empty VLPs" reduce CXCR4 diffusivity is potentially interesting. However, it is not supported by the data owing to insufficient controls. The authors correctly discuss the limitations of that observation in the Discussion section (lines 702-704). However, they overinterpret the observation in the Results section (lines 509-512), suggesting non-specific interactions between empty VLPs, CD4 and CXCR4. I suggest either removing the sentence from the Results section or replacing it with a sentence similar to the one in the Discussion section.
2. In the case of the WHIM mutant CXCR4-R334X, the addition of "empty VLPs" did not cause a significant change in the diffusivity of CXCR4-R334X (Figure 4B). This result is in contrast with the addition of empty VLPs to WT CXCR4. However, the authors neither mention nor comment on that result in the results section. Please mention the result in the paper and comment on it in relation to the addition of empty VLPs to WT CXCR4.

Minor comments:

1. It would be helpful for the reader to combine thematically or experimentally linked figures, e.g., Figures 3 and 4.
2. Figures 3 and 4 are very similar. Please unify the colours in them and the order of the panels (e.g. Figure 3 panel A shows diffusivity of CXCR4, while Figure 4 panel A shows MSI of CXCR4-R334X).
3. Some parts of the Discussion section could be shortened, moved to the Introduction (e.g., lines 648-651), or entirely removed (e.g., lines 633-635 about GPCRs).
4. I suggest renaming "empty VLPs" to "Env(−) VLPs" (or similar). The name empty VLPs can mislead the reader into thinking that these are empty vesicles.
5. Line 492 - please rephrase "...lower expression of Env..." to "...lower expression of Env or its incorporation into the VLPs...".
6. Line 527 - The data on CXCL12 modulating CXCR4-R334X dynamics and clustering are not present in Figure 4 (or any other Figure). Please add them or rephrase the sentence with an appropriate reference. Make clear which results are yours.
7. Line 532 - Do the data in the paper really support a model in which CXCL12 binds to CXCR4-R334X? If not, please rephrase with an appropriate reference.
8. Line 695 - "...lipid rafts during HIV-1 (missing word?) and their ability to..." During what?

Significance

In summary, the work is presented in a clear fashion, and the main findings are properly highlighted. The paper is of interest to the retrovirology community and possibly to the broader virology community. The findings are not entirely surprising because it has been shown previously that the binding of Env to CD4 mediates CD4 clustering, which would also suggest clustering of the co-receptor. Nonetheless, the paper provides strong evidence that CXCR4 clusters and changes its dynamics in the presence of CD4 and X4-gp120. Moreover, the evidence that X4-gp120 clusters CXCR4-R334X is of high interest because it suggests a different binding mechanism for X4-gp120 from that of the natural ligand CXCL12, raising questions for further research. The diffusivity data with empty VLPs require additional controls to strengthen the evidence. My expertise is in virology and structural biology. I did not comment on the technical aspects of the light-microscopy experiments in the study because these are beyond my expertise.

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

The author investigates how the HIV-1 Env glycoprotein modulates the nanoscale organisation and dynamics of the CXCR4 co-receptor on CD4⁺ T cells. The author demonstrates that HIV-1 Env induces CXCR4 clustering distinct from that triggered by its natural ligand (CXCL12), implicating spatial receptor organization as a determinant of infection. This study investigates how HIV-1 Env (specifically X4-tropic gp120) alters the membrane organization and dynamics of the chemokine receptor CXCR4 and its WHIM-associated mutant, CXCR4R334X, in a CD4-dependent manner. Using single-particle tracking total internal reflection fluorescence microscopy (SPT-TIRF-M), the authors demonstrate that both soluble gp120 and virus-like particles (VLPs) displaying gp120 induce CXCR4 nanoclustering, reduce receptor diffusivity, and promote immobile nanoclusters of CXCR4 at the membrane of Jurkat T cells and primary CD4⁺ T cell blasts.The work offers new insights into the spatial organisation of receptors during HIV-1 entry and infection. The manuscript is well-written, and the findings are significant.

Major Comments: 1. For mechanistic basis of gp120-CXCR4 versus CXCL12-CXCR4 differences

Provide additional structural or biochemical evidence to support the claim that gp120 stabilises a distinct CXCR4 conformation compared to CXCL12.

If feasible, include molecular modelling, mutagenesis, or cross-linking experiments to corroborate the proposed conformational differences. 2. For Empty VLP effects on CXCR4 dynamics

Explore potential causes for the observed effects of Env-deficient VLPs. It's valuable to include additional controls such as particles from non-producer cells, lipid composition analysis, or blocking experiments to assess nonspecific interactions. 3. For Direct link between clustering and infection efficiency - Test whether disruption of CXCR4 clustering (e.g., using actin cytoskeleton inhibitors, membrane lipid perturbants, or clustering-deficient mutants) alters HIV-1 fusion or infection efficiency. 4. CD4/CXCR4 co-endocytosis hypothesis - Support the proposed model with direct evidence from live-cell imaging or co-localization experiments during viral entry. Clarification is needed on whether internalization is simultaneous or sequential for CD4 and CXCR4.

Minor Comments: 1. The conclusions rely solely on the HXB2 X4-tropic Env. It would strengthen the study to assess whether other X4 or dual-tropic strains induce similar receptor clustering and dynamics. 2. Given the observed clustering effects, it would be valuable to explore whether gp120-induced rearrangements alter epitope exposure to broadly neutralizing antibodies like 17b or 3BNC117. This would help connect the mechanistic insights to therapeutic relevance. 3 . TIRF imaging limits analysis to the cell substrate interface. It would be useful to clarify whether CXCR4 receptor clustering occurs elsewhere, such as at immunological synapses or during cell-to-cell contact. 4. In LVP experiments, it would be useful to report transduction efficiency (% GFP+ cells) alongside MSI data to relate VLP infectivity with receptor clustering functionally. 5. To ensure that differences in fusion events (Figure 7B) are attributable to target cell receptor properties, consider confirming that effector cells express similar levels of HIV-1 Env. Quantifying gp120 expression by flow cytometry or western blot would rule out the confounding effects of variable Env surface density 6. HIV-mediated receptor downregulation may occur more slowly than ligand-induced internalization. Including a 24-hour time point would help assess whether gp120 induces delayed CD4 or CXCR4 loss beyond the early effects shown and to better capture potential delayed downregulation induced by gp120. 7. Increase label font size in microscopy panels for improved readability. 8. Consider adding more references on ligand-induced co-endocytosis of CD4 and chemokine receptors during HIV-1 entry. For Statistical analysis. Biological replicates are adequate, and statistical tests are generally appropriate. For transparency, report n values, exact p-values, and the statistical test used in every figure legend and discussed in the results.

Referee cross-commenting

Overall, the manuscript provides compelling mechanistic insight into HIV-1 entry by demonstrating Env-induced CXCR4 clustering, including in WHIM mutant receptors. While the core findings are well supported and of high interest, clarifications regarding Env trimer densities, receptor internalization, and the contribution of empty VLPs would further strengthen the work.

Significance

Nature and significance of the advance

This work marks a conceptual and mechanistic breakthrough in understanding HIV-1 entry. It goes beyond the static view of Env-co-receptor interaction to show that nanoscale reorganization of CXCR4, distinct from chemokine-induced clustering, occurs during HIV-1 Env engagement and may be essential for infection Context within existing literature. Previous studies established Env-induced CD4 clustering (Yin et al., 2020) and chemokine-induced CXCR4 nanocluster formation (Martínez-Muñoz et al., 2018), but the exact nanoscale rearrangement of CXCR4 in the context of HIV-1 Env and physiological Env densities remains unquantified. This study addresses this gap using SPT-TIRF, STED microscopy, and functional assays.

Audience and influence

The findings will be of interest to researchers in HIV virology, membrane receptor biology, viral entry mechanisms, and therapeutic target development. The receptor-clustering aspect could also influence broader fields of study, such as GPCR organization and immune receptor signalling.

Reviewer expertise

I can evaluate HIV-1 entry mechanisms, viral glycoprotein-host-host-host receptor interactions, single-molecule fluorescence microscopy, and membrane protein dynamics. I am less equipped to evaluate the deep structural modelling aspects, though the in silico AlphaFold results are straightforward to interpret in context.

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

The authors examine the distribution of CXCR4 on the cell surface following exposure to gp120 and HIV virus-like particles (VLPs) using single particle tracking total internal reflection fluorescence (SPT-TIRF) microscopy. They show that gp120 and VLPs promote clustering of wild-type CXCR4 and CXCR4.R334X from a person with WHIM syndrome. The HIV Env-induced clustering involves heterodimeric interactions between CXCR4 and CD4 and spatial distribution and dynamics are distinct from that induced by CXCR4's natural ligand, CXCL12. The authors suggest the CD4-CXCR4 interaction may be targeted to specifically block HIV infection.

Major comments

This study is well described in both the main text and figures. Introduction provides adequate background and cites the literature appropriately. Materials and Methods are detailed. Authors are careful in their interpretations, statistical comparisons, and include necessary controls in each experiment. The Discussion presents a reasonable interpretation of the results. Overall, there are no major weaknesses with this manuscript.

Minor comments

Ln 477-497. NL4-3deltaIN and immature HIV virions are found to have less associated gp120 relative to wild-type particles. It is not obvious why this is the case for the deltaIN particles or genetically immature particles. Can the authors provide possible explanations? (A prior paper was cited, Chojnacki et al Science, 2012 but can the current authors provide their own interpretation.)

Significance

The current study builds on prior works that examined CXCR4 distribution, HIV pseudotyped infection in CXCR4.R334X cells, but goes beyond these studies in resolution and depth of analysis of CXCR4/CD4 nanoclustering, AF3 modeling of CXCR4/CD4 heterodimer, as well as demonstration of replication of HIV in CXCR4.R334X cells.

Audience:

Scientists interested in HIV-1, cell biologists and virologists interested in receptor nanoclustering

Reviewer expertise:

HIV-1 Envelope glycoproteins and entry assays, HIV broadly neutralizing antibodies, HIV vaccine design

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

The authors investigate the impact of surface bound HIV gp120 and VLPs on CXCR4 dynamics in Jurkat T cells expressing WT or WHIM syndrome mutated CXCR4, which has a defective response to CXCL12. Jurkat cells were transfected with CXCR4-AcGFP. Images were acquired and a single particle tracking routine was applied to generate information about nanoclustering and diffusion, and FRET was used to investigate CD4-CXCR4 proximity. They compare effects of soluble gp120 to immature and mature VLPs, which include varying degrees of gp120 clustering. They find that solid phase gp120 or VLP can increase CXCR4 clustering size and decrease diffusion in Jurkat cells. Surprisingly, VLP lacking gp120 could increase CXCR4 clustering and speed, which is paradoxical as there were no known ligands on the VLPs, but they likely carry many cellular proteins with potential interactions. The impact of CXCL12 and gp120 binding to CXCR4 was different in terms of clustering and receptor down-regulation.

While a single particle tracking routine was applied to the data, it's not clear how the signal from a single GFP was defined and if movement during the 100 ms acquisition time impacts this. My concern would be that the routine is tracking fluctuations, and these are related to single particle dynamics, it appears from the movies that the density or the GFP tagged receptors in the cells is too high to allow clear tracking of single molecules. SPT with GFP is very difficult due to bleaching and relatively low quantum yield. Current efforts in this direction that are more successful include using SNAP tags with very photostable organic fluorophores. The data likely does mean something is happening with the receptor, but they need to be more conservative about the interpretation.

Some of the paradoxical effects might be better understood through deeper analysis of the SPT data, particularly investigation of active transport and more detailed analysis of "immobile" objects. Comments on early figures illustrate how this could be approached. This would require selecting acquisitions where the GFP density is low enough for SPT and performing a more detailed analysis, but this may be difficult to do with GFP.

When the authors discuss clusters of <2 or >3, how do they calibrate the value of GFP and the impact of diffusion on the measurement. One way to approach this might be single molecules measurements of dilute samples on glass vs in a supported lipid bilayer to map the streams of true immobility to diffusion at >1 µm2/sec.

I understand that the CXCL12 or gp120 are attached to the substrate with fibronectin for adhesion. I'm less clear how how that VLPs are integrated. Were these added to cells already attached to FN? Fig 1A- The classification of particle tracks into mobile and immobile is overly simplistic description that goes back to bulk FRAP measurements and it not really applicable to single molecule tracking data, where it's rare to see anything that is immobile and alive. An alternative classification strategy uses sub-diffusion, normal diffusion and active diffusion (or active transport) to descriptions and particles can transition between these classes over the tracking period. Fig 1B- this data might be better displayed as histograms showing distributions within the different movement classes. Fig 1C,D- It would be helpful to see a plot of D vs MSI at a single particle level. In comparing C and D I'm surprised there is not a larger difference between CXCL12 and X4-gp120. It would also be very important to see the behaviour of X4-gp120 on the CXCR4 deficient Jurkat that would provide a picture of CD4 diffusion. The CXCR4 nanoclustering related to the X4-gp120 could be dominated by CD4 behaviour.

