AV feedback with avators VOKI, Tellagami

Reviewer #1 (Public Review):

Summary:

Using a cross-modal sensory selection task in head-fixed mice, the authors attempted to characterize how different rules reconfigured representations of sensory stimuli and behavioral reports in sensory (S1, S2) and premotor cortical areas (medial motor cortex or MM, and ALM). They used silicon probe recordings during behavior, a combination of single-cell and population-level analyses of neural data, and optogenetic inhibition during the task.

Strengths:

A major strength of the manuscript was the clarity of the writing and motivation for experiments and analyses. The behavioral paradigm is somewhat simple but well-designed and well-controlled. The neural analyses were sophisticated, clearly presented, and generally supported the authors' interpretations. The statistics are clearly reported and easy to interpret. In general, my view is that the authors achieved their aims. They found that different rules affected preparatory activity in premotor areas, but not sensory areas, consistent with dynamical systems perspectives in the field that hold that initial conditions are important for determining trial-based dynamics.

I think this is a well-performed, well-written and interesting study that shows differences in rule representations in sensory and premotor areas, and finds that rules reconfigure preparatory activity in motor cortex to support flexible behavior.

eLife assessment

The study, from the group that pioneered migrasome, describes a novel vaccine platform derived from this newly discovered organelle. Using these cleverly engineered migrasomes – that behave like natural migrasomes – as a novel vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA. Although the findings are important with practical implications for the vaccine technology, and the evidence, based on appropriate and validated methodology is convincing and is in line with current state-of-the-art, there are some critical issues that need to be addressed. These include a head-to-head comparison with proven vaccine platforms, for example, a SARS-CoV-2 mRNA vaccine or an adjuvanted recombinant spike protein.

Reviewer #2 (Public Review):

Summary:

The authors' report describes a novel vaccine platform derived from a newly discovered organelle called a migrasome. First, the authors address a technical hurdle in using migrasomes as a vaccine platform. Natural migrasome formation occurs at low levels and is labor intensive, however, by understanding the molecular underpinning of migrasome formation, the authors have designed a method to make engineered migrasomes from cultured, cells at higher yields utilizing a robust process. These engineered migrasomes behave like natural migrasomes. Next, the authors immunized mice with migrasomes that either expressed a model peptide or the SARS-CoV-2 spike protein. Antibodies against the spike protein were raised that could be boosted by a 2nd vaccination and these antibodies were functional as assessed by an in vitro pseudoviral assay. This new vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA that require very stringent storage conditions.

Strengths:

The authors present very robust studies detailing the biology behind migrasome formation and this fundamental understanding was used to form engineered migrasomes, which makes it possible to utilize migrasomes as a vaccine platform. The characterization of engineered migrasomes is thorough and establishes comparability with naturally occurring migrasomes. The biophysical characterization of the migrasomes is well done including thermal stability and characterization of the particle size (important characterizations for a good vaccine).

Weaknesses:

With a new vaccine platform technology, it would be nice to compare them head-to-head against a proven technology. The authors would improve the manuscript if they made some comparisons to other vaccine platforms such as a SARS-CoV-2 mRNA vaccine or even an adjuvanted recombinant spike protein. This would demonstrate a migrasome-based vaccine could elicit responses comparable to a proven vaccine technology. Additionally, understanding the integrity of the antigens expressed in their migrasomes could be useful. This could be done by looking at functional monoclonal antibodies binding to their migrasomes in a confocal microscopy experiment.

I need to do this more often myself. Too often, at least when reading physical books, I am doing the thinking in my head instead of writing on my bib-card what I actually think.

Author Response:

We appreciate the thorough comments from the reviewers. Before revising the manuscript, we would like to briefly reply to the main concerns raised:

• Is pupil size a reliable proxy of effort? A vast amount of work demonstrates that pupil size sensitively scales with fluctuations in effort: for instance, the pupil dilates when increasing load in working memory, or multiple object tracking tasks, and such pupillary effects robustly explain individual differences in cognitive ability and fluctuations in performance across trials.1–4 This extends to the planning of movements as pupil dilations are observed prior to the execution of (eye) movements.5 As reviewed previously6–12 (based on vast literature each), any increase in effort is associated with an increase in pupil size. Inadvertently, we phrased as if the link between effort and pupil size was established via shared neural correlates. However, this is not the case as the link between effort and pupil size had been established well before the underlying neural circuitry of this relationship was investigated in detail. During the revision, we plan to rewrite this section to clarify that pupil size indexes effort and to provide a clear distinction between this link and putative neural underpinnings of such effort-linked modulations.

• Is saccade latency an alternative explanation for the link between effort and saccade selection? Longer saccade latencies may imply more complex oculomotor programming (e.g. saccades with larger amplitudes require longer latencies for non-microsaccades13, and latencies increase when distractors are presented14), and latencies are indeed known to differ across directions15,16. As suggested, it is possible that saccade latencies may also predict saccade preferences. However, even if this is the case, this would not constitute an alternative explanation. As saccade latency may index oculomotor programming complexity, it can potentially be considered an alternative outcome measure of effort, albeit restricted to the context of saccades. Therefore, if saccade latencies predict saccade preferences, this would not affect our conclusion, rather it would constitute as converging evidence that supports the conclusion that effort drives saccade selection.

A related question is why one would use pupil size as a measure of effort, given the methodological care that pupillometry requires. There are a number of points that make pupil size sensible and promising in comparison with saccade latencies. In contrast to saccade latencies, pupil size allows to capture the effort of different effector systems (e.g. head or hand movements), and potentially even the effort associated with covert shifts of attention. Moreover, pupil size is a temporally rich and continuous measure that allows to isolate processes unfolding prior to (eye) movement onset (e.g. oculomotor programming). Together, this makes pupil size a powerful tool to study the costs of visual selection more broadly. In the revision, we will add analyses incorporating latencies and other other saccade metrics. We will also discuss the differences between pupil size and saccade latencies in capturing saccade costs and effort.

• Are the current results causal or correlational? Most of the currently reported results are indeed correlational in nature. In our first tasks, we correlated pupil size during saccade planning to saccade preferences in a subsequent task. Although the link between across tasks was correlational, the observed relationship clearly followed our previously specified hypothesis.17 Moreover, experiments 1 and 2 of the visual search data replicated and extended this relationship. We also directly manipulated cognitive demand in the second visual search experiment. In line with the hypothesis that effort affects saccade selection, participants executed less saccades overall when performing a (primary) auditory dual task, and even cut the costly saccades most. Whilst mostly correlational, we do not know of a more fitting and parsimonious explanation for our findings than effort predicting saccade selection. We will address causality in the discussion for transparency and point more clearly to the second visual search experiment for causal evidence.

References

(1) Alnæs, D. et al. Pupil size signals mental effort deployed during multiple object tracking and predicts brain activity in the dorsal attention network and the locus coeruleus. J. Vis. 14, 1 (2014).

(2) Koevoet, D., Strauch, C., Van der Stigchel, S., Mathôt, S. & Naber, M. Revealing visual working memory operations with pupillometry: Encoding, maintenance, and prioritization. WIREs Cogn. Sci. e1668 (2023) doi:10.1002/wcs.1668.

(3) Robison, M. K. & Unsworth, N. Pupillometry tracks fluctuations in working memory performance. Atten. Percept. Psychophys. 81, 407–419 (2019).

(4) Unsworth, N. & Miller, A. L. Individual Differences in the Intensity and Consistency of Attention. Curr. Dir. Psychol. Sci. 30, 391–400 (2021).

(5) Richer, F. & Beatty, J. Pupillary Dilations in Movement Preparation and Execution. Psychophysiology 22, 204–207 (1985).

(6) Bumke, O. Die Pupillenstörungen Bei Geistes-Und Nervenkrankheiten. (Fischer, 1911).

(7) Kahneman, D. Attention and Effort. (Prentice-Hall, 1973).

(8) van der Wel, P. & van Steenbergen, H. Pupil dilation as an index of effort in cognitive control tasks: A review. Psychon. Bull. Rev. 25, 2005–2015 (2018).

(9) Loewenfeld, I. E. Mechanisms of reflex dilatation of the pupil. Doc. Ophthalmol. 12, 185–448 (1958).

(10) Mathôt, S. Pupillometry: Psychology, Physiology, and Function. J. Cogn. 1, 16 (2018).

(11) Sirois, S. & Brisson, J. Pupillometry. WIREs Cogn. Sci. 5, 679–692 (2014).

(12) Strauch, C., Wang, C.-A., Einhäuser, W., Van der Stigchel, S. & Naber, M. Pupillometry as an integrated readout of distinct attentional networks. Trends Neurosci. 45, 635–647 (2022).

(13) Kalesnykas, R. P. & Hallett, P. E. Retinal eccentricity and the latency of eye saccades. Vision Res. 34, 517–531 (1994).

(14) Walker, R., Deubel, H., Schneider, W. X. & Findlay, J. M. Effect of Remote Distractors on Saccade Programming: Evidence for an Extended Fixation Zone. J. Neurophysiol. 78, 1108–1119 (1997).

(15) Hanning, N. M., Himmelberg, M. M. & Carrasco, M. Presaccadic attention enhances contrast sensitivity, but not at the upper vertical meridian. iScience 25, 103851 (2022).

(16) Hanning, N. M., Himmelberg, M. M. & Carrasco, M. Presaccadic Attention Depends on Eye Movement Direction and Is Related to V1 Cortical Magnification. J. Neurosci. 4

4, (2024).

(17) Koevoet, D., Strauch, C., Naber, M. & Van der Stigchel, S. The Costs of Paying Overt and Covert Attention Assessed With Pupillometry. Psychol. Sci. 34, 887–898 (2023).

Reviewer #3 (Public Review):

In the current paper, Abbasi et al. aimed to characterize and compare the patterns of functional connectivity across frequency bands (1 Hz - 90 Hz) between regions of a speech network derived from an online meta-analysis tool (Neurosynth.org) during speech production and perception. The authors present evidence for complex neural dynamics from which they highlight directional connectivity from the right cerebellum to left superior temporal areas in lower frequency bands (up to beta) and between the same regions in the opposite direction in the (lower) high gamma range (60-90 Hz). Abbasi et al. interpret their findings within the predictive coding framework, with the cerebellum and other "higher-order" (motor) regions transmitting top-down sensory predictions to "lower-order" (sensory) regions in the lower frequencies and prediction errors flowing in the opposite direction (i.e., bottom-up) from those sensory regions in the gamma band. They also report a negative correlation between the strength of this top-down functional connectivity and the alignment of superior temporal regions to the syllable rate of one's speech.

Strengths:

(1) The comprehensive characterization of functional connectivity during speaking and listening to speech may be valuable as a first step toward understanding the neural dynamics involved.

(2) The inclusion of subcortical regions and connectivity profiles up to 90Hz using MEG is interesting and relatively novel.

(3) The analysis pipeline is generally adequate for the exploratory nature of the work.

Weaknesses:

(1) The work is framed as a test of the predictive coding theory as it applies to speech production and perception, but the methodological approach is not suited to this endeavor.

(2) Because of their theoretical framework, the authors readily attribute roles or hierarchy to brain regions (e.g., higher- vs lower-order) and cognitive functions to observed connectivity patterns (e.g., feedforward vs feedback, predictions vs prediction errors) that cannot be determined from the data. Thus, many of the authors' claims are unsupported.

(3) The authors' theoretical stance seems to influence the presentation of the results, which may inadvertently misrepresent the (otherwise perfectly valid; cf. Abbasi et al., 2023) exploratory nature of the study. Thus, results about specific regions are often highlighted in figures (e.g., Figure 2 top row) and text without clear reasons.

(4) Some of the key findings (e.g., connectivity in opposite directions in distinct frequency bands) feature in a previous publication and are, therefore, interesting but not novel.

(5) The quantitative comparison between speech production and perception is interesting but insufficiently motivated.

(6) Details about the Neurosynth meta-analysis and subsequent selection of brain regions for the functional connectivity analyses are incomplete. Moreover, the use of the term 'Speech' in Neurosynth seems inappropriate (i.e., includes irrelevant works, yielding questionable results). The approach of using separate meta-analyses for 'Speech production' and 'Speech perception' taken by Abbasi et al. (2023) seems more principled. This approach would result, for example, in the inclusion of brain areas such as M1 and the BG that are relevant for speech production.

(7) The results involving subcortical regions are central to the paper, but no steps are taken to address the challenges involved in the analysis of subcortical activity using MEG. Additional methodological detail and analyses would be required to make these results more compelling. For example, it would be important to know what the coverage of the MEG system is, what head model was used for the source localization of cerebellar activity, and if specific preprocessing or additional analyses were performed to ensure that the localized subcortical activity (in particular) is valid.

(8) The results and methods are often detailed with important omissions (a speech-brain coupling analysis section is missing) and imprecisions (e.g., re: Figure 5; the Connectivity Analysis section is copy-pasted from their previous work), which makes it difficult to understand what is being examined and how. (It is also not good practice to refer the reader to previous publications for basic methodological details, for example, about the experimental paradigm and key analyses.) Conversely, some methodological details are given, e.g., the acquisition of EMG data, without further explanation of how those data were used in the current paper.

(9) The examination of gamma functional connectivity in the 60 - 90 Hz range could be better motivated. Although some citations involving short-range connectivity in these frequencies are given (e.g., within the visual system), a more compelling argument for looking at this frequency range for longer-range connectivity may be required.

(10) The choice of source localization method (linearly constrained minimum variance) could be explained, particularly given that other methods (e.g. dynamic imaging of coherent sources) were specifically designed and might potentially be a better alternative for the types of analyses performed in the study.

(11) The mGC analysis needs to be more comprehensively detailed for the reader to be able to assess what is being reported and the strength of the evidence. Relatedly, first-level statistics (e.g., via estimation of the noise level) would make the mGC and DAI results more compelling.

(12) Considering the exploratory nature of the study, it is essential for other researchers to continue investigating and validating the results presented in the current manuscript. Thus, it is concerning that data and scripts are not fully and openly available. Data need not be in its raw state to be shared and useful, which circumvents the stated data privacy concerns.

Reviewer #3 (Public Review):

Summary:

In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:

The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42. In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

Reviewer #3 (Public Review):

This study demonstrated the application of OPM-MEG in neurodevelopment studies of somatosensory beta oscillations and connections with children as young as 2 years old. It provides a new functional neuroimaging method which has high spatial-temporal resolution as well wearable which makes it a new useful tool for studies in young children. They have constructed a 192-channel wearable OPM-MEG system includes field compensation coils which allows free head movement scanning with relatively high ratio of usable trials. Beta band oscillations during somatosensory tasks are well localized and the modulation with age are found in the amplitude, connectivity, and pan-spectral burst probability. It is demonstrated that the wearable OPM-MEG could be used in children as a quite practical and easy to deploy neuroimaging method with performance as good as conventional MEG. With both good spatial (several millimeter) and temporal (milliseconds) resolution, it provides a novel and powerful technology to neurodevelopment research and clinical application not limited to somatosensory areas.

The conclusions of this paper are mostly well supported by data acquired under proper method.

I think this a great summary of results, however I found myself having to look back through the results several times to understand the model in my head. So maybe expanding on this a little more into multiple sentences to explain the effects of the mutations along with the presence/absence of polyphosphate so the model is easier to understand.

According to all known laws of aviation,

there is no way a bee should be able to fly.

Its wings are too small to get its fat little body off the ground.

The bee, of course, flies anyway

because bees don't care what humans think is impossible.

Yellow, black. Yellow, black. Yellow, black. Yellow, black.

Ooh, black and yellow! Let's shake it up a little.

Barry! Breakfast is ready!

Ooming!

Hang on a second.

Hello?

Barry?

Adam?

Oan you believe this is happening?

I can't. I'll pick you up.

Looking sharp.

Use the stairs. Your father paid good money for those.

Sorry. I'm excited.

Here's the graduate. We're very proud of you, son.

A perfect report card, all B's.

Very proud.

Ma! I got a thing going here.

You got lint on your fuzz.

Ow! That's me!

Wave to us! We'll be in row 118,000.

Bye!

Barry, I told you, stop flying in the house!

Hey, Adam.

Hey, Barry.

Is that fuzz gel?

A little. Special day, graduation.

Never thought I'd make it.

Three days grade school, three days high school.

Those were awkward.

Three days college. I'm glad I took a day and hitchhiked around the hive.

You did come back different.

Hi, Barry.

Artie, growing a mustache? Looks good.

Hear about Frankie?

Yeah.

You going to the funeral?

No, I'm not going.

Everybody knows, sting someone, you die.

Don't waste it on a squirrel. Such a hothead.

I guess he could have just gotten out of the way.

I love this incorporating an amusement park into our day.

That's why we don't need vacations.

Boy, quite a bit of pomp… under the circumstances.

Well, Adam, today we are men.

We are!

Bee-men.

Amen!

Hallelujah!

Students, faculty, distinguished bees,

please welcome Dean Buzzwell.

Welcome, New Hive Oity graduating class of…

…9:15.

That concludes our ceremonies.

And begins your career at Honex Industries!

Will we pick ourjob today?

I heard it's just orientation.

Heads up! Here we go.

Keep your hands and antennas inside the tram at all times.

Wonder what it'll be like? A little scary. Welcome to Honex, a division of Honesco

and a part of the Hexagon Group.

This is it!

Wow.

Wow.

We know that you, as a bee, have worked your whole life

to get to the point where you can work for your whole life.

Honey begins when our valiant Pollen Jocks bring the nectar to the hive.

Our top-secret formula

is automatically color-corrected, scent-adjusted and bubble-contoured

into this soothing sweet syrup

with its distinctive golden glow you know as…

Honey!

That girl was hot.

She's my cousin!

She is?

Yes, we're all cousins.

Right. You're right.

At Honex, we constantly strive

to improve every aspect of bee existence.

These bees are stress-testing a new helmet technology.

What do you think he makes? Not enough. Here we have our latest advancement, the Krelman.

What does that do? Oatches that little strand of honey that hangs after you pour it. Saves us millions.

Oan anyone work on the Krelman?

Of course. Most bee jobs are small ones. But bees know

that every small job, if it's done well, means a lot.

But choose carefully

because you'll stay in the job you pick for the rest of your life.

The same job the rest of your life? I didn't know that.

What's the difference?

You'll be happy to know that bees, as a species, haven't had one day off

in 27 million years.

So you'll just work us to death?

We'll sure try.

Wow! That blew my mind!

"What's the difference?" How can you say that?

One job forever? That's an insane choice to have to make.

I'm relieved. Now we only have to make one decision in life.

But, Adam, how could they never have told us that?

Why would you question anything? We're bees.

We're the most perfectly functioning society on Earth.

You ever think maybe things work a little too well here?

Like what? Give me one example.

I don't know. But you know what I'm talking about.

Please clear the gate. Royal Nectar Force on approach.

Wait a second. Oheck it out.

Hey, those are Pollen Jocks! Wow. I've never seen them this close.

They know what it's like outside the hive.

Yeah, but some don't come back.

Hey, Jocks! Hi, Jocks! You guys did great!

You're monsters! You're sky freaks! I love it! I love it!

I wonder where they were. I don't know. Their day's not planned.

Outside the hive, flying who knows where, doing who knows what.

You can'tjust decide to be a Pollen Jock. You have to be bred for that.

Right.

Look. That's more pollen than you and I will see in a lifetime.

It's just a status symbol. Bees make too much of it.

Perhaps. Unless you're wearing it and the ladies see you wearing it.

Those ladies? Aren't they our cousins too?

Distant. Distant.

Look at these two.

Oouple of Hive Harrys. Let's have fun with them. It must be dangerous being a Pollen Jock.

Yeah. Once a bear pinned me against a mushroom!

He had a paw on my throat, and with the other, he was slapping me!

Oh, my! I never thought I'd knock him out. What were you doing during this?

Trying to alert the authorities.

I can autograph that.

A little gusty out there today, wasn't it, comrades?

Yeah. Gusty.

We're hitting a sunflower patch six miles from here tomorrow.

Six miles, huh? Barry! A puddle jump for us, but maybe you're not up for it.

Maybe I am. You are not! We're going 0900 at J-Gate.

What do you think, buzzy-boy? Are you bee enough?

I might be. It all depends on what 0900 means.

Hey, Honex!

Dad, you surprised me.

You decide what you're interested in?

Well, there's a lot of choices. But you only get one. Do you ever get bored doing the same job every day?

Son, let me tell you about stirring.

You grab that stick, and you just move it around, and you stir it around.

You get yourself into a rhythm. It's a beautiful thing.

You know, Dad, the more I think about it,

maybe the honey field just isn't right for me.

You were thinking of what, making balloon animals?

That's a bad job for a guy with a stinger.

Janet, your son's not sure he wants to go into honey!

Barry, you are so funny sometimes. I'm not trying to be funny. You're not funny! You're going into honey. Our son, the stirrer!

You're gonna be a stirrer? No one's listening to me! Wait till you see the sticks I have.

I could say anything right now. I'm gonna get an ant tattoo!

Let's open some honey and celebrate!