Fig S1D- This data is really interesting. However, if both the CD4 and the gp120 have his tags they need to be careful as poly-His tags can bind weakly to cells and increasing valency could generate some background. So, they should make the control is fair here. Ideally, using non-his tagged person of sCD4 and gp120 would be needed ideal or they need a His-tagged Fab binding to gp120 that doesn't induce CXCR4 binding.

Fig S4- Panel D needs a scale bar. I can't figure out what I'm being shown without this.

Significance

The strengths are that its an important question and the reagents are well prepared and characterised. They are detecting quantitative effects that will likely be reproducible. The information generated is potentially useful for those studying HIV infection processes and strategies to prevent infection.

The major weakness is that the conditions for the SPT experiments are not ideal in that the density of particles is too high for SPT and the single molecule basis for assessing nanoclusters is not clear. This means that the data is getting at complex molecules phenomena and less likely be generating pure single molecules measurements.

eLife Assessment

This important study shows that a controlled pause in gene reading is required for early heart cells to form during development. The authors demonstrate that loss of this pause prevents the proper activation of the heart-producing program across animal and stem cell systems. The evidence is compelling, supported by careful genomic and functional analyses that clearly define the developmental block. Overall, this work will interest developmental biologists and inspire further studies on the origins of early heart defects.

Reviewer #1 (Public review):

This is a highly original and impactful study that significantly advances our understanding of transcriptional regulation, in particular RNAPII pausing, during early heart development. The Chen lab has a long history of producing influential studies in cardiac morphogenesis, and this manuscript represents another thorough and mechanistically insightful contribution. The authors have thoroughly addressed this Reviewer's concerns and incorporated all of my suggestions in the revised manuscript. In addition, their responses to the other reviewer's comments are also very clear. As it is, this work is of great interest to the readership of Elife, as well as to the general scientific community.

The authors reveal a fundamentally new role for Rtf1-a component of the PAF1 complex-in governing promoter-proximal RNAPII pausing in the context of myocardial lineage specification. While transcriptional pausing has been implicated in stress responses and inducible gene programs, its developmental relevance has remained poorly defined. This study fills that gap with rigorous in vivo evidence demonstrating that Rtf1-dependent pausing is indispensable for activating the cardiac gene program from the lateral plate mesoderm.

Importantly, the study also provides compelling therapeutic implications. Showing that CDK9 inhibition-using either flavopiridol or targeted knockdown-can restore promoter-proximal pausing and rescue cardiomyocyte formation in Rtf1-deficient embryos suggests that modulation of pause-release kinetics may represent a new avenue for correcting transcriptionally driven congenital heart defects. Given that many CDK inhibitors are clinically approved or in active development, this connection significantly elevates the translational impact of the findings.

In sum, this study is rigorous, innovative, and transformative in its implications for developmental biology and cardiac medicine. I strongly support its publication.

Reviewer #2 (Public review):

Summary:

Langenbacher at el. examine the requirement of Rtf1, a component of the PAF1C complex, which regulates transcriptional pausing in cardiac development. The authors first confirm that newly generated rtf1 mutant alleles recapitulate the defects in cardiac progenitor differentiation found using morpholinos from their previous work. The authors then show that conditional loss of Rtf1 in mouse embryos and depletion in mouse ESCs both demonstrates a failure to turn on cardiac progenitor and differentiation marker genes, supporting conservation of Rtf1 in promoting vertebrate cardiac progenitor development. The authors then employ bulk RNA-seq on flow-sorted hand2:GFP+ cells and multiomic single-cell RNA-seq on whole Rtf1-depleted zebrafish embryos at the 10-12 somite stage. These experiments corroborate that gene expression associated with cardiac progenitor differentiation is lost. Furthermore, analysis of differentiation trajectories suggests that the expression of genes associated with cardiac, blood, and endothelial progenitor differentiation is not initiated within the anterior lateral plate mesoderm. Structure-function analysis supports that the Rtf1 Plus3 domain is necessary for its function in promoting cardiac progenitor differentiation. ChIP-seq for RNA Pol II on 10-12 somite stage zebrafish embryos supports that Rtf1 is required for proper promoter pausing at the transcriptional start site. The transcriptional promoter pausing defect and cardiac differentiation can partially be rescued in zebrafish rtf1 mutants through pharmacological inhibition and depletion of Cdk9, a kinase that inhibits elongation. Thus, the authors have provided a clear analysis of the requirements and basic mechanism that Rf1 employs regulating cardiac progenitor development.

Strengths and weaknesses:

Overall, the data presented are strong and the message of the study is clear. The conclusions that Rtf1 is required for transcriptional pause release and promotes vertebrate cardiac progenitor differentiation are supported. Areas of strength include the complementary approaches in zebrafish and mouse embryos, and mouse embryonic stem cells, which together support the conserved requirement for Rtf1 in promoting cardiac differentiation. The bulk and single-cell RNA-sequencing analyses provide further support for this model via examining broader gene expression. In particular, the pseudotime analysis bolsters that there is a broader effect on differentiation of anterior lateral plate mesoderm derivatives. The structure-function analysis provides a relatively clean demonstration of the requirement of the Rtf1 Plus3 domain. The pharmacological and depletion epistasis of Cdk9 combined with the RNA Pol II ChIP-seq nicely support the mechanism implicating Cdk9 in the Rtf1-dependent RNA Pol II promoter pausing. Additionally, this is a revised manuscript. The authors were overall responsive to the previous critiques. The new analysis and revisions have helped to strengthen their hypothesis and improve the clarity of their study. While the revised manuscript is significantly improved, the lack of analysis from the multiomic analysis still represents a lost opportunity to provide further insight into Rtf1 mechanisms within this study. However, the authors have nevertheless achieved their goal for this study. The data sets reported will also be useful tools for further analysis and integration by the cardiovascular development community. Thus, the study will be of interest to scientists studying cardiovascular development and those broadly interested in epigenetic regulation controlling vertebrate development.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary:

The manuscript submitted by Langenbacher et al., entitled " Rtf1-dependent transcriptional pausing regulates cardiogenesis", describes very interesting and highly impactful observations about the function of Rtf-1 in cardiac development. Over the last few years, the Chen lab has published novel insights into the genes involved in cardiac morphogenesis. Here, they used the mouse model, the zebrafish model, cellular assays, single cell transcription, chemical inhibition, and pathway analysis to provide a comprehensive view of Rtf1 in RNAPII (Pol2) transcription pausing during cardiac development. They also conducted knockdown-rescue experiments to dissect the functions of Rtf1 domains. 

Strengths:

The most interesting discovery is the connection between Rtf1 and CDK9 in regulating Pol2 pausing as an essential step in normal heart development. The design and execution of these experiments also demonstrate a thorough approach to revealing a previously underappreciated role of Pol2 transcription pausing in cardiac development. This study also highlights the potential amelioration of related cardiac deficiencies using small molecule inhibitors against cyclin dependent kinases, many of which are already clinically approved, while many other specific inhibitors are at various preclinical stages of development for the treatment of other human diseases. Thus, this work is impactful and highly significant. 

We thank the reviewer for appreciating our work.

Reviewer #2 (Public Review): 

Summary: 

Langenbacher at el. examine the requirement of Rtf1, a component of the PAF1C, which regulates transcriptional pausing in cardiac development. The authors first confirm their previous morphant study with newly generated rtf1 mutant alleles, which recapitulate the defects in cardiac progenitor and diUerentiation gene expression observed previously in morphants. They then examine the conservation of Rtf1 in mouse embryos and embryonic stem cell-derived cardiomyocytes. Conditional loss of Rtf1 in mesodermal lineages and depletion in murine ESCs demonstrates a failure to turn on cardiac progenitor and diUerentiation marker genes, supporting conservation of Rtf1 in promoting cardiac development. The authors subsequently employ bulk RNA-seq on flow-sorted hand2:GFP+ cells and multiomic single-cell RNA-seq on whole Rtf1-depleted embryos at the 10-12 stage. These experiments corroborate that genes associated with cardiac and muscle development are lost. Furthermore, the diUerentiation trajectories suggest that the expression of genes associated with cardiac maturation is not initiated.  Structure-function analysis supports that the Plus3 domain is necessary for its function in promoting cardiac progenitor formation. ChIP-seq for RNA Pol II on 1012 somite stage embryos suggests that Rtf1 is required for proper promoter pausing. This defect can partially be rescued through use of a pharmacological inhibitor for Cdk9, which inhibits elongation, can partially restore elongation in rtf1 mutants.  

Strengths: 

Many aspects of the data are strong, which support the basic conclusions of the authors that Rtf1 is required for transcriptional pausing and has a conserved requirement in vertebrate cardiac development. Areas of strength include the genetic data supporting the conserved requirement for Rtf1 in promoting cardiac development, the complementary bulk and single-cell RNA-sequencing approaches providing some insight into the gene expression changes of the cardiac progenitors, the structure-function analysis supporting the requirement of the Plus3 domain, and the pharmacological epistasis combined with the RNA Pol II ChIP-seq, supporting the mechanism implicating Cdk9 in the Rtf1 dependent mechanism of RNA Pol II pausing. 

We thank the reviewer for the summary and for recognizing many strengths of our work. 

Weaknesses: 

While most of the basic conclusions are supported by the data, there are a number of analyses that are confusing as to why they chose to perform the experiments the way they did and some places where the interpretations presently do not support the interpretations. One of the conclusions is that the phenotype aUects the maturation of the cardiomyocytes and they are arresting in an immature state. However, this seems to be mostly derived from picking a few candidates from the single cell data in Fig. 6. If that were the case, wouldn't the expectation be to observe relatively normal expression of earlier marker genes required for specification, such as Nkx2.5 and Gata5/6? The in situ expression analysis from fish and mice (Fig. 2 and Fig. 3) and bulk RNA-seq (Fig. 5) seems to suggest that there are pretty early specification and diUerentiation defects. While some genes associated with cardiac development are not changed, many of these are not specific to cardiomyocyte progenitors and expressed broadly throughout the ALPM. Similarly, it is not clear why a consistent set of cardiac progenitor genes (for instance mef2ca, nkx2.5, and tbx20) was analyzed for all the experiments, in particular with the single cell analysis. 

A major conclusion of our study is that Rtf1 deficiency impairs myocardial lineage differentiation from mesoderm, as suggested by the reviewer. Thus, the main goal of this study is to understand how Rtf1 drives cardiac differentiation from the LPM, rather than the maturation of cardiomyocytes.  Multiple lines of evidence support this conclusion:

(a) In situ hybridization showed that Rtf1 mutant embryos do not have nkx2.5+ cardiac progenitor cells and subsequently fail to produce cardiomyocytes (Figs. 2, 3).

(b) RT-PCR analysis showed that knockdown of Rtf1 in mouse embryonic stem cells causes a dramatic reduction of cardiac gene expression and production of significantly fewer beating patches (Fig.4).

(c) Bulk RNA sequencing revealed significant downregulation of cardiac lineage genes, including nkx2.5 (Fig. 5).

(d) Single cell RNA sequencing clearly showed that lateral plate mesoderm (LPM) cells are significantly more abundant in Rtf1 morphant,s whereas cardiac progenitors are less abundant (Fig. 6 and Fig.6 Supplement 1-5). 

When feasible, we used cardiac lineage restricted markers in our assays. Nkx2.5 and tbx5a are not highlighted in the single cell analysis because their expression in our sc-seq dataset was too low to examine in the clustering/trajectory analysis.  In this revised manuscript, we provide violin plots showing the low expression levels of these genes in single cells from Rtf1 deficient embryos (Figure 6 Supplement 5).

The point of the multiomic analysis is confusing. RNA- and ATAC-seq were apparently done at the same time. Yet, the focus of the analysis that is presented is on a small part of the RNA-seq data. This data set could have been more thoroughly analyzed, particularly in light of how chromatin changes may be associated with the transcriptional pausing. This seems to be a lost opportunity. Additionally, how the single cell data is covered in Supplemental Fig. 2 and 3 is confusing. There is no indication of what the diUerent clusters are in the Figure or the legend. 

In this study, we performed single cell multiome analysis and used both scRNAseq and scATACseq datasets to generate reliable clustering.  The scRNAseq analysis reveals how Rtf1 deficiency impacts cardiac differentiation from mesoderm, which inspired us to investigate the underlying mechanism and led to the discovery of defects in Rtf1-dependent transcriptional pause release.

We agree with the reviewer that deep examination of Rtf1-dependent chromatin changes would provide additional insights into how Rtf1 influences early development and careful examination of the scATACseq dataset is certainly a good future direction.  

In this revised manuscript, we have revised Fig.6 Supplement 1 to include the predicted cell types and provide an additional excel file showing the annotation of all 39 clusters (Supplementary Table 2). 

While the effect of Rtf1 loss on cardiomyocyte markers is certainly dramatic, it is not clear how well the mutant fish have been analyzed and how specific the eUect is to this population. It is interpreted that the eUects on cardiomyocytes are not due to "transfating" of other cell fates, yet supplemental Fig. 4 shows numerous eUects on potentially adjacent cell populations. Minimally, additional data needs to be provided showing the live fish at these stages and marker analysis to support these statements. In some images, it is not clear the embryos are the same stage (one can see pigmentation in the eyes of controls that is not in the mutants/morphants), causing some concern about developmental delay in the mutants. 