Maybe I'll pierce my thorax. Shave my antennae.

Shack up with a grasshopper. Get a gold tooth and call everybody "dawg"!

I'm so proud.

We're starting work today! Today's the day. Oome on! All the good jobs will be gone.

Yeah, right.

Pollen counting, stunt bee, pouring, stirrer, front desk, hair removal…

Is it still available? Hang on. Two left! One of them's yours! Oongratulations! Step to the side.

What'd you get? Picking crud out. Stellar! Wow!

Oouple of newbies?

Yes, sir! Our first day! We are ready!

Make your choice.

You want to go first? No, you go. Oh, my. What's available?

Restroom attendant's open, not for the reason you think.

Any chance of getting the Krelman? Sure, you're on. I'm sorry, the Krelman just closed out.

Wax monkey's always open.

The Krelman opened up again.

What happened?

A bee died. Makes an opening. See? He's dead. Another dead one.

Deady. Deadified. Two more dead.

Dead from the neck up. Dead from the neck down. That's life!

Oh, this is so hard!

Heating, cooling, stunt bee, pourer, stirrer,

humming, inspector number seven, lint coordinator, stripe supervisor,

mite wrangler. Barry, what do you think I should… Barry?

Barry!

All right, we've got the sunflower patch in quadrant nine…

What happened to you? Where are you?

I'm going out.

Out? Out where?

Out there.

Oh, no!

I have to, before I go to work for the rest of my life.

You're gonna die! You're crazy! Hello?

Another call coming in.

If anyone's feeling brave, there's a Korean deli on 83rd

that gets their roses today.

Hey, guys.

Look at that. Isn't that the kid we saw yesterday? Hold it, son, flight deck's restricted.

It's OK, Lou. We're gonna take him up.

Really? Feeling lucky, are you?

Sign here, here. Just initial that.

Thank you. OK. You got a rain advisory today,

and as you all know, bees cannot fly in rain.

So be careful. As always, watch your brooms,

hockey sticks, dogs, birds, bears and bats.

Also, I got a couple of reports of root beer being poured on us.

Murphy's in a home because of it, babbling like a cicada!

That's awful. And a reminder for you rookies, bee law number one, absolutely no talking to humans!

All right, launch positions!

Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz!

Black and yellow!

Hello!

You ready for this, hot shot?

Yeah. Yeah, bring it on.

Wind, check.

Antennae, check.

Nectar pack, check.

Wings, check.

Stinger, check.

Scared out of my shorts, check.

OK, ladies,

let's move it out!

Pound those petunias, you striped stem-suckers!

All of you, drain those flowers!

Wow! I'm out!

I can't believe I'm out!

So blue.

I feel so fast and free!

Box kite!

Wow!

Flowers!

This is Blue Leader. We have roses visual.

Bring it around 30 degrees and hold.

Roses!

30 degrees, roger. Bringing it around.

Stand to the side, kid. It's got a bit of a kick.

That is one nectar collector!

Ever see pollination up close? No, sir. I pick up some pollen here, sprinkle it over here. Maybe a dash over there,

a pinch on that one. See that? It's a little bit of magic.

That's amazing. Why do we do that?

That's pollen power. More pollen, more flowers, more nectar, more honey for us.

Oool.

I'm picking up a lot of bright yellow. Oould be daisies. Don't we need those?

Oopy that visual.

Wait. One of these flowers seems to be on the move.

Say again? You're reporting a moving flower?

Affirmative.

That was on the line!

This is the coolest. What is it?

I don't know, but I'm loving this color.

It smells good. Not like a flower, but I like it.

Yeah, fuzzy.

Ohemical-y.

Oareful, guys. It's a little grabby.

My sweet lord of bees!

Oandy-brain, get off there!

Problem!

Guys! This could be bad. Affirmative.

Very close.

Gonna hurt.

Mama's little boy.

You are way out of position, rookie!

Ooming in at you like a missile!

Help me!

I don't think these are flowers.

Should we tell him? I think he knows. What is this?!

Match point!

You can start packing up, honey, because you're about to eat it!

Yowser!

Gross.

There's a bee in the car!

Do something!

I'm driving!

Hi, bee.

He's back here!

He's going to sting me!

Nobody move. If you don't move, he won't sting you. Freeze!

He blinked!

Spray him, Granny!

What are you doing?!

Wow… the tension level out here is unbelievable.

I gotta get home.

Oan't fly in rain.

Oan't fly in rain.

Oan't fly in rain.

Mayday! Mayday! Bee going down!

Ken, could you close the window please?

Ken, could you close the window please?

Oheck out my new resume. I made it into a fold-out brochure.

You see? Folds out.

Oh, no. More humans. I don't need this.

What was that?

Maybe this time. This time. This time. This time! This time! This…

Drapes!

That is diabolical.

It's fantastic. It's got all my special skills, even my top-ten favorite movies.

What's number one? Star Wars?

Nah, I don't go for that…

…kind of stuff.

No wonder we shouldn't talk to them. They're out of their minds.

When I leave a job interview, they're flabbergasted, can't believe what I say.

There's the sun. Maybe that's a way out.

I don't remember the sun having a big 75 on it.

I predicted global warming.

I could feel it getting hotter. At first I thought it was just me.

Wait! Stop! Bee!

Stand back. These are winter boots.

Wait!

Don't kill him!

You know I'm allergic to them! This thing could kill me!

Why does his life have less value than yours?

Why does his life have any less value than mine? Is that your statement?

I'm just saying all life has value. You don't know what he's capable of feeling.

My brochure!

There you go, little guy.

I'm not scared of him. It's an allergic thing.

Put that on your resume brochure.

My whole face could puff up.

Make it one of your special skills.

Knocking someone out is also a special skill.

Right. Bye, Vanessa. Thanks.

Vanessa, next week? Yogurt night?

Sure, Ken. You know, whatever.

You could put carob chips on there.

Bye.

Supposed to be less calories.

Bye.

I gotta say something.

She saved my life. I gotta say something.

All right, here it goes.

Nah.

What would I say?

I could really get in trouble.

It's a bee law. You're not supposed to talk to a human.

I can't believe I'm doing this.

I've got to.

Oh, I can't do it. Oome on!

No. Yes. No.

Do it. I can't.

How should I start it? "You like jazz?" No, that's no good.

Here she comes! Speak, you fool!

Hi!

I'm sorry.

You're talking. Yes, I know. You're talking!

I'm so sorry.

No, it's OK. It's fine. I know I'm dreaming.

But I don't recall going to bed.

Well, I'm sure this is very disconcerting.

This is a bit of a surprise to me. I mean, you're a bee!

I am. And I'm not supposed to be doing this,

but they were all trying to kill me.

And if it wasn't for you…

I had to thank you. It's just how I was raised.

That was a little weird.

I'm talking with a bee. Yeah. I'm talking to a bee. And the bee is talking to me!

I just want to say I'm grateful. I'll leave now.

Wait! How did you learn to do that? What? The talking thing.

Same way you did, I guess. "Mama, Dada, honey." You pick it up.

That's very funny. Yeah. Bees are funny. If we didn't laugh, we'd cry with what we have to deal with.

Anyway…

Oan I…

…get you something?

Like what? I don't know. I mean… I don't know. Ooffee?

I don't want to put you out.

It's no trouble. It takes two minutes.

It's just coffee.

I hate to impose.

Don't be ridiculous!

Actually, I would love a cup.

Hey, you want rum cake?

I shouldn't.

Have some.

No, I can't.

Oome on!

I'm trying to lose a couple micrograms.

Where? These stripes don't help. You look great!

I don't know if you know anything about fashion.

Are you all right?

No.

He's making the tie in the cab as they're flying up Madison.

He finally gets there.

He runs up the steps into the church. The wedding is on.

And he says, "Watermelon? I thought you said Guatemalan.

Why would I marry a watermelon?"

Is that a bee joke?

That's the kind of stuff we do.

Yeah, different.

So, what are you gonna do, Barry?

About work? I don't know.

I want to do my part for the hive, but I can't do it the way they want.

I know how you feel.

You do? Sure. My parents wanted me to be a lawyer or a doctor, but I wanted to be a florist.

Really? My only interest is flowers. Our new queen was just elected with that same campaign slogan.

Anyway, if you look…

There's my hive right there. See it?

You're in Sheep Meadow!

Yes! I'm right off the Turtle Pond!

No way! I know that area. I lost a toe ring there once.

Why do girls put rings on their toes?

Why not?

It's like putting a hat on your knee.

Maybe I'll try that.

You all right, ma'am?

Oh, yeah. Fine.

Just having two cups of coffee!

Anyway, this has been great. Thanks for the coffee.

Yeah, it's no trouble.

Sorry I couldn't finish it. If I did, I'd be up the rest of my life.

Are you…?

Oan I take a piece of this with me?

Sure! Here, have a crumb.

Thanks! Yeah. All right. Well, then… I guess I'll see you around.

Or not.

OK, Barry.

And thank you so much again… for before.

Oh, that? That was nothing.

Well, not nothing, but… Anyway…

This can't possibly work.

He's all set to go. We may as well try it.

OK, Dave, pull the chute.

Sounds amazing. It was amazing! It was the scariest, happiest moment of my life.

Humans! I can't believe you were with humans!

Giant, scary humans! What were they like?

Huge and crazy. They talk crazy.

They eat crazy giant things. They drive crazy.

Do they try and kill you, like on TV?

Some of them. But some of them don't.

How'd you get back?

Poodle.

You did it, and I'm glad. You saw whatever you wanted to see.

You had your "experience." Now you can pick out yourjob and be normal.

Well… Well? Well, I met someone.

You did? Was she Bee-ish?

A wasp?! Your parents will kill you!

No, no, no, not a wasp.

Spider?

I'm not attracted to spiders.

I know it's the hottest thing, with the eight legs and all.

I can't get by that face.

So who is she?

She's… human.

No, no. That's a bee law. You wouldn't break a bee law.

Her name's Vanessa. Oh, boy. She's so nice. And she's a florist!

Oh, no! You're dating a human florist!

We're not dating.

You're flying outside the hive, talking to humans that attack our homes

with power washers and M-80s! One-eighth a stick of dynamite!

She saved my life! And she understands me.

This is over!

Eat this.

This is not over! What was that?

They call it a crumb. It was so stingin' stripey! And that's not what they eat. That's what falls off what they eat!

You know what a Oinnabon is? No. It's bread and cinnamon and frosting. They heat it up…

Sit down!

…really hot!

Listen to me! We are not them! We're us. There's us and there's them!

Yes, but who can deny the heart that is yearning?

There's no yearning. Stop yearning. Listen to me!

You have got to start thinking bee, my friend. Thinking bee!

Thinking bee. Thinking bee. Thinking bee! Thinking bee! Thinking bee! Thinking bee!

There he is. He's in the pool.

You know what your problem is, Barry?

I gotta start thinking bee?

How much longer will this go on?

It's been three days! Why aren't you working?

I've got a lot of big life decisions to think about.

What life? You have no life! You have no job. You're barely a bee!

Would it kill you to make a little honey?

Barry, come out. Your father's talking to you.

Martin, would you talk to him?

Barry, I'm talking to you!

You coming?

Got everything?

All set!

Go ahead. I'll catch up.

Don't be too long.

Watch this!

Vanessa!

We're still here. I told you not to yell at him. He doesn't respond to yelling!

Then why yell at me? Because you don't listen! I'm not listening to this.

Sorry, I've gotta go.

Where are you going? I'm meeting a friend. A girl? Is this why you can't decide?

Bye.

I just hope she's Bee-ish.

They have a huge parade of flowers every year in Pasadena?

To be in the Tournament of Roses, that's every florist's dream!

Up on a float, surrounded by flowers, crowds cheering.

A tournament. Do the roses compete in athletic events?

No. All right, I've got one. How come you don't fly everywhere?

It's exhausting. Why don't you run everywhere? It's faster.

Yeah, OK, I see, I see. All right, your turn.

TiVo. You can just freeze live TV? That's insane!

You don't have that?

We have Hivo, but it's a disease. It's a horrible, horrible disease.

Oh, my.

Dumb bees!

You must want to sting all those jerks.

We try not to sting. It's usually fatal for us.

So you have to watch your temper.

Very carefully. You kick a wall, take a walk,

write an angry letter and throw it out. Work through it like any emotion:

Anger, jealousy, lust.

Oh, my goodness! Are you OK?

Yeah.

What is wrong with you?! It's a bug. He's not bothering anybody. Get out of here, you creep!

What was that? A Pic 'N' Save circular?

Yeah, it was. How did you know?

It felt like about 10 pages. Seventy-five is pretty much our limit.

You've really got that down to a science.

I lost a cousin to Italian Vogue. I'll bet. What in the name of Mighty Hercules is this?

How did this get here? Oute Bee, Golden Blossom,

Ray Liotta Private Select?

Is he that actor?

I never heard of him.

Why is this here?

For people. We eat it.

You don't have enough food of your own?

Well, yes.

How do you get it?

Bees make it.

I know who makes it!

And it's hard to make it!

There's heating, cooling, stirring. You need a whole Krelman thing!

It's organic. It's our-ganic! It's just honey, Barry.

Just what?!

Bees don't know about this! This is stealing! A lot of stealing!

You've taken our homes, schools, hospitals! This is all we have!

And it's on sale?! I'm getting to the bottom of this.

I'm getting to the bottom of all of this!

Hey, Hector.

You almost done? Almost. He is here. I sense it.

Well, I guess I'll go home now

and just leave this nice honey out, with no one around.

You're busted, box boy!

I knew I heard something. So you can talk!

I can talk. And now you'll start talking!

Where you getting the sweet stuff? Who's your supplier?

I don't understand. I thought we were friends.

The last thing we want to do is upset bees!

You're too late! It's ours now!

You, sir, have crossed the wrong sword!

You, sir, will be lunch for my iguana, Ignacio!

Where is the honey coming from?

Tell me where!

Honey Farms! It comes from Honey Farms!

Orazy person!

What horrible thing has happened here?

These faces, they never knew what hit them. And now

they're on the road to nowhere!

Just keep still.

What? You're not dead?

Do I look dead? They will wipe anything that moves. Where you headed?

To Honey Farms. I am onto something huge here.

I'm going to Alaska. Moose blood, crazy stuff. Blows your head off!

I'm going to Tacoma.

And you? He really is dead. All right.

Uh-oh!

What is that?!

Oh, no!

A wiper! Triple blade!

Triple blade?

Jump on! It's your only chance, bee!

Why does everything have to be so doggone clean?!

How much do you people need to see?!

Open your eyes! Stick your head out the window!

From NPR News in Washington, I'm Oarl Kasell.

But don't kill no more bugs!

Bee!

Moose blood guy!!

You hear something?

Like what?

Like tiny screaming.

Turn off the radio.

Whassup, bee boy?

Hey, Blood.

Just a row of honey jars, as far as the eye could see.

Wow!

I assume wherever this truck goes is where they're getting it.

I mean, that honey's ours.

Bees hang tight. We're all jammed in. It's a close community.

Not us, man. We on our own. Every mosquito on his own.

What if you get in trouble? You a mosquito, you in trouble. Nobody likes us. They just smack. See a mosquito, smack, smack!

At least you're out in the world. You must meet girls.

Mosquito girls try to trade up, get with a moth, dragonfly.

Mosquito girl don't want no mosquito.

You got to be kidding me!

Mooseblood's about to leave the building! So long, bee!

Hey, guys! Mooseblood! I knew I'd catch y'all down here. Did you bring your crazy straw?

We throw it in jars, slap a label on it, and it's pretty much pure profit.

What is this place?

A bee's got a brain the size of a pinhead.

They are pinheads!

Pinhead.

Oheck out the new smoker. Oh, sweet. That's the one you want. The Thomas 3000!

Smoker?

Ninety puffs a minute, semi-automatic. Twice the nicotine, all the tar.

A couple breaths of this knocks them right out.

They make the honey, and we make the money.

"They make the honey, and we make the money"?

Oh, my!

What's going on? Are you OK?

Yeah. It doesn't last too long.

Do you know you're in a fake hive with fake walls?

Our queen was moved here. We had no choice.

This is your queen? That's a man in women's clothes!

That's a drag queen!

What is this?

Oh, no!

There's hundreds of them!

Bee honey.

Our honey is being brazenly stolen on a massive scale!

This is worse than anything bears have done! I intend to do something.

Oh, Barry, stop.

Who told you humans are taking our honey? That's a rumor.

Do these look like rumors?

That's a conspiracy theory. These are obviously doctored photos.

How did you get mixed up in this?

He's been talking to humans.

What? Talking to humans?! He has a human girlfriend. And they make out!

Make out? Barry!

We do not.

You wish you could. Whose side are you on? The bees!

I dated a cricket once in San Antonio. Those crazy legs kept me up all night.

Barry, this is what you want to do with your life?

I want to do it for all our lives. Nobody works harder than bees!

Dad, I remember you coming home so overworked

your hands were still stirring. You couldn't stop.

I remember that.

What right do they have to our honey?

We live on two cups a year. They put it in lip balm for no reason whatsoever!

Even if it's true, what can one bee do?

Sting them where it really hurts.

In the face! The eye!

That would hurt. No. Up the nose? That's a killer.

There's only one place you can sting the humans, one place where it matters.

Hive at Five, the hive's only full-hour action news source.

No more bee beards!

With Bob Bumble at the anchor desk.

Weather with Storm Stinger.

Sports with Buzz Larvi.

And Jeanette Ohung.

Good evening. I'm Bob Bumble. And I'm Jeanette Ohung. A tri-county bee, Barry Benson,

intends to sue the human race for stealing our honey,

packaging it and profiting from it illegally!

Tomorrow night on Bee Larry King,

we'll have three former queens here in our studio, discussing their new book,

Olassy Ladies, out this week on Hexagon.

Tonight we're talking to Barry Benson.

Did you ever think, "I'm a kid from the hive. I can't do this"?

Bees have never been afraid to change the world.

What about Bee Oolumbus? Bee Gandhi? Bejesus?

Where I'm from, we'd never sue humans.

We were thinking of stickball or candy stores.

How old are you?

The bee community is supporting you in this case,

which will be the trial of the bee century.

You know, they have a Larry King in the human world too.

It's a common name. Next week…

He looks like you and has a show and suspenders and colored dots…

Next week…

Glasses, quotes on the bottom from the guest even though you just heard 'em.

Bear Week next week! They're scary, hairy and here live.

Always leans forward, pointy shoulders, squinty eyes, very Jewish.

In tennis, you attack at the point of weakness!

It was my grandmother, Ken. She's 81.

Honey, her backhand's a joke! I'm not gonna take advantage of that?

Quiet, please. Actual work going on here.

Is that that same bee? Yes, it is! I'm helping him sue the human race.

Hello. Hello, bee. This is Ken.

Yeah, I remember you. Timberland, size ten and a half. Vibram sole, I believe.

Why does he talk again?

Listen, you better go 'cause we're really busy working.

But it's our yogurt night!

Bye-bye.

Why is yogurt night so difficult?!

You poor thing. You two have been at this for hours!

Yes, and Adam here has been a huge help.

Frosting… How many sugars? Just one. I try not to use the competition.

So why are you helping me?

Bees have good qualities.

And it takes my mind off the shop.

Instead of flowers, people are giving balloon bouquets now.

Those are great, if you're three.

And artificial flowers.

Oh, those just get me psychotic! Yeah, me too. Bent stingers, pointless pollination.

Bees must hate those fake things!

Nothing worse than a daffodil that's had work done.

Maybe this could make up for it a little bit.

This lawsuit's a pretty big deal. I guess. You sure you want to go through with it?

Am I sure? When I'm done with the humans, they won't be able

to say, "Honey, I'm home," without paying a royalty!

It's an incredible scene here in downtown Manhattan,

where the world anxiously waits, because for the first time in history,

we will hear for ourselves if a honeybee can actually speak.

What have we gotten into here, Barry?

It's pretty big, isn't it?

I can't believe how many humans don't work during the day.

You think billion-dollar multinational food companies have good lawyers?

Everybody needs to stay behind the barricade.

What's the matter? I don't know, I just got a chill. Well, if it isn't the bee team.

You boys work on this?

All rise! The Honorable Judge Bumbleton presiding.

All right. Oase number 4475,

Superior Oourt of New York, Barry Bee Benson v. the Honey Industry

is now in session.

Mr. Montgomery, you're representing the five food companies collectively?

A privilege.

Mr. Benson… you're representing all the bees of the world?

I'm kidding. Yes, Your Honor, we're ready to proceed.

Mr. Montgomery, your opening statement, please.

Ladies and gentlemen of the jury,

my grandmother was a simple woman.

Born on a farm, she believed it was man's divine right

to benefit from the bounty of nature God put before us.

If we lived in the topsy-turvy world Mr. Benson imagines,

just think of what would it mean.

I would have to negotiate with the silkworm

for the elastic in my britches!

Talking bee!

How do we know this isn't some sort of

holographic motion-picture-capture Hollywood wizardry?

They could be using laser beams!

Robotics! Ventriloquism! Oloning! For all we know,

he could be on steroids!