Single cell RNA sequencing showed an increased abundance of LPM cells and a reduced abundance of cardiac progenitors in Rtf1 morphants (Fig. 6 and Fig.6 Supplement 1-5). The reclustering of anterior lateral plate mesoderm (ALPM) cells and their derivatives further showed that cells representing undiRerentiated ALPM were increased whereas cells representing all three ALPM derivatives were reduced. These findings indicate a defect in ALPM diRerentiation. 

The reviewer questioned whether we examined stage-matched embryos. In our assay, Rtf1 mutant embryos were collected from crosses of Rtf1 heterozygotes. Each clutch from these crosses consists of ¼ embryos showing rtf1 mutant phenotypes and ¾ embryos showing wild type phenotypes which were used as control. Mutants and their wild type siblings were fixed or analyzed at the same time.

The reviewer questioned the specificity of the Rtf1 deficient cardiac phenotype and pointed out that Rtf1 mutant embryos do not have pigment cells around the eye.  Rtf1 is a ubiquitously expressed transcriptional regulator.  Previous studies in zebrafish have shown that Rtf1 deficiency significantly impacts embryonic development. Rtf1 deficiency causes severe defects in cardiac lineage and neural crest cell development; consequently, Rtf1 deficient embryos do not have cardiomyocytes and pigmentation (Langenbacher et al., 2011, Akanuma et al., 2007, and Jurynec et al., 2019).  We now provide an image showing a 2-day-old Rtf1 mutant embryo and their wild type sibling to illustrate the cardiac, neural crest, and somitogenesis defects caused by loss of Rtf1 activity (Fig. 2 Supplement 1).

With respect to the transcriptional pausing defects in the Rtf1 deficient embryos, it is not clear from the data how this eUect relates to the expression of the cardiac markers. This could have been directly analyzed with some additional sequencing, such as PRO-seq, which would provide a direct analysis of transcriptional elongation. 

We showed that Rtf1 deficiency results in a nearly genome-wide decrease in promoterproximal pausing and downregulation of cardiac makers. Attenuating transcriptional pause release could restore cardiomyocyte formation in Rtf1 deficient embryos. In this revised manuscript, we provide additional RNAseq data showing that the expression levels of critical cardiac development genes such as nkx2.5, tbx5a, tbx20, mef2ca, mef2cb, ttn.2, and ryr2b are significantly rescued.  We agree with the reviewer that further analyses using the PRO-seq approach could provide additional insights, but it is beyond the scope of this manuscript. 

Some additional minor issues include the rationale that sequence conservation suggests an important requirement of a gene (line 137), which there are many examples this isn't the case, referencing figures panels out of order in Figs. 4, 7, and 8) as described in the text, and using the morphants for some experiments, such as the rescue, that could have been done in a blinded manner with the mutants. 

We have clarified the rationale in this revised manuscript and made the eRort to reference figures in order. 

The reviewer commented that rescue experiments “could have been done in a blinded manner with the mutants”. This was indeed how the flavopiridol rescue and cdk9 knockdown experiments were carried out. Embryos from crosses of Rtf1 heterozygotes were collected, fixed after treatment and subjected to in situ hybridization. Embryos were then scored for cardiac phenotype and genotyped (Fig.8 d-g). Morpholino knockdown was used in genomic experiments because our characterization of rtf1 morphants showed that they faithfully recapitulate the rtf1 mutant phenotype during the timeframe of interest (Fig. 2).

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

This reviewer has a few suggestions below, aimed at improving the clarity and impact of the current study. Once these items are addressed, the manuscript should be of interest to the Elife reader. 

Item 1. Strengthening the interaction between Rfh1 and CDK9 on Pol2 pausing. 

The authors have convincingly shown that the chemical inhibition of CDK9 by flavopiridol can partially rescue the expression of cardiac genes in the zebrafish model. Although flavopiridol is FDA approved and has been a classical inhibitor for the dissection of CDK9 function, it also inhibits related CDKs (such as Flavopiridol (Alvocidib) competes with ATP to inhibit CDKs including CDK1, CDK2, CDK4, CDK6, and CDK9 with IC50 values in the 20-100 nM range) Therefore, this study could be more impactful if the authors can provide evidence on which of these CDKs may be most relevant during Rtf1-dependent cardiogenesis. To determine whether the observed cardiac defect indicates a preferential role for CDK9, or that other CDKs may also be able to provide partial rescue may be clarified using additional, more selective small molecules (e.g., BAY1251152, LDC000067 are commercially available). 

The reviewer raised a reasonable concern about the specificity of flavopiridol. We thank the reviewer for the insightful suggestion and share the concern about specificity. To address this question, we have used an orthogonal testing through morpholino inhibition where we directly targeted CDK9 and observed the same level of rescue, supporting a critical role of transcription pausing in cardiogenesis.

Item 2. Differences between CRISPR lines and morphants 

Much of the work presented used Rtf1 morphants while the authors have already generated 2 CRISPR lines. What is the diUerence between morphants and mutants? The authors should comment on the similarities and/or differences between using morphants or mutants in their study and whether the same Rtf1- CDK9 connection also occurs in the CRISPR lines. 

The morphology of our mutants (rtf1^LA2678 and rtf1^LA2679) resembles the morphants and the previously reported ENU-induced rtf1^KT641 allele. Extensive in situ hybridization analysis showed that the morphants faithfully recapitulate the mutant phenotypes (Fig.2). We have performed rescue experiments (flavopiridol and CDK9 morpholino) using Rtf1 mutant embryos and found that inhibiting Cdk9 restores cardiomyocyte formation (Fig.8). 

Item 3. Discuss the therapeutic relevance of study 

The authors have already generated a mouse model of Rtf1 Mesp1-Cre knockout where cardiac muscle development is severely derailed (Fig 3B). Thus, a demonstration of a conserved role for CDK9 inhibitor in rescuing cardiogenesis using mouse cells or the mouse model will provide important information on a conserved pathway function relevant to mammalian heart development. In the Discussion, how this underlying mechanistic role may be useful in the treatment of congenital heart disease should be provided.  

Thank you for the insight. We have incorporated your comments in the discussion. 

Item 4. Insights into the role of CDK9-Rtf1 in response to stress versus in cardiogenesis. 

In the Discussion, the authors commented on the role of additional stress-related stimuli such as heat shock and inflammation that have been linked to CDK9 activity. However, the current ms provides the first, endogenous role of Pol2 pausing in a critical developmental step during normal cardiogenesis. The authors should emphasize the novelty and significance of their work by providing a paragraph on the state of knowledge on the molecular mechanisms governing cardiogenesis, then placing their discovery within this framework. This minor addition will also clarify the significance of this work to the broad readership of eLife. 

Thank you for the suggestion. We have incorporated your comments and elaborate on the novelty and significance of our work in the discussion. 

Reviewer #2 (Recommendations For The Authors): 

(1) It is diUicult to assess what the overt defects are in the embryos at any stages. Images of live images were not included in the supplement. Do these have a small, malformed heart tube later or are the embryos just deteriorating due to broad defects? 

The Rtf1 deficient embryos do not produce nkx2.5+ cardiac progenitors. Consequently, we never observed a heart tube or detected cells expressing cardiomyocyte marker genes such as myl7. This finding is consistent with previous reports using rtf1 morphants and rtf^1KT64, an ENU-induced point mutation allele (Langenbacher et al., 2011 and Akanuma, 2007). In this revised manuscript, we provide a live image of 2-day-old wild type and rtf1^{LA2679/LA2679} embryos (Fig. 2 Supplement 1). After two days, rtf1 mutant embryos undergo broad cell death. 

(2) Fig. 2, although the in situs are convincing, there is not a quantitative assessment of expression changes for these genes. This could have been done for the bulk or single cell RNA-seq experiments, but was not and these genes weren't not included in the heat maps. A quantitative assessment of these genes would benefit the study. 

The top 40 most significantly diRerentially expressed genes are displayed in the heatmap presented in Fig.5d. The complete diRerential gene expression analysis results for our hand2 FACS-based comparison of rtf1 morphants and controls is presented in Supplementary Data File 1.  In this revised manuscript, we provide a new supplemental figure with violin plots showing the expression levels of genes of interest in our single cell sequencing dataset (Fig.6 Supplement 5).

(3) It doesn't not appear that any statistical tests were used for the comparisons in Fig. 2.

We now provide the statistical data in the legend and Fig.2 b, d, f, h and i.

(4) It's not clear the magnifications and orientations of the embryos in Fig. 3b are the same. 

Embryos shown in Fig.3b are at the same magnification. However, because Rtf1 mutant embryos display severe morphological defects, the orientation of mutant embryos was adjusted to examine the cardiac tissue.

(5) The n's for analysis of MLC2v in WT Rtf1 CKO embryos in Fig. 3b are only 1. At least a few more embryos should be analyzed to confirm that the phenotype is consistent. 

We have revised the figure and present the number of embryos analyzed and statistics in Fig.3c. 

(6) A number of figure panels are referred to out of order in the text. Fig. 4E-G are before Fig. 4C, D, Fig. 7C  before 7B, Fig. 8D-I before 8A ,B. In general, it is easier for the reader if the figures panels are presented in the order they are referred to in the text. 

Revised as suggested.

(7) While additional genes can be included, it is not clear why the same sets of genes are not examined in the bulk or single-cell RNA-seq as with the in situs or expression was analyzed in embryos. I suggest including the genes like nkx2.5, tbx20, myl7, in all the sequencing analysis. 

We used the same set of genes in all analyses when possible. However, the low expression of genes such as nkx2.5 and myl7 in our sc-seq dataset preclude them from the clustering/trajectory analysis. In this revised manuscript, we present violin plots showing their expression in wild type and rtf1 morphants (Fig. 6 Supplement 5).

(8) If a multiomic approach was used, why wasn't its analysis incorporated more into the manuscript? In general, a clearer presentation and deeper analysis of the single cell data would benefit the study. The integration of the RNA and ATAC would benefit the analysis.

As addressed in our response to the reviewer’s public review, both datasets were used in clustering. Examining changes in chromatin accessibility is certainly interesting, but beyond the scope of this study. 

(9) Many of the markers analyzed are not cardiac specific or it is not clear they are expressed in cardiac progenitors at the stage of the analysis. Hand2 has broader expression. Additional confirmation of some of the genes through in situ would help the interpretations. 

Markers used for the in situ hybridization analysis (myl7, mef2ca, nkx2.5, tbx5a, and tbx20) are known for their critical role in heart development. For sc-seq trajectory analyses, most displayed genes (sema3e, bmp6, ttn.2, mef2cb, tnnt2a, ryr2b, and myh7bb) were identified based on their diRerential expression along the LPM-cardiac progenitor pseudotime trajectory. Rather than selecting genes based on their cardiac specificity, our goal was to examine the progressive gene expression changes associated with cardiac progenitor formation and compare gene expression of wild type and rtf1 deficient embryos.

(10) Additional labels of the cell clusters are needed for Supplemental Figs. 2 and 3. 

The cluster IDs were presented on Supplementary Figures 2 and 3. In this revised version, we added predicted cell types to the UMAP (revised Fig.6 Supplement 1) and provided an excel file with this information (revised Supplementary Table 2). 

(11) On lines 101-102, the interpretation from the previous data is that diUerentiation of the LPM requires Rtf1. However, later from the single cell data the interpretation based on the markers is that Rtf1 loss aUects maturation. However, it is not clear this interpretation is correct or what changed from the single cell data. If that were the case, one would expect to see maintenance of more early marks and subsequent loss of maturation markers, which does not appear to the be the case from the presented data.

Our data suggests that cardiac progenitor formation is not accomplished by simultaneously switching on all cardiac marker genes. Our pseudotime trajectory analysis highlights tnnt2a, ryr2b, and myh7bb as genes that increase in expression in a lagged manner compared to mef2cb (Fig. 6). Thus, the abnormal activation of mef2cb without subsequent upregulation of tnnt2a, ryr2b, and myh7bb in rtf1 morphants suggests a requirement for rtf1 in the progressive gene expression changes required for proper cardiac progenitor diRerentiation. Our single cell experiment focuses on the process of cardiac progenitor diRerentiation and does not provide insights into cardiomyocyte maturation. We have edited the text to clarify these interpretations. 

(12) The interpretation that there is not "transfating" is not supported by the shown data. Analysis of markers in other tissues, again with in situ, to show spatially would benefit the study. 

As stated in our response to the reviewer’s public review, we observed a dramatic increase of ALPM cells, but a decrease of ALPM derivatives including the cardiac lineage. We did not observe the expansion of one ALPM-derived subpopulation at the expense of the others. These observations suggest a defect in ALPM diRerentiation and argue against the notion that the region of the ALPM that would normally give rise to cardiac progenitors is instead diRerentiating into another cell type.