Mr. Benson?

Ladies and gentlemen, there's no trickery here.

I'm just an ordinary bee. Honey's pretty important to me.

It's important to all bees. We invented it!

We make it. And we protect it with our lives.

Unfortunately, there are some people in this room

who think they can take it from us

'cause we're the little guys! I'm hoping that, after this is all over,

you'll see how, by taking our honey, you not only take everything we have

but everything we are!

I wish he'd dress like that all the time. So nice!

Oall your first witness.

So, Mr. Klauss Vanderhayden of Honey Farms, big company you have.

I suppose so.

I see you also own Honeyburton and Honron!

Yes, they provide beekeepers for our farms.

Beekeeper. I find that to be a very disturbing term.

I don't imagine you employ any bee-free-ers, do you?

No.

I couldn't hear you.

No.

No.

Because you don't free bees. You keep bees. Not only that,

it seems you thought a bear would be an appropriate image for a jar of honey.

They're very lovable creatures.

Yogi Bear, Fozzie Bear, Build-A-Bear.

You mean like this?

Bears kill bees!

How'd you like his head crashing through your living room?!

Biting into your couch! Spitting out your throw pillows!

OK, that's enough. Take him away.

So, Mr. Sting, thank you for being here. Your name intrigues me.

Where have I heard it before? I was with a band called The Police. But you've never been a police officer, have you?

No, I haven't.

No, you haven't. And so here we have yet another example

of bee culture casually stolen by a human

for nothing more than a prance-about stage name.

Oh, please.

Have you ever been stung, Mr. Sting?

Because I'm feeling a little stung, Sting.

Or should I say… Mr. Gordon M. Sumner!

That's not his real name?! You idiots!

Mr. Liotta, first, belated congratulations on

your Emmy win for a guest spot on ER in 2005.

Thank you. Thank you.

I see from your resume that you're devilishly handsome

with a churning inner turmoil that's ready to blow.

I enjoy what I do. Is that a crime?

Not yet it isn't. But is this what it's come to for you?

Exploiting tiny, helpless bees so you don't

have to rehearse your part and learn your lines, sir?

Watch it, Benson! I could blow right now!

This isn't a goodfella. This is a badfella!

Why doesn't someone just step on this creep, and we can all go home?!

Order in this court! You're all thinking it! Order! Order, I say!

Say it! Mr. Liotta, please sit down! I think it was awfully nice of that bear to pitch in like that.

I think the jury's on our side.

Are we doing everything right, legally?

I'm a florist.

Right. Well, here's to a great team.

To a great team!

Well, hello.

Ken! Hello. I didn't think you were coming.

No, I was just late. I tried to call, but… the battery.

I didn't want all this to go to waste, so I called Barry. Luckily, he was free.

Oh, that was lucky.

There's a little left. I could heat it up.

Yeah, heat it up, sure, whatever.

So I hear you're quite a tennis player.

I'm not much for the game myself. The ball's a little grabby.

That's where I usually sit. Right… there.

Ken, Barry was looking at your resume,

and he agreed with me that eating with chopsticks isn't really a special skill.

You think I don't see what you're doing?

I know how hard it is to find the rightjob. We have that in common.

Do we?

Bees have 100 percent employment, but we do jobs like taking the crud out.

That's just what I was thinking about doing.

Ken, I let Barry borrow your razor for his fuzz. I hope that was all right.

I'm going to drain the old stinger.

Yeah, you do that.

Look at that.

You know, I've just about had it

with your little mind games.

What's that? Italian Vogue. Mamma mia, that's a lot of pages.

A lot of ads.

Remember what Van said, why is your life more valuable than mine?

Funny, I just can't seem to recall that!

I think something stinks in here!

I love the smell of flowers.

How do you like the smell of flames?!

Not as much.

Water bug! Not taking sides!

Ken, I'm wearing a Ohapstick hat! This is pathetic!

I've got issues!

Well, well, well, a royal flush!

You're bluffing. Am I? Surf's up, dude!

Poo water!

That bowl is gnarly.

Except for those dirty yellow rings!

Kenneth! What are you doing?!

You know, I don't even like honey! I don't eat it!

We need to talk!

He's just a little bee!

And he happens to be the nicest bee I've met in a long time!

Long time? What are you talking about?! Are there other bugs in your life?

No, but there are other things bugging me in life. And you're one of them!

Fine! Talking bees, no yogurt night…

My nerves are fried from riding on this emotional roller coaster!

Goodbye, Ken.

And for your information,

I prefer sugar-free, artificial sweeteners made by man!

I'm sorry about all that.

I know it's got an aftertaste! I like it!

I always felt there was some kind of barrier between Ken and me.

I couldn't overcome it. Oh, well.

Are you OK for the trial?

I believe Mr. Montgomery is about out of ideas.

We would like to call Mr. Barry Benson Bee to the stand.

Good idea! You can really see why he's considered one of the best lawyers…

Yeah.

Layton, you've gotta weave some magic

with this jury, or it's gonna be all over.

Don't worry. The only thing I have to do to turn this jury around

is to remind them of what they don't like about bees.

You got the tweezers? Are you allergic? Only to losing, son. Only to losing.

Mr. Benson Bee, I'll ask you what I think we'd all like to know.

What exactly is your relationship

to that woman?

We're friends.

Good friends? Yes. How good? Do you live together?

Wait a minute…

Are you her little…

…bedbug?

I've seen a bee documentary or two. From what I understand,

doesn't your queen give birth to all the bee children?

Yeah, but…

So those aren't your real parents!

Oh, Barry…

Yes, they are!

Hold me back!

You're an illegitimate bee, aren't you, Benson?

He's denouncing bees!

Don't y'all date your cousins?

Objection! I'm going to pincushion this guy! Adam, don't! It's what he wants!

Oh, I'm hit!!

Oh, lordy, I am hit!

Order! Order!

The venom! The venom is coursing through my veins!

I have been felled by a winged beast of destruction!

You see? You can't treat them like equals! They're striped savages!

Stinging's the only thing they know! It's their way!

Adam, stay with me. I can't feel my legs. What angel of mercy will come forward to suck the poison

from my heaving buttocks?

I will have order in this court. Order!

Order, please!

The case of the honeybees versus the human race

took a pointed turn against the bees

yesterday when one of their legal team stung Layton T. Montgomery.

Hey, buddy.

Hey.

Is there much pain?

Yeah.

I…

I blew the whole case, didn't I?

It doesn't matter. What matters is you're alive. You could have died.

I'd be better off dead. Look at me.

They got it from the cafeteria downstairs, in a tuna sandwich.

Look, there's a little celery still on it.

What was it like to sting someone?

I can't explain it. It was all…

All adrenaline and then… and then ecstasy!

All right.

You think it was all a trap?

Of course. I'm sorry. I flew us right into this.

What were we thinking? Look at us. We're just a couple of bugs in this world.

What will the humans do to us if they win?

I don't know.

I hear they put the roaches in motels. That doesn't sound so bad.

Adam, they check in, but they don't check out!

Oh, my.

Oould you get a nurse to close that window?

Why? The smoke. Bees don't smoke.

Right. Bees don't smoke.

Bees don't smoke! But some bees are smoking.

That's it! That's our case!

It is? It's not over?

Get dressed. I've gotta go somewhere.

Get back to the court and stall. Stall any way you can.

And assuming you've done step correctly, you're ready for the tub.

Mr. Flayman.

Yes? Yes, Your Honor!

Where is the rest of your team?

Well, Your Honor, it's interesting.

Bees are trained to fly haphazardly,

and as a result, we don't make very good time.

I actually heard a funny story about…

Your Honor, haven't these ridiculous bugs

taken up enough of this court's valuable time?

How much longer will we allow these absurd shenanigans to go on?

They have presented no compelling evidence to support their charges

against my clients, who run legitimate businesses.

I move for a complete dismissal of this entire case!

Mr. Flayman, I'm afraid I'm going

to have to consider Mr. Montgomery's motion.

But you can't! We have a terrific case.

Where is your proof? Where is the evidence?

Show me the smoking gun!

Hold it, Your Honor! You want a smoking gun?

Here is your smoking gun.

What is that?

It's a bee smoker!

What, this? This harmless little contraption?

This couldn't hurt a fly, let alone a bee.

Look at what has happened

to bees who have never been asked, "Smoking or non?"

Is this what nature intended for us?

To be forcibly addicted to smoke machines

and man-made wooden slat work camps?

Living out our lives as honey slaves to the white man?

What are we gonna do? He's playing the species card. Ladies and gentlemen, please, free these bees!

Free the bees! Free the bees!

Free the bees!

Free the bees! Free the bees!

The court finds in favor of the bees!

Vanessa, we won!

I knew you could do it! High-five!

Sorry.

I'm OK! You know what this means?

All the honey will finally belong to the bees.

Now we won't have to work so hard all the time.

This is an unholy perversion of the balance of nature, Benson.

You'll regret this.

Barry, how much honey is out there?

All right. One at a time.

Barry, who are you wearing?

My sweater is Ralph Lauren, and I have no pants.

What if Montgomery's right? What do you mean? We've been living the bee way a long time, 27 million years.

Oongratulations on your victory. What will you demand as a settlement?

First, we'll demand a complete shutdown of all bee work camps.

Then we want back the honey that was ours to begin with,

every last drop.

We demand an end to the glorification of the bear as anything more

than a filthy, smelly, bad-breath stink machine.

We're all aware of what they do in the woods.

Wait for my signal.

Take him out.

He'll have nauseous for a few hours, then he'll be fine.

And we will no longer tolerate bee-negative nicknames…

But it's just a prance-about stage name!

…unnecessary inclusion of honey in bogus health products

and la-dee-da human tea-time snack garnishments.

Oan't breathe.

Bring it in, boys!

Hold it right there! Good.

Tap it.

Mr. Buzzwell, we just passed three cups, and there's gallons more coming!

I think we need to shut down! Shut down? We've never shut down. Shut down honey production!

Stop making honey!

Turn your key, sir!

What do we do now?

Oannonball!

We're shutting honey production!

Mission abort.

Aborting pollination and nectar detail. Returning to base.

Adam, you wouldn't believe how much honey was out there.

Oh, yeah?

What's going on? Where is everybody?

Are they out celebrating? They're home. They don't know what to do. Laying out, sleeping in.

I heard your Uncle Oarl was on his way to San Antonio with a cricket.

At least we got our honey back.

Sometimes I think, so what if humans liked our honey? Who wouldn't?

It's the greatest thing in the world! I was excited to be part of making it.

This was my new desk. This was my new job. I wanted to do it really well.

And now…

Now I can't.

I don't understand why they're not happy.

I thought their lives would be better!

They're doing nothing. It's amazing. Honey really changes people.

You don't have any idea what's going on, do you?

What did you want to show me? This. What happened here?

That is not the half of it.

Oh, no. Oh, my.

They're all wilting.

Doesn't look very good, does it?

No.

And whose fault do you think that is?

You know, I'm gonna guess bees.

Bees?

Specifically, me.

I didn't think bees not needing to make honey would affect all these things.

It's notjust flowers. Fruits, vegetables, they all need bees.

That's our whole SAT test right there.

Take away produce, that affects the entire animal kingdom.

And then, of course…

The human species?

So if there's no more pollination,

it could all just go south here, couldn't it?

I know this is also partly my fault.

How about a suicide pact?

How do we do it?

I'll sting you, you step on me. Thatjust kills you twice. Right, right.

Listen, Barry… sorry, but I gotta get going.

I had to open my mouth and talk.

Vanessa?

Vanessa? Why are you leaving? Where are you going?

To the final Tournament of Roses parade in Pasadena.

They've moved it to this weekend because all the flowers are dying.

It's the last chance I'll ever have to see it.

Vanessa, I just wanna say I'm sorry. I never meant it to turn out like this.

I know. Me neither.

Tournament of Roses. Roses can't do sports.

Wait a minute. Roses. Roses?

Roses!

Vanessa!

Roses?!

Barry?

Roses are flowers! Yes, they are. Flowers, bees, pollen!

I know. That's why this is the last parade.

Maybe not. Oould you ask him to slow down?

Oould you slow down?

Barry!

OK, I made a huge mistake. This is a total disaster, all my fault.

Yes, it kind of is.

I've ruined the planet. I wanted to help you

with the flower shop. I've made it worse.

Actually, it's completely closed down.

I thought maybe you were remodeling.

But I have another idea, and it's greater than my previous ideas combined.

I don't want to hear it!

All right, they have the roses, the roses have the pollen.

I know every bee, plant and flower bud in this park.

All we gotta do is get what they've got back here with what we've got.

Bees.

Park.

Pollen!

Flowers.

Repollination!

Across the nation!

Tournament of Roses, Pasadena, Oalifornia.

They've got nothing but flowers, floats and cotton candy.

Security will be tight.

I have an idea.

Vanessa Bloome, FTD.

Official floral business. It's real.

Sorry, ma'am. Nice brooch.

Thank you. It was a gift.

Once inside, we just pick the right float.

How about The Princess and the Pea?

I could be the princess, and you could be the pea!

Yes, I got it.

Where should I sit?

What are you?

I believe I'm the pea.

The pea?

It goes under the mattresses.

Not in this fairy tale, sweetheart. I'm getting the marshal. You do that! This whole parade is a fiasco!

Let's see what this baby'll do.

Hey, what are you doing?!

Then all we do is blend in with traffic…

…without arousing suspicion.

Once at the airport, there's no stopping us.

Stop! Security.

You and your insect pack your float? Yes. Has it been in your possession the entire time?

Would you remove your shoes?

Remove your stinger. It's part of me. I know. Just having some fun. Enjoy your flight.

Then if we're lucky, we'll have just enough pollen to do the job.

Oan you believe how lucky we are? We have just enough pollen to do the job!

I think this is gonna work.

It's got to work.

Attention, passengers, this is Oaptain Scott.

We have a bit of bad weather in New York.

It looks like we'll experience a couple hours delay.

Barry, these are cut flowers with no water. They'll never make it.

I gotta get up there and talk to them.

Be careful.

Oan I get help with the Sky Mall magazine?

I'd like to order the talking inflatable nose and ear hair trimmer.

Oaptain, I'm in a real situation.

What'd you say, Hal? Nothing. Bee!

Don't freak out! My entire species…

What are you doing?

Wait a minute! I'm an attorney! Who's an attorney? Don't move.

Oh, Barry.

Good afternoon, passengers. This is your captain.

Would a Miss Vanessa Bloome in 24B please report to the cockpit?

And please hurry!

What happened here?

There was a DustBuster, a toupee, a life raft exploded.

One's bald, one's in a boat, they're both unconscious!

Is that another bee joke? No! No one's flying the plane!

This is JFK control tower, Flight 356. What's your status?

This is Vanessa Bloome. I'm a florist from New York.

Where's the pilot?

He's unconscious, and so is the copilot.

Not good. Does anyone onboard have flight experience?

As a matter of fact, there is.

Who's that? Barry Benson. From the honey trial?! Oh, great.

Vanessa, this is nothing more than a big metal bee.

It's got giant wings, huge engines.

I can't fly a plane.

Why not? Isn't John Travolta a pilot? Yes. How hard could it be?

Wait, Barry! We're headed into some lightning.

This is Bob Bumble. We have some late-breaking news from JFK Airport,

where a suspenseful scene is developing.

Barry Benson, fresh from his legal victory…

That's Barry!

…is attempting to land a plane, loaded with people, flowers

and an incapacitated flight crew.

Flowers?!

We have a storm in the area and two individuals at the controls

with absolutely no flight experience.

Just a minute. There's a bee on that plane.

I'm quite familiar with Mr. Benson and his no-account compadres.

They've done enough damage.

But isn't he your only hope?

Technically, a bee shouldn't be able to fly at all.

Their wings are too small…

Haven't we heard this a million times?

"The surface area of the wings and body mass make no sense."

Get this on the air!

Got it.

Stand by.

We're going live.

The way we work may be a mystery to you.

Making honey takes a lot of bees doing a lot of small jobs.

But let me tell you about a small job.

If you do it well, it makes a big difference.

More than we realized. To us, to everyone.

That's why I want to get bees back to working together.

That's the bee way! We're not made of Jell-O.

We get behind a fellow.

Black and yellow! Hello! Left, right, down, hover.

Hover? Forget hover. This isn't so hard. Beep-beep! Beep-beep!

Barry, what happened?!

Wait, I think we were on autopilot the whole time.

That may have been helping me. And now we're not! So it turns out I cannot fly a plane.

All of you, let's get behind this fellow! Move it out!

Move out!

Our only chance is if I do what I'd do, you copy me with the wings of the plane!

Don't have to yell.

I'm not yelling! We're in a lot of trouble.

It's very hard to concentrate with that panicky tone in your voice!

It's not a tone. I'm panicking!

I can't do this!

Vanessa, pull yourself together. You have to snap out of it!

You snap out of it.

You snap out of it.

You snap out of it!

You snap out of it!

You snap out of it!

You snap out of it!

You snap out of it!

You snap out of it!

Hold it!

Why? Oome on, it's my turn.

How is the plane flying?

I don't know.

Hello?

Benson, got any flowers for a happy occasion in there?

The Pollen Jocks!

They do get behind a fellow.

Black and yellow. Hello. All right, let's drop this tin can on the blacktop.

Where? I can't see anything. Oan you?

No, nothing. It's all cloudy.

Oome on. You got to think bee, Barry.

Thinking bee. Thinking bee. Thinking bee! Thinking bee! Thinking bee!

Wait a minute. I think I'm feeling something.

What? I don't know. It's strong, pulling me. Like a 27-million-year-old instinct.

Bring the nose down.

Thinking bee! Thinking bee! Thinking bee!

What in the world is on the tarmac? Get some lights on that! Thinking bee! Thinking bee! Thinking bee!

Vanessa, aim for the flower. OK. Out the engines. We're going in on bee power. Ready, boys?

Affirmative!

Good. Good. Easy, now. That's it.

Land on that flower!

Ready? Full reverse!

Spin it around!

Not that flower! The other one!

Which one?

That flower.

I'm aiming at the flower!

That's a fat guy in a flowered shirt. I mean the giant pulsating flower

made of millions of bees!

Pull forward. Nose down. Tail up.

Rotate around it.

This is insane, Barry! This's the only way I know how to fly. Am I koo-koo-kachoo, or is this plane flying in an insect-like pattern?

Get your nose in there. Don't be afraid. Smell it. Full reverse!

Just drop it. Be a part of it.

Aim for the center!

Now drop it in! Drop it in, woman!

Oome on, already.

Barry, we did it! You taught me how to fly!

Yes. No high-five! Right. Barry, it worked! Did you see the giant flower?

What giant flower? Where? Of course I saw the flower! That was genius!

Thank you. But we're not done yet. Listen, everyone!

This runway is covered with the last pollen

from the last flowers available anywhere on Earth.

That means this is our last chance.

We're the only ones who make honey, pollinate flowers and dress like this.

If we're gonna survive as a species, this is our moment! What do you say?

Are we going to be bees, orjust Museum of Natural History keychains?

We're bees!

Keychain!

Then follow me! Except Keychain.

Hold on, Barry. Here.

You've earned this.

Yeah!

I'm a Pollen Jock! And it's a perfect fit. All I gotta do are the sleeves.

Oh, yeah.

That's our Barry.

Mom! The bees are back!

If anybody needs to make a call, now's the time.

I got a feeling we'll be working late tonight!

Here's your change. Have a great afternoon! Oan I help who's next?

Would you like some honey with that? It is bee-approved. Don't forget these.

Milk, cream, cheese, it's all me. And I don't see a nickel!

Sometimes I just feel like a piece of meat!

I had no idea.

Barry, I'm sorry. Have you got a moment?

Would you excuse me? My mosquito associate will help you.

Sorry I'm late.

He's a lawyer too?

I was already a blood-sucking parasite. All I needed was a briefcase.

Have a great afternoon!

Barry, I just got this huge tulip order, and I can't get them anywhere.

No problem, Vannie. Just leave it to me.

You're a lifesaver, Barry. Oan I help who's next?

All right, scramble, jocks! It's time to fly.

Thank you, Barry!

That bee is living my life!

Let it go, Kenny.

When will this nightmare end?!

Let it all go.

Beautiful day to fly.

Sure is.

Between you and me, I was dying to get out of that office.

You have got to start thinking bee, my friend.

Thinking bee! Me? Hold it. Let's just stop for a second. Hold it.

I'm sorry. I'm sorry, everyone. Oan we stop here?

I'm not making a major life decision during a production number!

All right. Take ten, everybody. Wrap it up, guys.

I had virtually no rehearsal for that.

Creating scenarios in her head, delusional and desperate/needy. not in a bad way

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Author response:

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

eLife assessment

Both reviewers positively received the manuscript, in general. The agreement was that the manuscript presented valuable findings, using solid techniques and approaches, that shed additional light into how the canine distemper virus hemagglutinin might engage cellular receptors and how that engagement impacts host tropism. While both reviewers appreciated the X-ray crystallographic data, they also felt that the AFM experiments could have been performed at a higher standard and that the interpretation of the results ensuing from those AFM experiments could have been explained more thoroughly and in simpler terms. An additional missed opportunity of the current manuscript is the lack of comparison of the crystal structure to that of the already published cryo-EM structure, for context.