(13) The rationale that sequence conservation means a gene is important (lines 137-139) is not really true. There are examples a lot of highly conserved genes whose mutants don't have defects. 

We have revised the text to avoid confusion. 

(14) The data showing that the 8 bp mutations do not aUect the RNA transcript is not shown or at least indicated in Fig. 7. It would seem that this experiment could have been done in the mutant embryos, in which case the experiment would have been semi-blinded as the genotyping would occur after imaging. 

The modified Rtf1 wt RNA (Rtf1 wt* in revised Fig. 7) robustly rescued nkx2.5 expression in rtf1 deficient embryos, demonstrating that the 8 bp modifications do not negatively impact the activity of the injected RNA. As stated previously, morpholino knockdown was used in some experiments because our characterization of rtf1 morphants showed that they faithfully recapitulate the rtf1 mutant phenotype during the timeframe of interest.

(15) Using a technique like PRO-seq at the same stage as the ChIP-seq would complement the ChIP-seq and allow a more detailed analysis of the transcriptional pausing on specific genes observed in WT and mutant embryos. 

As stated in our response to the reviewer’s public review, we appreciate the suggestion but PRO-seq is beyond the scope of this study.

“Here” = accepting that the old story might not come back.

Again, not a physical place — a different way of orienting yourself.

Life may get materially harder

Stability isn’t guaranteed

Growth may stop or reverse

Climate shocks are normal, not exceptional

Community matters more than status

“There” = the world we were trained to believe in.

It’s not a physical place. It’s a mindset + system.

Go to school → get a good job → keep climbing

Growth is always good

Convenience = progress

Politics can fix things eventually

If you work hard enough, life will improve

Most of daily life still runs on this logic:

mortgages

careers

productivity

status

resumes

elections as salvation

You can’t opt out of There.

There is the normal world we all still operate in—jobs, bills, career, elections, productivity. Here is the part of me that’s accepted that the old ‘everything keeps getting better’ story might not come back, so I’m trying to build a more grounded life: community, skills, relationships, resilience

There as playing a game you know is ending

Here as learning skills for the next game

Collapse awareness is the mental shift from:

“Things are broken, but they’ll probably be fixed soon”

to:

“Some things may not be fixed, and I should plan my life accordingly”

eLife Assessment

In the gram-positive model organism Bacillus subtilis, the membrane associated ParA family member MinD, concentrates the division inhibitor MinC at cell poles where it prevents aberrant division events. This important study presents compelling data suggesting that polar localization of MinCD is largely due to differences in diffusion rates between monomeric and dimeric MinD. This finding is exciting as it negates the necessity for a third, localization determinant, in this system as has been proposed by previous investigations.

Reviewer #1 (Public review):

The authors used fluorescence microscopy, image analysis, and mathematical modeling to study the effects of membrane affinity and diffusion rates of MinD monomer and dimer states on MinD gradient formation in B. subtilis. To test these effects, the authors experimentally examined MinD mutants that lock the protein in specific states, including Apo monomer (K16A), ATP-bound monomer (G12V) and ATP-bound dimer (D40A, hydrolysis defective), and compared to wild-type MinD. Overall, the experimental results support the conclusions that reversible membrane binding of MinD is critical for the formation of the MinD gradient, but the binding affinities between monomers and dimers are similar.

The modeling part is a new attempt to use the Monte Carlo method to test the conditions for the formation of the MinD gradient in B. subtilis. The modeling results provide good support for the observations and find that the MinD gradient is sensitive to different diffusion rates between monomers and dimers. This simulation is based on several assumptions and predictions, which raises new questions that need to be addressed experimentally in the future.

Reviewer #3 (Public review):

This important study by Bohorquez et al examines the determinants necessary for concentrating the spatial modulator of cell division, MinD, at the future site of division and the cell poles. Proper localization of MinD is necessary to bring the division inhibitor, MinC, in proximity to the cell membrane and cell poles where it prevents aberrant assembly of the division machinery. In contrast to E. coli, in which MinD oscillates from pole-to-pole courtesy of a third protein MinE, how MinD localization is achieved in B. subtilis-which does not encode a MinE analog-has remained largely a mystery. The authors present compelling data indicating that MinD dimerization is dispensable for membrane localization but required for concentration at the cell poles. Dimerization is also important for interactions between MinD and MinC, leading to the formation of large protein complexes. Computational modeling, specifically a Monte Carlo simulation, supports a model in which differences in diffusion rates between MinD monomers and dimers lead to concentration of MinD at cell poles. Once there, interaction with MinC increases the size of the complex, further reinforcing diffusion differences. Notably, interactions with MinJ-which has previously been implicated in MinCD localization, are dispensable for concentrating MinD at cell poles although MinJ may help stabilize the MinCD complex at those locations.

[Editor's note: The editors and reviewers have no further comments and encourage the authors to proceed with a Version of Record.]

Author response:

The following is the authors’ response to the previous reviews

Public Review:

Reviewer #1 (Public review):

The authors used fluorescence microscopy, image analysis, and mathematical modeling to study the effects of membrane affinity and diffusion rates of MinD monomer and dimer states on MinD gradient formation in B. subtilis. To test these effects, the authors experimentally examined MinD mutants that lock the protein in specific states, including Apo monomer (K16A), ATP-bound monomer (G12V) and ATP-bound dimer (D40A, hydrolysis defective), and compared to wild-type MinD. Overall, the experimental results support the conclusions that reversible membrane binding of MinD is critical for the formation of the MinD gradient, but the binding affinities between monomers and dimers are similar.

The modeling part is a new attempt to use the Monte Carlo method to test the conditions for the formation of the MinD gradient in B. subtilis. The modeling results provide good support for the observations and find that the MinD gradient is sensitive to different diffusion rates between monomers and dimers. This simulation is based on several assumptions and predictions, which raises new questions that need to be addressed experimentally in the future.  

Reviewer #3 (Public review):

This important study by Bohorquez et al examines the determinants necessary for concentrating the spatial modulator of cell division, MinD, at the future site of division and the cell poles. Proper localization of MinD is necessary to bring the division inhibitor, MinC, in proximity to the cell membrane and cell poles

where it prevents aberrant assembly of the division machinery. In contrast to E. coli, in which MinD 50 oscillates from pole-to-pole courtesy of a third protein MinE, how MinD localization is achieved in B. 51 subtilis-which does not encode a MinE analog-has remained largely a mystery. The authors present 52 compelling data indicating that MinD dimerization is dispensable for membrane localization but required 53 for concentration at the cell poles. Dimerization is also important for interactions between MinD and MinC, 54 leading to the formation of large protein complexes. Computational modeling, specifically a Monte Carlo 55 simulation, supports a model in which differences in diffusion rates between MinD monomers and dimers 56 lead to concentration of MinD at cell poles. Once there, interaction with MinC increases the size of the 57 complex, further reinforcing diffusion differences. Notably, interactions with MinJ-which has previously 58 been implicated in MinCD localization, are dispensable for concentrating MinD at cell poles although MinJ may help stabilize the MinCD complex at those locations.

Comments on revisions:  

I believe the authors put respectable effort into revisions and addressing reviewer comments, particularly 64      those that focused on the strengths of the original conclusions. The language in the current version of the manuscript is more precise and the overall product is stronger.  

We are happy to learn that the reviewer considers our manuscript ready for publication.  

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):  

The author has adequately answered the questions that were raised in my previous comments. There are only few minor revisions needed for improvement.  

Line 48−49: 'These proteins ensure that cell division occurs at midcell and not close to nascent division sites or cell poles'  

delete 'nascent division site'  

This has now been corrected as suggested.

Line 64−65: 'MinC inhibits polymerization of FtsZ by direct protein-protein interactions and needs to bind to the Walker A-type ATPase MinD for its recruitment to septa or the polar regions of the cell'

delete 'septa or', because MinD recruits MinC to the cell poles to block polar division, not septal formation.  

This has now been corrected as suggested.

Supplemental information:

Some parameters in Table S1 are missing definitions. If these parameters relate to terms described in the "Methods" section, please add the corresponding parameter symbols after the terms.  

We would like to thank the reviewer for pointing this out. We have improved Table S1 and corrected the related parameters in the Methods section (lines 605-619).

eLife Assessment

Ge et al here report a structural study of the native tripartite multidrug efflux pump complexes from Escherichia coli that identifies a novel accessory subunit, YbjP, the structure of the native TolC-YbjP-AcrABZ complex, as well as structures of the AcrB protein in L, T, and O conformations. The strength of the structural data is compelling, and the importance of the findings is potentially fundamental. However, additional analysis and comparison with pre-existing data would help to put the obtained data and its impact in the proper context, and the inclusion of functional data would help to substantiate some claims that are currently incompletely supported.

Reviewer #1 (Public review):

Summary:

This manuscript investigates the biological mechanism underlying the assembly and transport of the AcrAB-TolC efflux pump complex. By combining endogenous protein purification with cryo-EM analysis, the authors show that the AcrB trimer adopts three distinct conformations simultaneously and identify a previously uncharacterized lipoprotein, YbjP, as a potential additional component of the complex. The work aims to advance our understanding of the AcrAB-TolC efflux system in near-native conditions and may have broader implications for elucidating its physiological mechanism.

Strengths:

Overall, the manuscript is clearly presented, and several of the datasets are of high quality. The use of natively isolated complexes is a major strength, as it minimizes artifacts associated with reconstituted systems and enables the discovery of a novel subunit. The authors also distinguish two major assemblies-the TolC-YbjP sub-complex and the complete pump-which appear to correspond to the closed and open channel states, respectively. The conceptual advance is potentially meaningful, and the findings could be of broad interest to the field.

Weaknesses:

(1) As the identification of YbjP is a key contribution of this work, a deeper comparison with functional "anchor" proteins in other efflux pumps is needed. Including an additional supplementary figure illustrating these structural comparisons would be valuable.

(2) The observation of the LTO states in the presence of TolC represents an important extension of previous findings. A more detailed discussion comparing these LTO states to those reported in earlier structural and biochemical studies would improve the clarity and significance of this point.

Reviewer #2 (Public review):

Summary:

This manuscript reports the high-resolution cryo-EM structures of the endogenous TolC-YbjP-AcrABZ complex and a TolC-YbjP subcomplex from E. coli, identifying a novel accessory subunit. This work is an impressive effort that provides valuable structural insights into this native complex.

Strengths:

(1) The study successfully determines the structure of the complete, endogenously purified complex, marking a significant achievement.

(2) The identification of a previously unknown accessory subunit is an important finding.

(3) The use of cryo-EM to resolve the complex, including potential post-translational modifications such as N-palmitoyl and S-diacylglycerol, is a notable highlight.

Weaknesses:

(1) Clarity and Interpretation: Several points need clarification. Additionally, the description of the sample preparation method, which is a key strength, is currently misplaced and should be introduced earlier.

(2) Data Presentation: The manuscript would benefit significantly from improved figures.

(3) Supporting Evidence: The inclusion of the protein purification profile as a supplementary figure is essential. Furthermore, a discussion comparing the endogenous AcrB structure to those obtained in other systems (e.g., liposomes) and commenting on observed lipid densities would strengthen the overall analysis.

Reviewer #3 (Public review):

Summary:

The manuscript "Structural mechanisms of pump assembly and drug transport in the AcrAB-TolC efflux system" by Ge et al. describes the identification of a previously uncharacterized lipoprotein, YbjP, as a novel partner of the well-studied Enterobacterial tripartite efflux pump AcrAB-TolC. The authors present cryo-electron microscopy structures of the TolC-YbjP subcomplex and the complete AcrABZ-TolC-YbjP assembly. While the identification and structural characterization of YbjP are potentially novel, the stated focus of the manuscript-mechanisms of pump assembly and drug transport - is not sufficiently addressed. The manuscript requires reframing to emphasize the principal novelty associated with YbjP and significant development of the other aspects, especially the claimed novelty of the AcrB drug-efflux cycle.

Strengths:

The reported association of YbjP with AcrAB-TolC is novel; however, a recent deposition of a preceding and much more detailed manuscript to the BioRxiv server (Horne et al., https://doi.org/10.1101/2025.03.19.644130) removes much of the immediate novelty.

Weaknesses:

While the identification of YbjP is novel, the authors do not appear to acknowledge the precedence of another work (Horne et al., 2025), and it is not cited within the correct context in the manuscript.

Several results presented in the TolC-YbjP section do not represent new findings regarding TolC structure itself. The structure and gating behaviour of TolC should be more thoroughly introduced in the Introduction, including prior work describing channel opening and conformational transitions. The current manuscript does not discuss the mechanistic role of helices H3/H4 and H7/H8 in channel dilation, despite implying that YbjP binding may influence these features. Only the original closed TolC structure is cited, and the manuscript does not address prior mutational studies involving the D396 region, though this residue is specifically highlighted in the presented structures.

The manuscript provides only a general structural alignment between the closed TolC-YbjP subcomplex and the open TolC observed in the full pump assembly. However, multiple open, closed, and intermediate conformations of AcrAB-TolC have already been reported. Thus, YbjP alone cannot be assumed to account for TolC channel gating. A systematic comparison with existing structures is necessary to determine whether YbjP contributes any distinct allosteric modulation.