Thank you very much for constructive comments of the editor and reviewers. Following your comments, we have changed the text related to the AFM experiments with simpler terms as follows.

“When CDV-H was loaded onto a mica substrate and scanned with a cantilever to acquire images of attached molecules, the CDV-H dimer was observed as two globules clustered together in most cases, but sometimes, each domain moved independently (Fig. 7B and Supplementary Movie). Time-course analysis of the dynamics of the representative CDV-H dimer showed that CDV-H could adopt both associated and dissociated forms (Fig. 7C). The distances between the domains were calculated by measuring those between the centers of mass of each domain. Finally, the distribution of distances between each head domain in the CDV-H dimers showed approximately 15 nm as a major peak (Fig. 7D). This is a reasonable length for the linker between the head domain dimers.” in Page 11, Lines 8-17.

With regards to the structural comparison between cryo-EM structure published in Proc. Natl. Acad. Sci. U. S. A. (2023) 120, e2208866120 and our crystal structure, we have compared these structures for Cα on page 6 and added the following text. “A recent cryo-EM structure of the wild-type CDV-H ectodomain revealed that the head dimer is located on one side of the stalk region in solution (Proc. Natl. Acad. Sci. U. S. A. (2023) 120, e2208866120)” in Page 14, Lines 22-24.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Fukuhara, Maenaka, and colleagues report a crystal structure of the canine distemper virus (CDV) attachment hemagglutinin protein globular domain. The structure shows a dimeric organization of the viral protein and describes the detailed amino-acid side chain interactions between the two protomers. The authors also use their best judgement to comment on predicted sites for the two cellular receptors - Nectin-4 and SLAM - and thus speculate on the CDV host tropism. A complementary AFM study suggests a breathing movement at the hemagglutinin dimer interface.

Strengths:

The study of CDV and related Paramyxoviruses is significant for human/animal health and is very timely. The crystallographic data seem to be of good quality.

Thank you very much for the constructive comment of the reviewer.

Weaknesses:

While the recent CDV hemagglutinin cryo-EM structure is mentioned, it is not compared to the present crystal structure, and thus the context of the present study is poorly justified. Additionally, the results of the AFM experiment are not unexpected. Indeed, other paramyxoviral RBP/G proteins also show movement at the protomer interface.

Thank you very much for constructive comments of the reviewer. When we submitted our manuscript to e-life, cryo-EM structure just published in Proc. Natl. Acad. Sci. U. S. A. (2023) 120, e2208866120 a week ago was not able to be available. Following the comment of the reviewer, we have added the text about the structural comparison between the cryo-EM structure and our crystal structure. We also have changed the text related to the AFM experiments to tone down the movement of the protomer interfaceas follows.

“This observation raises the possibility that each head domain of CDV-H also dissociates and moves flexibly, as shown in the structure of Nipah virus (NiV)-G protein, previously (Science (2022) 375, 1373–1378).” in Page 11, Lines 4-6.

Reviewer #2 (Public Review):

Summary:

The authors solved the crystal structure of CDV H-protein head domain at 3,2 A resolution to better understand the detailed mechanism of membrane fusion triggering. The structure clearly showed that the orientation of the H monomers in the homodimer was similar to that of measles virus H and different from other paramyxoviruses. The authors used the available co-crystal strictures of the closely related measles virus H structures with the SLAM and Nectin4 receptors to map the receptor binding site on CDV H. The authors also confirmed which N-linked sites were glycosylated in the CDV H protein and showed that both wildtype and vaccine strains of CDV H have the same glycosylation pattern. The authors documented that the glycans cover a vast majority of the H surface while leaving the receptor binding site exposed, which may in part explain the long-term success of measles virus and CDV vaccines. Finally, the authors used HS-AFM to visualize the real-time dynamic characteristics of CDV-H under physiological conditions. This analysis indicated that homodimers may dissociate into monomers, which has implications for the model of fusion triggering.

The structural data and analysis were thorough and well-presented. However, the HS-AFM data, while very exciting, was not presented in a manner that could be easily grasped by readers of this manuscript. I have some suggestions for improvement.

(1) The authors claim their structure is very similar to the recently published croy-EM structure of CDV H. Can the authors provide us with a quantitative assessment of this statement?

Thank you very much for constructive comments of the reviewer. When we submitted our manuscript to e-life, cryo-EM structure just published in Proc. Natl. Acad. Sci. U. S. A. (2023) 120, e2208866120 a week ago was not able to be available. Following the comment of the reviewer, we have added the text about the structural comparison between the cryo-EM structure and our crystal structure. We also have changed the text related to the AFM experiments to tone down the movement of the protomer interface as follows.

“This observation raises the possibility that each head domain of CDV-H also dissociates and moves flexibly, as shown in the structure of Nipah virus (NiV)-G protein, previously (Science (2022) 375, 1373–1378).” in Page 11, Lines 4-6.

(2) The results for the HS-AFM are difficult to follow and it is not clear how the authors came to their conclusions. Can the authors better explain this data and justify their conclusions based on it?

Thank you very much for constructive comments of the reviewer. Following your comments, we have changed the text related to the AFM experiments with simpler terms as follows.

“When CDV-H was loaded onto a mica substrate and scanned with a cantilever to acquire images of attached molecules, the CDV-H dimer was observed as two globules clustered together in most cases, but sometimes, each domain moved independently (Fig. 7B and Supplementary Movie). Time-course analysis of the dynamics of the representative CDV-H dimer showed that CDV-H could adopt both associated and dissociated forms (Fig. 7C). The distances between the domains were calculated by measuring those between the centers of mass of each domain. Finally, the distribution of distances between each head domain in the CDV-H dimers showed approximately 15 nm as a major peak (Fig. 7D). This is a reasonable length for the linker between the head domain dimers.” in Page 11, Lines 8-17.

(3) The fusion triggering model in Figure 8 is ambiguous as to when H-F interactions are occurring and when they may be disrupted. The authors should clarify this point in their model.

Thank you very much for constructive comments of the reviewer. Following your comments, we have changed the Figure 8 and its legend.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) AFM experiments with SLAM or Nectin-4 immobilized on the cantilever would be much more informative.

Thank you very much for the constructive comment of the reviewer. We will try this experiment in the next paper.

(2) The authors should compare their crystal structure to that of the reported cryo-EM structure.

With regards to the structural comparison between cryo-EM structure published in Proc. Natl. Acad. Sci. U. S. A. (2023) 120, e2208866120 and our crystal structure, we have added the text.

(3) Figure 1D - why does the beta2 MG negative control have such a high SPR signal?

Thank you very much for the constructive comment of the reviewer. The immobilization levels for b 2-microglobulin (beta2 MG), CDV-OP-H and CDV-5VD-H were similar, 1204.7 RU, 1235.7 RU, and 1504.5 RU, respectively. We applied relatively high concentrations (5 mM) of dNectin4 and hNectin4 onto the chip to determine low-affinity dissociation constants. Then, the signals for beta2 MG (negative control) were high. In other SPR experiments for cell surface receptors, such high signals for beta2 MG were often observed in our previous paper, Kuroki et al., J. Immunol. 2019 Dec 15;203(12):3386-3394. doi: 10.4049/jimmunol.1900562. Therefore, we think that these SPR signals are not unusual.

(4) Figure 1C - please indicate the Ve volume for the peak and add in Ve for standard.

Thank you very much for the constructive comment of the reviewer. We have indicated the Ve volume for the peak and added in Ve for standard in Figure 1C.

(5) The authors mention that one of the chains in the asymmetric unit was better resolved than the other. Please show regions of the atomic model fit regions of the electron density to convince the reader of the quality of your data.

Thank you very much for the constructive comment of the reviewer. We have added new Supplementary figure 2 for comparison of electron density maps of chains A and B.

(6) Table 2 indicates that the difference between Rw and Rf values is larger than 5% which indicates slight overfitting during refinement. Please provide details of your refinement strategy and attempt simulated annealing as a strategy to reduce this delta.

Thank you very much for the constructive comment of the reviewer. We further introduced TLS and NCS parameters for the refinement. Consequently, the R/Rfree factors became 0.2645/0.3092. Simulated annealing had been already carried out. All the refinement statistics in the table 2 are updated.

Reviewer #2 (Recommendations For The Authors):

(1) The authors' fusion triggering model was difficult to follow. For example, this sentence was difficult to understand: "The other possible models may include the monomer-dimer-tetramer transition facilitated by receptor binding for the fusion."

Thank you very much for the constructive comment of the reviewer. Following your comments, we have removed the above sentences and have added the detail mechanism of the proposed model in Discussion. Furthermore, we have changed the Figure 8 and its legend for readers to understand more clearly.

(2) Figure 5A is not called out in the main text.

Thank you very much for the constructive comment of the reviewer. Following your comments, we have added the text as follows.

“the crystal structure of MeV-H in complex with hNectin-4 showed that the H-SLAM interaction consists of three main sites (Fig. 5A) (Nat. Struct. Mol. Biol. (2013) 20, 67–72).” in Page 11, Lines 4-6.

(3) Page 9, Line 4: interspaces? Perhaps interphases.

Thank you very much for the constructive comment of the reviewer. We have changed the term “interspaces” to “internal spaces”.

(4) Page 12, penultimate line: The authors mention "epitopes for anti-MeV-H Abs." Do they mean anti-CDV-H Abs?

Thank you very much for the constructive comment of the reviewer. Following your comments, we have changed the “anti-MeV-H Abs” to “anti-morbillivirus H neutralizing antibodies”.

(5) The paper will benefit from an English language editor to help clarify what the authors are trying to convey.

Thank you very much for the constructive comment of the reviewer.

We have asked a English proof reading company to check.

Reviewer #2 (Public Review):

The authors solved the crystal structure of CDV H-protein head domain at 3,2 A resolution to better understand the detailed mechanism of membrane fusion triggering. The structure clearly showed that the orientation of the H monomers in the homodimer was similar to that of measles virus H and different from other paramyxoviruses. The authors used the available co-crystal strictures of the closely related measles virus H structures with the SLAM and Nectin4 receptors to map the receptor binding site on CDV H. The authors also confirmed which N-linked sites were glycosylated in the CDV H protein and showed that both wildtype and vaccine strains of CDV H have the same glycosylation pattern. The authors documented that the glycans cover a vast majority of the H surface while leaving the receptor binding site exposed, which may in part explain the long-term success of measles virus and CDV vaccines. Finally, the authors used HS-AFM to visualize the real-time dynamic characteristics of CDV-H under physiological conditions. This analysis indicated that homodimers may dissociate into monomers, which has implications for the model of fusion triggering.