The analysis of AcrB peristaltic action is superficial, poorly substantiated and importantly, not novel. Several references to the ATP-synthase cycle have been provided, but this has been widely established already some 20 years ago - e.g. https://www.science.org/doi/10.1126/science.1131542.

The most significant limitation of the study is the absence of functional characterization of YbjP in vivo or in vitro. While the structural association between YbjP and TolC is interesting, the biological role of YbjP remains unclear. Moreover, the manuscript does not examine structural differences between the presented complex and previously solved AcrAB-TolC or MexAB-OprM assemblies that might support a mechanistic model.

Author response:

Public Reviews:

Reviewer #1 (Public review): 

Summary: 

This manuscript investigates the biological mechanism underlying the assembly and transport of the AcrAB-TolC efflux pump complex. By combining endogenous protein purification with cryo-EM analysis, the authors show that the AcrB trimer adopts three distinct conformations simultaneously and identify a previously uncharacterized lipoprotein, YbjP, as a potential additional component of the complex. The work aims to advance our understanding of the AcrAB-TolC efflux system in near-native conditions and may have broader implications for elucidating its physiological mechanism. 

Strengths: 

Overall, the manuscript is clearly presented, and several of the datasets are of high quality. The use of natively isolated complexes is a major strength, as it minimizes artifacts associated with reconstituted systems and enables the discovery of a novel subunit. The authors also distinguish two major assemblies-the TolC-YbjP sub-complex and the complete pump-which appear to correspond to the closed and open channel states, respectively. The conceptual advance is potentially meaningful, and the findings could be of broad interest to the field. 

Weaknesses: 

(1) As the identification of YbjP is a key contribution of this work, a deeper comparison with functional "anchor" proteins in other efflux pumps is needed. Including an additional supplementary figure illustrating these structural comparisons would be valuable. 

We appreciate this helpful suggestion. We will expand the comparative analysis between YbjP and established anchoring or accessory components in other efflux pumps, and we will add a new supplementary figure illustrating these structural relationships.

(2) The observation of the LTO states in the presence of TolC represents an important extension of previous findings. A more detailed discussion comparing these LTO states to those reported in earlier structural and biochemical studies would improve the clarity and significance of this point. 

We agree. In the revised manuscript we will expand our discussion of the LTO conformations, including a direct comparison with previously reported structural and biochemical observations, to better contextualize the significance of our findings.

Reviewer #2 (Public review): 

Summary: 

This manuscript reports the high-resolution cryo-EM structures of the endogenous TolC-YbjP-AcrABZ complex and a TolC-YbjP subcomplex from E. coli, identifying a novel accessory subunit. This work is an impressive effort that provides valuable structural insights into this native complex. 

Strengths: 

(1) The study successfully determines the structure of the complete, endogenously purified complex, marking a significant achievement. 

(2) The identification of a previously unknown accessory subunit is an important finding. 

(3) The use of cryo-EM to resolve the complex, including potential post-translational modifications such as N-palmitoyl and S-diacylglycerol, is a notable highlight. 

Weaknesses: 

(1) Clarity and Interpretation: Several points need clarification. Additionally, the description of the sample preparation method, which is a key strength, is currently misplaced and should be introduced earlier. 

Thank you for pointing this out. We will reorganize the text to introduce the sample preparation strategy earlier and clarify the points that may cause ambiguity.

(2) Data Presentation: The manuscript would benefit significantly from improved figures. 

We agree and will revise the figures to improve clarity, consistency, and readability. Additional schematic illustrations will also be included where appropriate.

(3) Supporting Evidence: The inclusion of the protein purification profile as a supplementary figure is essential. Furthermore, a discussion comparing the endogenous AcrB structure to those obtained in other systems (e.g., liposomes) and commenting on observed lipid densities would strengthen the overall analysis. 

We appreciate these suggestions. We will add the purification profile and expand the comparison between our endogenous AcrB structure and previously reported structures from reconstituted systems, including a more detailed discussion of lipid densities.

Reviewer #3 (Public review): 

Summary: 

The manuscript "Structural mechanisms of pump assembly and drug transport in the AcrAB-TolC efflux system" by Ge et al. describes the identification of a previously uncharacterized lipoprotein, YbjP, as a novel partner of the well-studied Enterobacterial tripartite efflux pump AcrAB-TolC. The authors present cryo-electron microscopy structures of the TolC-YbjP subcomplex and the complete AcrABZ-TolC-YbjP assembly. While the identification and structural characterization of YbjP are potentially novel, the stated focus of the manuscript-mechanisms of pump assembly and drug transport - is not sufficiently addressed. The manuscript requires reframing to emphasize the principal novelty associated with YbjP and significant development of the other aspects, especially the claimed novelty of the AcrB drug-efflux cycle. 

Strengths: 

The reported association of YbjP with AcrAB-TolC is novel; however, a recent deposition of a preceding and much more detailed manuscript to the BioRxiv server (Horne et al., https://doi.org/10.1101/2025.03.19.644130) removes much of the immediate novelty. 

Weaknesses: 

While the identification of YbjP is novel, the authors do not appear to acknowledge the precedence of another work (Horne et al., 2025), and it is not cited within the correct context in the manuscript. 

We thank the reviewer for rasising this important point regarding the independent nature of our work.

Our study indeed progressed independently. The process began with our purification of an endogenous protein sample containing the AcrAB-TolC efflux pump. During our cryo-EM analysis, we observed an unassigned density in the map, for which we built a preliminary main-chain model. A subsequent search of structural databases, including AlphaFold predictions, allowed us to identify this density as the protein YbjP. It was only after this identification that we became aware of the related preprint by Horne et al. on BioRxvi (Posted March 19, 2025).

Therefore, our structural determination of YbjP was conducted entirely independently. We fully acknowledge and respect the work by Horne et al. and have already cited their reprint in our manuscript. While their detailed structural data, maps, and coordinates are not yet publicly available, we have described their findings appropriately. We agree that our manuscript can better reflect this context and will carefully check for any missing citations to ensure that their contribution is properly and clearly acknowledged.

We also believe that the two studies are mutually complementary and collectively reinforce the emerging understanding of YbjP.

Several results presented in the TolC-YbjP section do not represent new findings regarding TolC structure itself.

We agree that the TolC features we describe are consistent with previously reported structural characteristics. However, these observations could only be confirmed in the context of the newly determined TolC–YbjP subcomplex, which was not available prior to this study. We will clarify this point in the revision to avoid overstating novelty.

The structure and gating behaviour of TolC should be more thoroughly introduced in the Introduction, including prior work describing channel opening and conformational transitions.

We appreciate this suggestion and agree that a more comprehensive overview of TolC gating and conformational transitions will strengthen the Introduction. We will revise the text to incorporate relevant prior structural and functional studies.

The current manuscript does not discuss the mechanistic role of helices H3/H4 and H7/H8 in channel dilation, despite implying that YbjP binding may influence these features.

Thank you for this comment. The primary novel contributions of this manuscript are the identification of YbjP and the structural characterization of AcrB in three distinct states. The discussion of the dilation mechanism, while included because we observed the closed TolC-YbjP state, is a secondary point. In the revised manuscript, we will expand this discussion as suggested.

Only the original closed TolC structure is cited, and the manuscript does not address prior mutational studies involving the D396 region, though this residue is specifically highlighted in the presented structures. 

We appreciate the reviewer drawing attention to this oversight. We will add citations to the relevant mutational and mechanistic studies, including those involving the D396 region, and more clearly discuss these findings in relation to our structural observations.

The manuscript provides only a general structural alignment between the closed TolC-YbjP subcomplex and the open TolC observed in the full pump assembly. However, multiple open, closed, and intermediate conformations of AcrAB-TolC have already been reported. Thus, YbjP alone cannot be assumed to account for TolC channel gating. A systematic comparison with existing structures is necessary to determine whether YbjP contributes any distinct allosteric modulation. 

We agree with the reviewer’s assessment and appreciate the constructive suggestion. In our revised manuscript, we will expand the structural comparison to include previously reported open, closed, and intermediate AcrAB–TolC conformations. This expanded analysis will more clearly position our findings within the existing structural framework.

The analysis of AcrB peristaltic action is superficial, poorly substantiated and importantly, not novel. Several references to the ATP-synthase cycle have been provided, but this has been widely established already some 20 years ago - e.g. https://www.science.org/doi/10.1126/science.1131542. 

We thank the reviewer for this comment. We fully acknowledge the foundational studies that established the AcrB functional cycle and its analogy to the ATP-synthase mechanism. While previous work indeed defined the LTO (Loose, Tight, Open) cycle of AcrB, those structures were obtained using AcrB in isolation. In contrast, our endogenous sample, which includes the native constraints of AcrA from above and the presence of AcrZ, reveals conformational changes in the transmembrane and porter domains that differ from those previously reported. We interpret these differences as reflecting a more physiologically relevant mechanism. In our revision, we will provide a detailed discussion to contextualize these distinctions within the existing literature.

The most significant limitation of the study is the absence of functional characterization of YbjP in vivo or in vitro. While the structural association between YbjP and TolC is interesting, the biological role of YbjP remains unclear.

We agree that the lack of functional characterization is a limitation of the present work. Our study focuses on structural elucidation and structural analysis. Although the recent preprint you mentioned suggests that YbjP deletion may not produce a strong phenotype, we are still interested in conducting additional experiments to explore its potential roles in future work. We will revise the text to clearly acknowledge this limitation.

Moreover, the manuscript does not examine structural differences between the presented complex and previously solved AcrAB-TolC or MexAB-OprM assemblies that might support a mechanistic model.

We thank the reviewer for this suggestion. We will incorporate a more detailed comparative analysis with existing AcrAB–TolC and MexAB–OprM structures and highlight similarities and differences that may inform mechanistic interpretation.

Range of downloads wrt the event organising by Adrian Segar

[[Event Crowdsourcing by Adrian Segar]] 2019

[[The Power of Participation by Adrian Segar]] 2015

eLife Assessment

This important study identifies PRRT2 as an auxiliary regulator of Nav channel slow inactivation, proposing that PRRT2 facilitates entry into, and delays recovery from, the slow-inactivated state. The evidence provided is compelling and well executed, though the work would be bolstered by additional studies of Nav1.6, as well as structural studies to directly investigate the molecular basis of gating modulation. Overall, this study will be of interest to ion channel biophysicists and neurophysiologists, particularly those studying channelopathies.

Reviewer #1 (Public review):

Summary:

The manuscript by Lu and colleagues demonstrates convincingly that PRRT2 interacts with brain voltage-gated sodium channels to enhance slow inactivation in vitro and in vivo. The work is interesting and rigorously conducted. The relevance to normal physiology and disease pathophysiology (e.g., PRRT2-related genetic neurodevelopmental disorders) seems high. Some simple additional experiments could elevate the impact and make the study more complete.

Strengths:

Experiments are conducted rigorously, including experimenter blinding and appropriate controls. Data presentation is excellent and logical. The paper is well written for a general scientific audience.

Weaknesses:

There are a few missing experiments and one place where data are over-interpreted.

(1) An in vitro study of Nav1.6 is conspicuously absent. In addition to being a major brain Na channel, Nav1.6 is predominant in cerebellar Purkinje neurons, which the authors note lack PRRT2 expression. They speculate that the absence of PRRT2 in these neurons facilitates the high firing rate. This hypothesis would be strengthened if PRRT2 also enhanced slow inactivation of Nav1.6. If a stable Nav1.6 cell were not available, then simple transient co-transfection experiments would suffice.

(2) To further demonstrate the physiological impact of enhanced slow inactivation, the authors should consider a simple experiment in the stable cell line experiments (Figure 1) to test pulse frequency dependence of peak Na current. One would predict that PRRT2 expression will potentiate 'run down' of the channels, and this finding would be complementary to the biophysical data.

(3) The study of one K channel is limited, and the conclusion from these experiments represents an over-interpretation. I suggest removing these data unless many more K channels (ideally with measurable proxies for slow inactivation) were tested. These data do not contribute much to the story.

(4) In Figure 2, the authors should confirm that protein is indeed expressed in cells expressing each truncated PRRT2 construct. Absent expression should be ruled out as an explanation for absent enhancement of slow inactivation.

Reviewer #2 (Public review):

Summary:

As a member of DspB subfamily, PRRT2 is primarily expressed in the nervous system and has been associated with various paroxysmal neurological disorders. Previous studies have shown that PRRT2 directly interacts with Nav1.2 and Nav1.6, modulating channel properties and neuronal excitability.

In this study, Lu et al. reported that PRRT2 is a physiological regulator of Nav channel slow inactivation, promoting the development of Nav slow inactivation and impeding the recovery from slow inactivation. This effect can be replicated by the C-terminal region (256-346) of PRRT2, and is highly conserved across species from zebrafish, mouse, to human PRRT2. TRARG1 and TMEM233, the other two DspB family members, showed similar effects on Nav1.2 slow inactivation. Co-IP data confirms the interaction between Nav channels and PRRT2. Prrt2-mutant mice, which lack PRRT2 expression, require lower stimulation thresholds for evoking after-discharges when compared to WT mice.

Strengths:

(1) This study is well designed, and data support the conclusion that PRRT2 is a potent regulator of slow inactivation of Nav channels.

(2) This study reveals similar effects on Nav1.2 slow inactivation by PRRT2, TMEM233, and TRARG1, indicating a common regulation of Nav channels by DspB family members (Supplemental Figure 2). A recent study has shown that TMEM233 is essential for ExTxA (a plant toxin)-mediated inhibition on fast inactivation of Nav channels; and PRRT2 and TRARG1 could replicate this effect (Jami S, et al. Nat Commun 2023). It is possible that all three DspB members regulate Nav channel properties through the same mechanism, and exploring molecules that target PRRT2/TRARG1/TMEM233 might be a novel strategy for developing new treatments of DspB-related neurological diseases.

Weaknesses:

(1) Previously, the authors have reported that PRRT2 reduces Nav1.2 current density and alters biophysical properties of both Nav1.2 and Nav1.6 channels, including enhanced steady-state inactivation, slower recovery, and stronger use-dependent inhibition (Lu B, et al. Cell Rep 2021, Fig 3 & S5). All those changes are expected to alter neuronal excitability and should be discussed.

(2) In this study, the fast inactivation kinetics was examined by a single stimulus at 0 mV, which may not be sufficient for the conclusion. Inactivation kinetics at more voltage potentials should be added.

(3) It is a little surprising that there is no difference in Nav1.2 current density in axon-blebs between WT and Prrt2-mutant mice (Figure 7B). PRRT2 significantly shifts steady-state slow inactivation curve to hyperpolarizing direction, at -70 mV, nearly 70% of Nav1.2 channels are inactivated by slow inactivation in cells expressing PRRT2 when compared to less than 10% in cells expressing GFP (Figure supplement 1B); with a holding potential of -70 mV, I would expect that most of Nav channels are inactivated in axon-blebs from WT mice but not in axon-blebs from Prrt2-mutant mice, and therefore sodium current density should be different in Figure 7B, which was not. Any explanation?

(3) Besides Nav channels, PRRT2 has been shown to act on Cav2.1 channels as well as molecules involved in neurotransmitter release, which may also contribute to abnormal neuronal activity in Prrt2-mutant mice. These should be mentioned when discussing PRRT2's role in neuronal resilience.

Reviewer #3 (Public review):

This paper reveals that the neuronal protein PRRT2, previously known for its association with paroxysmal dyskinesia and infantile seizures, modulates the slow inactivation of voltage-gated sodium ion (Nav) channels, a gating process that limits excitability during prolonged activity. Using electrophysiology, molecular biology, and mouse models, the authors show that PRRT2 accelerates entry of Nav channels into the slow-inactivated state and slows their recovery, effectively dampening excessive excitability. The effect seems evolutionarily conserved, requires the C-terminal region of PRRT2, and is recapitulated in cortical neurons, where PRRT2 deficiency leads to hyper-responsiveness and reduced cortical resilience in vivo. These findings extend the functional repertoire of PRRT2, identifying it as a physiological brake on neuronal excitability. The work provides a mechanistic link between PRRT2 mutations and episodic neurological phenotypes.

Comments:

(1) The precise structural interface and the molecular basis of gating modulation remain inferred rather than demonstrated.

(2) The in vivo phenotype reflects a complex circuit outcome and does not isolate slow-inactivation defects per se.

(3) Expression of PRRT2 in muscle or heart is low, so the cross isoform claims are likely of limited physiological significance.

(4) The mechanistic separation between the trafficking of PRRT2 and its gating effects is not clearly resolved.

(5) Additional studies with Nav1.6 should be carried out.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript by Lu and colleagues demonstrates convincingly that PRRT2 interacts with brain voltage-gated sodium channels to enhance slow inactivation in vitro and in vivo. The work is interesting and rigorously conducted. The relevance to normal physiology and disease pathophysiology (e.g., PRRT2-related genetic neurodevelopmental disorders) seems high. Some simple additional experiments could elevate the impact and make the study more complete.

Strengths:

Experiments are conducted rigorously, including experimenter blinding and appropriate controls. Data presentation is excellent and logical. The paper is well written for a general scientific audience.

Weaknesses:

There are a few missing experiments and one place where data are over-interpreted.

(1) An in vitro study of Nav1.6 is conspicuously absent. In addition to being a major brain Na channel, Nav1.6 is predominant in cerebellar Purkinje neurons, which the authors note lack PRRT2 expression. They speculate that the absence of PRRT2 in these neurons facilitates the high firing rate. This hypothesis would be strengthened if PRRT2 also enhanced slow inactivation of Nav1.6. If a stable Nav1.6 cell were not available, then simple transient co-transfection experiments would suffice.

We thank the reviewer for this suggestion. In the revised manuscript, we will examine whether PRRT2 modulates slow inactivation of Nav1.6 channels using heterologous co-expression experiments.

(2) To further demonstrate the physiological impact of enhanced slow inactivation, the authors should consider a simple experiment in the stable cell line experiments (Figure 1) to test pulse frequency dependence of peak Na current. One would predict that PRRT2 expression will potentiate 'run down' of the channels, and this finding would be complementary to the biophysical data.

We agree that examining pulse frequency-dependent changes in peak sodium current would provide a functional readout linking PRRT2-mediated enhancement of slow inactivation to use-dependent channel availability. In the revision, we will include a pulse-train protocol to quantify use-dependent attenuation (“run-down”) of peak sodium current across stimulation trains and will compare this adaptation between control and PRRT2-expressing conditions.

(3) The study of one K channel is limited, and the conclusion from these experiments represents an over-interpretation. I suggest removing these data unless many more K channels (ideally with measurable proxies for slow inactivation) were tested. These data do not contribute much to the story.

We agree with the reviewer’s assessment. To avoid over-interpretation and to maintain focus on PRRT2-dependent regulation of Nav channel slow inactivation, we will remove potassium channel dataset and the associated conclusions from the revised manuscript.

(4) In Figure 2, the authors should confirm that protein is indeed expressed in cells expressing each truncated PRRT2 construct. Absent expression should be ruled out as an explanation for the enhancement of slow inactivation.

We appreciate the reviewer’s concern regarding expression of the truncated PRRT2 constructs in the Nav1.2 stable cell line, particularly PRRT2(1-266), which shows little effect on slow inactivation of Nav1.2 channels. In the revision, we will include expression controls for each truncation construct in the Nav1.2-expressing cells to rule out lack of expression as an explanation for the observed functional differences.

Reviewer #2 (Public review):

Summary:

As a member of DspB subfamily, PRRT2 is primarily expressed in the nervous system and has been associated with various paroxysmal neurological disorders. Previous studies have shown that PRRT2 directly interacts with Nav1.2 and Nav1.6, modulating channel properties and neuronal excitability.

In this study, Lu et al. reported that PRRT2 is a physiological regulator of Nav channel slow inactivation, promoting the development of Nav slow inactivation and impeding the recovery from slow inactivation. This effect can be replicated by the C-terminal region (256-346) of PRRT2, and is highly conserved across species from zebrafish, mouse, to human PRRT2. TRARG1 and TMEM233, the other two DspB family members, showed similar effects on Nav1.2 slow inactivation. Co-IP data confirms the interaction between Nav channels and PRRT2. Prrt2-mutant mice, which lack PRRT2 expression, require lower stimulation thresholds for evoking after-discharges when compared to WT mice.

Strengths:

(1) This study is well designed, and data support the conclusion that PRRT2 is a potent regulator of slow inactivation of Nav channels.

(2) This study reveals similar effects on Nav1.2 slow inactivation by PRRT2, TMEM233, and TRARG1, indicating a common regulation of Nav channels by DspB family members (Supplemental Figure 2). A recent study has shown that TMEM233 is essential for ExTxA (a plant toxin)-mediated inhibition on fast inactivation of Nav channels; and PRRT2 and TRARG1 could replicate this effect (Jami S, et al. Nat Commun 2023). It is possible that all three DspB members regulate Nav channel properties through the same mechanism, and exploring molecules that target PRRT2/TRARG1/TMEM233 might be a novel strategy for developing new treatments of DspB-related neurological diseases.

Weaknesses:

(1) Previously, the authors have reported that PRRT2 reduces Nav1.2 current density and alters biophysical properties of both Nav1.2 and Nav1.6 channels, including enhanced steady-state inactivation, slower recovery, and stronger use-dependent inhibition (Lu B, et al. Cell Rep 2021, Fig 3 & S5). All those changes are expected to alter neuronal excitability and should be discussed.

We agree that PRRT2 has been reported to exert multiple effects on Nav channels which are all expected to influence neuronal excitability (Fruscione et al., 2018; Lu et al., 2021; Valente et al., 2023). In the revised manuscript, we will expand the Discussion to integrate these prior findings and to clarify how these PRRT2-dependent changes may interact with (and potentially converge on) modulation of slow inactivation to shape neuronal excitability.

(2) In this study, the fast inactivation kinetics was examined by a single stimulus at 0 mV, which may not be sufficient for the conclusion. Inactivation kinetics at more voltage potentials should be added.

We thank the reviewer for this suggestion. In the revision, we will extend our analysis of Nav1.2 fast-inactivation kinetics across a range of test potentials (e.g., -20, -10, 0, +10 and +20 mV) in the presence and absence of PRRT2.

(3) It is a little surprising that there is no difference in Nav1.2 current density in axon-blebs between WT and Prrt2-mutant mice (Figure 7B). PRRT2 significantly shifts steady-state slow inactivation curve to hyperpolarizing direction, at -70 mV, nearly 70% of Nav1.2 channels are inactivated by slow inactivation in cells expressing PRRT2 when compared to less than 10% in cells expressing GFP (Figure supplement 1B); with a holding potential of -70 mV, I would expect that most of Nav channels are inactivated in axon-blebs from WT mice but not in axon-blebs from Prrt2-mutant mice, and therefore sodium current density should be different in Figure 7B, which was not. Any explanation?

We appreciate the reviewer for raising this point. In our axonal bleb recordings, although the holding potential was -70 mV, sodium current density was measured after a hyperpolarizing pre-pulse (-110 mV) to relieve inactivation immediately prior to the test depolarization (as described in the Methods). Thus, the current density measurement in Figure 7B reflects the maximal available current following this recovery step, rather than the steady-state availability at -70 mV. In the revision, we will state this explicitly in the Results and/or figure legend to avoid confusion.

(4) Besides Nav channels, PRRT2 has been shown to act on Cav2.1 channels as well as molecules involved in neurotransmitter release, which may also contribute to abnormal neuronal activity in Prrt2-mutant mice. These should be mentioned when discussing PRRT2's role in neuronal resilience.

We agree with the reviewer. In the revised manuscript, we will broaden the Discussion to acknowledge PRRT2 functions beyond Nav channels, including reported roles in Cav2.1 regulation and neurotransmitter release. We will frame the in vivo phenotypes in Prrt2-mutant mice as likely arising from convergent mechanisms—altered intrinsic excitability together with changes in synaptic transmission.

Reviewer #3 (Public review):

This paper reveals that the neuronal protein PRRT2, previously known for its association with paroxysmal dyskinesia and infantile seizures, modulates the slow inactivation of voltage-gated sodium ion (Nav) channels, a gating process that limits excitability during prolonged activity. Using electrophysiology, molecular biology, and mouse models, the authors show that PRRT2 accelerates entry of Nav channels into the slow-inactivated state and slows their recovery, effectively dampening excessive excitability. The effect seems evolutionarily conserved, requires the C-terminal region of PRRT2, and is recapitulated in cortical neurons, where PRRT2 deficiency leads to hyper-responsiveness and reduced cortical resilience in vivo. These findings extend the functional repertoire of PRRT2, identifying it as a physiological brake on neuronal excitability. The work provides a mechanistic link between PRRT2 mutations and episodic neurological phenotypes.

Comments:

(1) The precise structural interface and the molecular basis of gating modulation remain inferred rather than demonstrated.

We thank the reviewer for this comment. In the revision, we will make it explicit that our structural modeling are based on prediction rather than evidential. We will also expand the Limitations section to highlight that direct structural and biochemical mapping of the PRRT2-Nav interface (e.g., through targeted mutagenesis, crosslinking, and/or structural determination) will be required to define the binding interface and establish the molecular basis of gating modulation.

(2) The in vivo phenotype reflects a complex circuit outcome and does not isolate slow-inactivation defects per se.

We agree with the reviewer. In the revision, we will refine the Discussion to avoid over-attributing the in vivo phenotype to slow-inactivation defects alone and to explicitly state that impaired slow inactivation in Prrt2-mutant mice represents one plausible contributing mechanism to reduced cortical resilience, alongside other PRRT2-dependent process.

(3) Expression of PRRT2 in muscle or heart is low, so the cross-isoform claims are likely of limited physiological significance.

We thank the review for your comment about physiological relevance. In the revised manuscript, we will clarify that our Nav isoform panel was designed to assess mechanistic generality at the channel level rather than to imply broad in vivo relevance across tissues. We will also expand the Discussion to emphasize that any therapeutic strategy involving PRRT2 delivery should consider its consistent effect on slow inactivation across multiple Nav isoforms.

(4) The mechanistic separation between the trafficking effect of PRRT2 and its gating effects is not clearly resolved.

We appreciate the reviewer for raising this important point. In the revision, we will expand the Discussion to clarify why we interpret the effect of PRRT2 on slow inactivation as a gating modulation rather than a secondary consequence of altered channel abundance or localization. First, our slow inactivation measurements are expressed as the fraction of available channels after depolarization conditioning relative to baseline availability within the same cell (post-/pre-conditioning), which minimizes confounding by differences in initial surface expression. Second, the slow inactivation of Nav channel occurs on a rapid, activity-dependent timescale (seconds), whereas remarkable changes in trafficking and surface abundance generally develop over longer intervals (minutes to hours).

(5) Additional studies with Nav1.6 should be carried out.

We thank the reviewer’s suggestion. We will include Nav1.6 slow inactivation experiments in the revised manuscript.

Well duh. Anytime you need to release your device(s) to whatever authority it's compromised. Everywhere. The journalists in this case seem unaware of opsec need

eLife Assessment

This important study fills a major geographic and temporal gap in understanding Paleocene mammal evolution in Asia and proposes an intriguing "brawn before bite" hypothesis grounded in diverse analytical approaches. However, the findings are incomplete because limitations in sampling design - such as the use of worn or damaged teeth, the pooling of different tooth positions, and the lack of independence among teeth from the same individuals - introduce uncertainties that weaken support for the reported disparity patterns. The taxonomic focus on predominantly herbivorous clades also narrows the ecological scope of the results. Clarifying methodological choices, expanding the ecological context, and tempering evolutionary interpretations would substantially strengthen the study.

Reviewer #1 (Public review):

Summary:

This work provides valuable new insights into the Paleocene Asian mammal recovery and diversification dynamics during the first ten million years post-dinosaur extinction. Studies that have examined the mammalian recovery and diversification post-dinosaur extinction have primarily focused on the North American mammal fossil record, and it's unclear if patterns documented in North America are characteristic of global patterns. This study examines dietary metrics of Paleocene Asian mammals and found that there is a body size disparity increase before dietary niche expansion and that dietary metrics track climatic and paleobotanical trends of Asia during the first 10 million years after the dinosaur extinction.

Strengths:

The Asian Paleocene mammal fossil record is greatly understudied, and this work begins to fill important gaps. In particular, the use of interdisciplinary data (i.e., climatic and paleobotanical) is really interesting in conjunction with observed dietary metric trends.

Weaknesses:

While this work has the potential to be exciting and contribute greatly to our understanding of mammalian evolution during the first 10 million years post-dinosaur extinction, the major weakness is in the dental topographic analysis (DTA) dataset.

There are several specimens in Figure 1 that have broken cusps, deep wear facets, and general abrasion. Thus, any values generated from DTA are not accurate and cannot be used to support their claims. Furthermore, the authors analyze all tooth positions at once, which makes this study seem comprehensive (200 individual teeth), but it's unclear what sort of noise this introduces to the study. Typically, DTA studies will analyze a singular tooth position (e.g., Pampush et al. 2018 Biol. J. Linn. Soc.), allowing for more meaningful comparisons and an understanding of what value differences mean. Even so, the dataset consists of only 48 specimens. This means that even if all the specimens were pristinely preserved and generated DTA values could be trusted, it's still only 48 specimens (representing 4 different clades) to capture patterns across 10 million years. For example, the authors note that their results show an increase in OPCR and DNE values from the middle to the late Paleocene in pantodonts. However, if a singular tooth position is analyzed, such as the lower second molar, the middle and late Paleocene partitions are only represented by a singular specimen each. With a sample size this small, it's unlikely that the authors are capturing real trends, which makes the claims of this study highly questionable.

Reviewer #2 (Public review):

Summary:

This study uses dental traits of a large sample of Chinese mammals to track evolutionary patterns through the Paleocene. It presents and argues for a 'brawn before bite' hypothesis - mammals increased in body size disparity before evolving more specialized or adapted dentitions. The study makes use of an impressive array of analyses, including dental topographic, finite element, and integration analyses, which help to provide a unique insight into mammalian evolutionary patterns.

Strengths:

This paper helps to fill in a major gap in our knowledge of Paleocene mammal patterns in Asia, which is especially important because of the diversification of placentals at that time. The total sample of teeth is impressive and required considerable effort for scanning and analyzing. And there is a wealth of results for DTA, FEA, and integration analyses. Further, some of the results are especially interesting, such as the novel 'brawn before bite' hypothesis and the possible link between shifts in dental traits and arid environments in the Late Paleocene. Overall, I enjoyed reading the paper, and I think the results will be of interest to a broad audience.

Weaknesses:

I have four major concerns with the study, especially related to the sampling of teeth and taxa, that I discuss in more detail below. Due to these issues, I believe that the study is incomplete in its support of the 'brawn before bite' hypothesis. Although my concerns are significant, many of them can be addressed with some simple updates/revisions to analyses or text, and I try to provide constructive advice throughout my review.

(1) If I understand correctly, teeth of different tooth positions (e.g., premolars and molars), and those from the same specimen, are lumped into the same analyses. And unless I missed it, no justification is given for these methodological choices (besides testing for differences in proportions of tooth positions per time bin; L902). I think this creates some major statistical concerns. For example, DTA values for premolars and molars aren't directly comparable (I don't think?) because they have different functions (e.g., greater grinding function for molars). My recommendation is to perform different disparity-through-time analyses for each tooth position, assuming the sample sizes are big enough per time bin. Or, if the authors maintain their current methods/results, they should provide justification in the main text for that choice.

Also, I think lumping teeth from the same specimen into your analyses creates a major statistical concern because the observations aren't independent. In other words, the teeth of the same individual should have relatively similar DTA values, which can greatly bias your results. This is essentially the same issue as phylogenetic non-independence, but taken to a much greater extreme.

It seems like it'd be much more appropriate to perform specimen-level analyses (e.g., Wilson 2013) or species-level analyses (e.g., Grossnickle & Newham 2016) and report those results in the main text. If the authors believe that their methods are justified, then they should explain this in the text.

(2) Maybe I misunderstood, but it sounds like the sampling is almost exclusively clades that are primarily herbivorous/omnivorous (Pantodonta, Arctostylopida, Anagalida, and maybe Tillodonta), which means that the full ecomorphological diversity of the time bins is not being sampled (e.g., insectivores aren't fully sampled). Similarly, the authors say that they "focused sampling" on those major clades and "Additional data were collected on other clades ... opportunistically" (L628). If they favored sampling of specific clades, then doesn't that also bias their results?

If the study is primarily focused on a few herbivorous clades, then the Introduction should be reframed to reflect this. You could explain that you're specifically tracking herbivore patterns after the K-Pg.

(3) There are a lot of topics lacking background information, which makes the paper challenging to read for non-experts. Maybe the authors are hindered by a short word limit. But if they can expand their main text, then I strongly recommend the following:

(a) The authors should discuss diets. Much of the data are diet correlates (DTA values), but diets are almost never mentioned, except in the Methods. For example, the authors say: "An overall shift towards increased dental topographic trait magnitudes ..." (L137). Does that mean there was a shift toward increased herbivory? If so, why not mention the dietary shift? And if most of the sampled taxa are herbivores (see above comment), then shouldn't herbivory be a focal point of the paper?

(b) The authors should expand on "we used dentitions as ecological indicators" (L75). For non-experts, how/why are dentitions linked to ecology? And, again, why not mention diet? A strong link between tooth shape and diet is a critical assumption here (and one I'm sure that all mammalogists agree with), but the authors don't provide justification (at least in the Introduction) for that assumption. Many relevant papers cited later in the Methods could be cited in the Introduction (e.g., Evans et al. 2007).

(c) Include a better introduction of the sample, such as explicitly stating that your sample only includes placentals (assuming that's the case) and is focused on three major clades. Are non-placentals like multituberculates or stem placentals/eutherians found at Chinese Paleocene fossil localities and not sampled in the study, or are they absent in the sampled area?

(d) The way in which "integration" is being used should be defined. That is a loaded term which has been defined in different ways. I also recommend providing more explanation on the integration analyses and what the results mean.

If the authors don't have space to expand the main text, then they should at least expand on the topics in the supplement, with appropriate citations to the supplement in the main text.

(4) Finally, I'm not convinced that the results fully support the 'brawn before bite' hypothesis. I like the hypothesis. However, the 'brawn before ...' part of the hypothesis assumes that body size disparity (L63) increased first, and I don't think that pattern is ever shown. First, body size disparity is never reported or plotted (at least that I could find) - the authors just show the violin plots of the body sizes (Figures 1B, S6A). Second, the authors don't show evidence of an actual increase in body size disparity. Instead, they seem to assume that there was a rapid diversification in the earliest Paleocene, and thus the early Paleocene bin has already "reached maximum saturation" (L148). But what if the body size disparity in the latest Cretaceous was the same as that in the Paleocene? (Although that's unlikely, note that papers like Clauset & Redner 2009 and Grossnickle & Newham 2016 found evidence of greater body size disparity in the latest Cretaceous than is commonly recognized.) Similarly, what if body size disparity increased rapidly in the Eocene? Wouldn't that suggest a 'BITE before brawn' hypothesis? So, without showing when an increase in body size diversity occurred, I don't think that the authors can make a strong argument for 'brawn before [insert any trait]".

Although it's probably well beyond the scope of the study to add Cretaceous or Eocene data, the authors could at least review literature on body size patterns during those times to provide greater evidence for an earliest Paleocene increase in size disparity.

Author response:

eLife Assessment

This important study fills a major geographic and temporal gap in understanding Paleocene mammal evolution in Asia and proposes an intriguing "brawn before bite" hypothesis grounded in diverse analytical approaches. However, the findings are incomplete because limitations in sampling design - such as the use of worn or damaged teeth, the pooling of different tooth positions, and the lack of independence among teeth from the same individuals - introduce uncertainties that weaken support for the reported disparity patterns. The taxonomic focus on predominantly herbivorous clades also narrows the ecological scope of the results. Clarifying methodological choices, expanding the ecological context, and tempering evolutionary interpretations would substantially strengthen the study.

We thank Dr. Rasmann for the constructive evaluation of our manuscript. Considering the reviewers’ comments, we plan to implement revisions to our study focusing on (1) expansion of the fossil sample description, including a detailed account of the process of excluding extremely worn or damaged teeth from all analyses, (2) expanded reporting of the analyses done on individual tooth positions, and tempering the interpretation of the pooled samples in light of the issues raised by reviewers, (3) providing a more comprehensive introduction that includes an overview of the Paleocene mammal faunas in south China, which unevenly samples certain clades whereas others are extremely rare, and why the current available fossil samples would not permit a whole-fauna analysis to be adequately conducted across the three land mammal age time bins of the Paleocene in China. We believe these revisions would substantially strengthen the study’s robustness and impact for understanding the ecomorphological evolution of the earliest abundant placental mammals during the Paleocene in Asia.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This work provides valuable new insights into the Paleocene Asian mammal recovery and diversification dynamics during the first ten million years post-dinosaur extinction. Studies that have examined the mammalian recovery and diversification post-dinosaur extinction have primarily focused on the North American mammal fossil record, and it's unclear if patterns documented in North America are characteristic of global patterns. This study examines dietary metrics of Paleocene Asian mammals and found that there is a body size disparity increase before dietary niche expansion and that dietary metrics track climatic and paleobotanical trends of Asia during the first 10 million years after the dinosaur extinction.

Strengths:

The Asian Paleocene mammal fossil record is greatly understudied, and this work begins to fill important gaps. In particular, the use of interdisciplinary data (i.e., climatic and paleobotanical) is really interesting in conjunction with observed dietary metric trends.

Weaknesses:

While this work has the potential to be exciting and contribute greatly to our understanding of mammalian evolution during the first 10 million years post-dinosaur extinction, the major weakness is in the dental topographic analysis (DTA) dataset.

There are several specimens in Figure 1 that have broken cusps, deep wear facets, and general abrasion. Thus, any values generated from DTA are not accurate and cannot be used to support their claims. Furthermore, the authors analyze all tooth positions at once, which makes this study seem comprehensive (200 individual teeth), but it's unclear what sort of noise this introduces to the study. Typically, DTA studies will analyze a singular tooth position (e.g., Pampush et al. 2018 Biol. J. Linn. Soc.), allowing for more meaningful comparisons and an understanding of what value differences mean. Even so, the dataset consists of only 48 specimens. This means that even if all the specimens were pristinely preserved and generated DTA values could be trusted, it's still only 48 specimens (representing 4 different clades) to capture patterns across 10 million years. For example, the authors note that their results show an increase in OPCR and DNE values from the middle to the late Paleocene in pantodonts. However, if a singular tooth position is analyzed, such as the lower second molar, the middle and late Paleocene partitions are only represented by a singular specimen each. With a sample size this small, it's unlikely that the authors are capturing real trends, which makes the claims of this study highly questionable.

We thank Reviewer 1 for their careful review of our manuscript. A major external limitation of the application of DTA to fossil samples is the availability of specimens. Whereas a typical study design using extant or geologically younger/more abundant fossil species would preferably sample much larger quantities of teeth from each treatment group (time bins, in our case), the rarity of well-preserved Paleocene mammalian dentitions in Asia necessitates the analysis of small samples in order to make observations regarding major trends in a region and time period otherwise impossible to study (see Chow et al. 1977). That said, we plan to clarify methodological details in response to the reviewer’s comments, including a more comprehensive explanation of our criteria for exclusion of broken tooth crowns from the analyses. We also plan to expand our results reporting on individual tooth position analysis, potentially including resampling and/or simulation analyses to assess the effect of small and uneven samples on our interpretation of results. Lastly, we plan to revise the discussion and conclusion accordingly, including more explicit distinction between well-supported findings that emerge from various planned sensitivity analyses, versus those that are more speculative and tentative in nature.

Chow, M., Zhang, Y., Wang, B., and Ding, S. (1977). Paleocene mammalian fauna from the Nanxiong Basin, Guangdong Province. Paleontol. Sin. New Ser. C 20, 1–100.

Reviewer #2 (Public review):

Summary:

This study uses dental traits of a large sample of Chinese mammals to track evolutionary patterns through the Paleocene. It presents and argues for a 'brawn before bite' hypothesis - mammals increased in body size disparity before evolving more specialized or adapted dentitions. The study makes use of an impressive array of analyses, including dental topographic, finite element, and integration analyses, which help to provide a unique insight into mammalian evolutionary patterns.

Strengths:

This paper helps to fill in a major gap in our knowledge of Paleocene mammal patterns in Asia, which is especially important because of the diversification of placentals at that time. The total sample of teeth is impressive and required considerable effort for scanning and analyzing. And there is a wealth of results for DTA, FEA, and integration analyses. Further, some of the results are especially interesting, such as the novel 'brawn before bite' hypothesis and the possible link between shifts in dental traits and arid environments in the Late Paleocene. Overall, I enjoyed reading the paper, and I think the results will be of interest to a broad audience.

Weaknesses:

I have four major concerns with the study, especially related to the sampling of teeth and taxa, that I discuss in more detail below. Due to these issues, I believe that the study is incomplete in its support of the 'brawn before bite' hypothesis. Although my concerns are significant, many of them can be addressed with some simple updates/revisions to analyses or text, and I try to provide constructive advice throughout my review.

(1) If I understand correctly, teeth of different tooth positions (e.g., premolars and molars), and those from the same specimen, are lumped into the same analyses. And unless I missed it, no justification is given for these methodological choices (besides testing for differences in proportions of tooth positions per time bin; L902). I think this creates some major statistical concerns. For example, DTA values for premolars and molars aren't directly comparable (I don't think?) because they have different functions (e.g., greater grinding function for molars). My recommendation is to perform different disparity-through-time analyses for each tooth position, assuming the sample sizes are big enough per time bin. Or, if the authors maintain their current methods/results, they should provide justification in the main text for that choice.

We thank Reviewer 2 for raising several issues worthy of clarification. Separate analyses for individual tooth positions were performed but not emphasized in the first version of the study. In our revised version we plan to highlight the nuances of the results from premolar versus molar partition analyses.

Also, I think lumping teeth from the same specimen into your analyses creates a major statistical concern because the observations aren't independent. In other words, the teeth of the same individual should have relatively similar DTA values, which can greatly bias your results. This is essentially the same issue as phylogenetic non-independence, but taken to a much greater extreme.

It seems like it'd be much more appropriate to perform specimen-level analyses (e.g., Wilson 2013) or species-level analyses (e.g., Grossnickle & Newham 2016) and report those results in the main text. If the authors believe that their methods are justified, then they should explain this in the text.

We plan to emphasize individual tooth position analyses in our revisions, and provide a stronger justification for our current treatment of multiple teeth from the same individual specimens as independent samples. We recognize the statistical nonindependence raised by Reviewer 2, but we would point out that from an ecomorphological perspective, it is unclear to us that the heterodont dentition of these early Cenozoic placental mammals should represent a single ecological signal (and thus warrant using only a single tooth position as representative of an individual’s DTA values). We plan to closely examine the nature of nonindependence in the DTA data within individuals, to assess a balanced approach to maximize information content from the relatively small and rare fossil samples used, while minimizing signal nonindependence across the dentition.

(2) Maybe I misunderstood, but it sounds like the sampling is almost exclusively clades that are primarily herbivorous/omnivorous (Pantodonta, Arctostylopida, Anagalida, and maybe Tillodonta), which means that the full ecomorphological diversity of the time bins is not being sampled (e.g., insectivores aren't fully sampled). Similarly, the authors say that they "focused sampling" on those major clades and "Additional data were collected on other clades ... opportunistically" (L628). If they favored sampling of specific clades, then doesn't that also bias their results?

If the study is primarily focused on a few herbivorous clades, then the Introduction should be reframed to reflect this. You could explain that you're specifically tracking herbivore patterns after the K-Pg.

We plan to revise the introduction section to more accurately reflect the emphasis on those clades. However, we would note that conventional dietary ecomorphology categories used to characterize later branching placental mammals are likely to be less informative when applied to their Paleocene counterparts. Although there are dental morphological traits that began to characterize major placental clades during the Paleocene, distinctive dietary ecologies have not been demonstrated for most of the clade representatives studied. Thus, insectivory was probably not restricted to “Insectivora”, nor carnivory to early Carnivmorpha or “Creodonta”, each of which represented less than 5% of the taxonomic richness during the Paleocene in China (Wang et al. 2007).

Wang, Y., Meng, J., Ni, X., and Li, C. (2007). Major events of Paleogene mammal radiation in China. Geol. J. 42, 415–430.

(3) There are a lot of topics lacking background information, which makes the paper challenging to read for non-experts. Maybe the authors are hindered by a short word limit. But if they can expand their main text, then I strongly recommend the following:

(a) The authors should discuss diets. Much of the data are diet correlates (DTA values), but diets are almost never mentioned, except in the Methods. For example, the authors say: "An overall shift towards increased dental topographic trait magnitudes ..." (L137). Does that mean there was a shift toward increased herbivory? If so, why not mention the dietary shift? And if most of the sampled taxa are herbivores (see above comment), then shouldn't herbivory be a focal point of the paper?

We plan to revise the text to make clearer connections between DTA and dietary inferences, and at the same time advise caution in making one-to-one linkages between them. Broadly speaking, dental indices such as DTA are phenotypic traits, and as in other phenotypic traits, the strength of structure-function relationships needs to be explicitly established before dietary ecological inferences can be confidently made. There is, to date, no consistent connection between dental topology and tooth use proxies and biomechanical traits in extant non-herbivorous species (e.g., DeSantis et al. 2017, Tseng and DeSantis 2024), and in our analyses, FEA and DTA generally did not show strong correlations to each other. Thus, we plan to continue to exercise care in interpreting DTA data as dietary data.

DeSantis LRG, Tseng ZJ, Liu J, Hurst A, Schubert BW, Jiangzuo Q. Assessing niche conservatism using a multiproxy approach: dietary ecology of extinct and extant spotted hyenas. Paleobiology. 2017;43(2):286-303. doi:10.1017/pab.2016.45

Tseng ZJ, DeSantis LR. Relationship between tooth macrowear and jaw morphofunctional traits in representative hypercarnivores. PeerJ. 2024 Nov 11;12:e18435.

(b) The authors should expand on "we used dentitions as ecological indicators" (L75). For non-experts, how/why are dentitions linked to ecology? And, again, why not mention diet? A strong link between tooth shape and diet is a critical assumption here (and one I'm sure that all mammalogists agree with), but the authors don't provide justification (at least in the Introduction) for that assumption. Many relevant papers cited later in the Methods could be cited in the Introduction (e.g., Evans et al. 2007).

Thank you for this suggestion. We plan to expand the introduction section to better contextualize the methodological basis for the work presented.

(c) Include a better introduction of the sample, such as explicitly stating that your sample only includes placentals (assuming that's the case) and is focused on three major clades. Are non-placentals like multituberculates or stem placentals/eutherians found at Chinese Paleocene fossil localities and not sampled in the study, or are they absent in the sampled area?

We thank Reviewer 2 for raising this important point worthy of clarification. Multituberculates are completely absent from the first two land mammal ages in the Paleocene of Asia, and non-placentals are rare in general (Wang et al. 2007). We plan to provide more context for the taxonomic sampling choices made in the study.

Wang, Y., Meng, J., Ni, X., and Li, C. (2007). Major events of Paleogene mammal radiation in China. Geol. J. 42, 415–430.

(d) The way in which "integration" is being used should be defined. That is a loaded term which has been defined in different ways. I also recommend providing more explanation on the integration analyses and what the results mean.

If the authors don't have space to expand the main text, then they should at least expand on the topics in the supplement, with appropriate citations to the supplement in the main text.

We plan to clarify our usage of “integration” to enable readers to accurately interpret what we mean by it.

(4) Finally, I'm not convinced that the results fully support the 'brawn before bite' hypothesis. I like the hypothesis. However, the 'brawn before ...' part of the hypothesis assumes that body size disparity (L63) increased first, and I don't think that pattern is ever shown. First, body size disparity is never reported or plotted (at least that I could find) - the authors just show the violin plots of the body sizes (Figures 1B, S6A). Second, the authors don't show evidence of an actual increase in body size disparity. Instead, they seem to assume that there was a rapid diversification in the earliest Paleocene, and thus the early Paleocene bin has already "reached maximum saturation" (L148). But what if the body size disparity in the latest Cretaceous was the same as that in the Paleocene? (Although that's unlikely, note that papers like Clauset & Redner 2009 and Grossnickle & Newham 2016 found evidence of greater body size disparity in the latest Cretaceous than is commonly recognized.) Similarly, what if body size disparity increased rapidly in the Eocene? Wouldn't that suggest a 'BITE before brawn' hypothesis? So, without showing when an increase in body size diversity occurred, I don't think that the authors can make a strong argument for 'brawn before [insert any trait]".

Although it's probably well beyond the scope of the study to add Cretaceous or Eocene data, the authors could at least review literature on body size patterns during those times to provide greater evidence for an earliest Paleocene increase in size disparity.

We plan to provide a broader discussion and any supporting evidence from the Cretaceous and Eocene to either make a stronger case for “brawn before bite”, or to refine what we mean by brawn/size/size disparity.

System design is the process of defining a system’s architecture, components, interfaces, and interactions in a structured way so the whole system meets its goals and requirements.

Put another way:

it’s planning how parts fit and work together, not just building them;

it creates a blueprint showing how elements communicate, behave, and support the system’s purpose;

it ensures the system will perform, scale, and remain reliable as conditions change.

System design is about setting constraints, incentives, and feedback so that any reasonable behavior leads to acceptable outcomes — without prescribing each individual action.

Design the conditions, not the conduct.

应该在/conf.d中创建.conf文件，在使用NM时会优先读取最新文件

DOI: 10.1016/j.xcrm.2025.102506

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:005557

What is this?

DOI: 10.1016/j.cell.2025.11.023

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:017537

What is this?

DOI: 10.1016/j.cell.2025.11.023

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:016263

What is this?

DOI: 10.1016/j.cell.2025.11.023

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:009088

What is this?

DOI: 10.1016/j.cell.2025.11.023

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:IMSR_JAX:007567

What is this?

statement by government from appeal

When I read that “society produces conformity,” it feels like the world around me is constantly telling me who I’m supposed to be. It pushes certain names, labels, and traditions on me, even if they don’t fit who I really am.

society” as a vague force that pressures equally unclear “individuals.” It assumes everyone feels the same pressure and only has two choices: give in or “be themselves.” In reality, social pressure comes from specific sources like friends, family, media, or rules and people respond in many different ways.

Writers should name specific groups instead of pretending the whole world agrees.

College papers require a thesis that someone could disagree with, and paragraphs that flow logically from one to the next.This is the main difference between high school and college writing. The thesis isn’t just a summary it drives the whole argument and keeps the reader interested. The organic structure makes the essay feel like a real conversation or exploration.

High school essays often use three main reasons to support the thesis, each getting its own body paragraph.This shows how formulaic high school essays can be. The order of the reasons doesn’t really matter, so it’s predictable and safe but not very creative.

This section explains that a college thesis shouldn’t just state a fact it should be something someone could disagree with. It makes the essay more interesting and gives it a purpose.

Shows that many students waste space with vague intros

Emphasizes clarity and purpose in introductions. Thought I like this it makes writing feel focused and interesting.

This shows a common writing habit: students often begin with weak or unclear introductions because they think a paper needs a “slow start.” It reminds us that strong, clear opening sentences make a paper more interesting and show confidence in your ideas.