The structural data and analysis were thorough and well-presented. The HS-AFM data, while very exciting, needs to be further validated, perhaps by alternate approaches to further support the authors' model describing the molecular dynamics of fusion triggering.

Reviewer #2 (Public Review):

Immunogenic cell death (ICD) can lead to the release of factors such as DAMPs which promote an adaptive immune response. In the context of cancer, there is clear evidence of anti-tumour benefits as a result of ICD, perhaps induced by chemotherapy.

Lilong et al used TCGA data to explore whether a previously published 34 gene 'ICD-related' signature could stratify bladder cancer patients by prognosis and ultimately predict patient survival. The gene signature contains many genes involved in inflammation and immunity (IFNg, IL6, TNF, IL17A, TLR4, CD8B, etc) and those related to ICD (such as CALR, HMGB1, HSP, NLRP3, etc). The authors divide patients into 'ICD-high' and '-low' based on the expression of this gene set and find that 'ICD-high' is associated with longer survival in TCGA bladder cancer data. The authors further argue that ICD-high group responds better to PD1 therapies. From this 34-gene signature, it appears that LASSO regularisation and Cox analysis identifies a four-gene 'risk' signature (CALR, IL1R1, IFNB1, IFNG) which is associated with shorter patient survival and lower immunotherapy response rates. This is the primary finding. Their methodology is very similar to a publication in 2021 in Frontiers in Immunology instead in the context of head and neck squamous cell carcinoma. This paper is not referenced.

In terms of the strengths of the work, it is certainly plausible that the author's four gene signature has an association with survival in bladder cancer, at least based on the two datasets studied. However, the relatedness of their findings to ICD is unconvincing, and glaring omissions from the manuscript in terms of methods limit confidence in the work. The authors show a potential association with bladder cancer patient survival and their four gene signatures, but substantial revisions are required for this to be appropriately evidenced.

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

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Reply to the reviewers

Reply to the Reviewers

We sincerely thank the Referees for providing important and constructive comments. We have addressed their concerns point-by-point as described below.

Associated to Reviewer#1's comments

*- Diploid embryos are used as controls. Gynogenetic diploids seem to be better controls to ensure that the observed phenotypes are not related to loss of heterozygosity. To limit the amount of work, the use of gynogenetic diploids could be restricted to spindle polarity and centrosome number experiments. *

Response 1-1

__[Experimental plan] __Following the reviewer's suggestion, we will conduct immunostaining of a-tubulin and centrin (for visualizing the spindles and centrioles, respectively) in gynogenetic diploids that will be generated by applying heat shock to gynogenetic haploid embryos during the 1st - 2nd cleavage stage. We will observe the head area of gynogenetic diploid larvae at 3-dpf when the haploid counterparts suffer particularly drastic centrosome loss and spindle monopolarization.

• *

• *

*- As the authors discuss, it would be necessary to rescue centrosome loss to establish a causal relationship between centrosome loss and haploid viability. I certainly acknowledge that this is difficult (if not impossible), but it currently limits the significance of the results. *

Response 1-2

We agree that rescuing centrosome loss would provide an important advancement in understanding the cause of haploid syndrome in the context of our study. However, as the reviewer also pointed out in the above comment, this poses a significant technical challenge. As described in Discussion in the original manuscript, we have attempted to restore normal centrosome number through cell cycle modulations. However, we have not found a condition that rescues centrosome loss without damaging larval viability. As an alternative approach, we have also tried to induce centriole amplification by injecting mRNA encoding plk4, an essential centriole duplication inducer. However, this caused earlier embryonic death, precluding us from observing its effects on larval morphology after 1 dpf. The main challenge is that any treatment to increase centrosome number can cause centrosome overduplication, which is as deleterious to development as centrosome loss. Efforts to identify a key factor enabling the rescue of centrosome loss in haploid larvae are underway in our laboratory, which requires new explorations over several years and is beyond the scope of the present study. Reflecting on the reviewer's comment, we added a new sentence explaining the situation on this issue (line 395, page 19). To further discuss possible contributions of centrosome loss and mitotic defects to haploidy-linked embryonic defects, we also added a citation of a previous study reporting that depletion of centrosomal proteins caused mitotic defects leading to embryonic defects similar to those observed in haploid embryos in zebrafish (Novorol et al., 2013 Open Biology; line 380, page 19).

__[Experimental plan] __Meanwhile, as a new trial to induce centriole amplification in a scalable and temporally controllable manner, we plan the following experiment, which can be conducted within the time range of the revision schedule: We will investigate the effects of low dose treatment of a plk4 inhibitor centrinone B on tissue growth and viability of haploid larvae. A recent study reported that centrinone B had complicated effects on the centriole duplication process, which is highly dose-sensitive (Tkach et al., 2022 Elife, PMID: 35758262). While it blocks centriole duplication at sufficiently high concentrations for blocking plk4 activities, it paradoxically causes centriole amplification at suboptimal conditions, presumably though over-stabilizing plk4 by blocking its autophosphorylation-dependent degradation (while its centriole duplicating function remains active). Since a previous study showed that centrinone B is also effective in zebrafish embryos (Rathbun et al., 2020 Current Biology, PMID: 32916112), we try to find optimal centrinone B treatment condition that potentially restores tissue growth or viability of haploid embryos. If we find such a rescuing condition, we will address the principle of the rescuing effects by investigating the possession of centrioles in mitotic cells in these haploid larvae.

*- Some experiments are not, or arguably, quantified/statistically analyzed. *

o Figure 2, Active caspase level. Larvae are sorted into three categories, and no statistical test is performed on the obtained contingency table. A Fisher'*s exact test here, or much better, the active caspase-3 levels should be quantified, instead of sorting larvae into categories. *

Response 1-3

We apologize that we showed only "zoomed-out" images of the immunostained embryos in the original figures (Fig. 2A), which precluded a clear presentation of the haploidy-associated aggravation of apoptosis and mitotic arrest. We could clearly distinguish cleaved caspase-3- and pH3-positive cells from non-specific background staining with an enlarged view of the same immunostaining data. Therefore, to quantitatively evaluate the extent of the haploidy-linked apoptosis and mitotic arrest, we compared the density of these cells within the right midbrain. This new quantification demonstrated a statistically significant increase in cleaved caspase-3- or pH3-positive cells in haploids compared to diploids.

In the revised manuscript, we added the enlarged views of cleaved-caspase and pH3 immunostaining (Fig. 2B) and new quantifications with statistical analyses (Fig. 2C). Accompanying these revisions, we omitted the categorization of the severeness of the apoptosis, which was pointed out to be subjective in the reviewer#2's comment (see Response 2-3). We rewrote the corresponding section of the manuscript to explain the new quantitative analyses (line 143, page 7).

o Same comment for 3E-F. Larvae are scored as Scarce, Mild or Severe. Looking at Fig S3A, I see one mild p53MO embryo, but the two others are not that different from 'severe' cases, which would completely change the contingency table. Again, a proper quantification would be better.

Response 1-4

We also quantified the frequency of cleaved caspase-3-positive cells in control and p53MO larvae (original Fig. 3E and F) as described in Response 1-3. While conducting the cell counting with enlarged images, we realized that staining quality within the inner larval layers of morphants was relatively poor in these experiments. This problem precluded us from counting cleaved caspase-3-positive cells within the inner larval layers. Therefore, we tentatively quantified only the surface larval layers of these morphants and found that cleaved caspase-3-positive cells were significantly reduced in haploids upon depletion of p53. We currently show this quantification in Fig. 3G of the revised manuscript. While this quantification confirmed the trend of p53MO-dependent decrease in apoptosis, we think it more appropriate to newly conduct the same experiment with better quality of the staining to apply the same standard of quantification for Fig. 3 as Fig. 2.

__[Experimental plan] __For the reason described above, we propose to re-conduct immunostaining of cleaved caspase-3 in control and p53MO-injected haploid larvae to improve the visibility of the inner layer of the larvae for better quality of the quantitation.

Meanwhile, we revised Fig. 3 by adding an enlarged view of immunostaining in Fig. 3F and omitting the subjective categorization shown in the original Fig. 3F and S3A. We plan to replace these data with new images and quantification to be obtained during the next revision. We also rewrote the main text to update these changes (line 166, page 8).

*o Figure 4D-E, no stats. *

Response 1-5

We conducted the ANOVA followed by the post-hoc Tukey test for new Fig. 4D and the Fisher exact test with Benjamini-Hochberg multiple testing correction for new Fig. 4E. Please note that statistical analyses were conducted after adding the data from original Fig. 6B-C following the reviewer's suggestion (see also Response 1-6).

*o Figure 6, Reversine treated haploid should be compared to haploid embryos (on the graphs and statistically). If no specific controls have been quantified for this experiment, data could be reused from previous figures, provided this is stated. *

Response 1-6

The live imaging data shown in original Fig. 4C-E and Fig. 6A-C were obtained within the same experimental series conducted in parallel at the same period under the same experimental condition. In the original manuscript, we separated them into two different figures according to the logical flow. However, following the reviewers' comments (see also Response 2-1), we realized it more appropriate to show them as a single figure panel as in the original experimental design. Therefore, we moved the reversine-treated haploid data from the original Fig. 6A-C to Fig. 4C-E to facilitate direct comparison among conditions with statistical analyses (see also Response 1-5).

*o Rescue by p53MO and Reversine, it would be nice to also include diploid measurements on the graphs, so that the reader can appreciate the extent of the rescue. *

Response 1-7

Following the reviewer's comment, we added control MO-injected or DMSO-treated diploid larval data in the corresponding graphs in Fig. 3I and 6G, respectively. Please refer to Response 2-6 for further discussion on the extent of the rescue.

Minor comments:

*- Lines 221-223, authors claim that centriole loss and spindle monopolarization commence earlier in the eyes and brain than in skin. I am note sure I see this in Fig. S5. It could as well be that the defect is less pronounced in skin. *

Response 1-8

We rewrote the manuscript to include the possible interpretation suggested by the reviewer on the result (line 225, page 11).

• *

- Lines 227-229, authors claim that 'The developmental stage when haploid larvae suffered the gradual aggravation of centrosome loss corresponded to the stage when larval cell size gradually decreased through successive cell divisions'. I did not get that. Doesn'*t cell size decrease since the first division? Fig 5D shows that cell size decreases all along development. *

Response 1-9

We agree that the original sentence implies, against our intention, that cell size does not decrease before the developmental stage mentioned here. To correct this problem, we rewrote the corresponding part of Discussion as below (line 230, page 11):

"Since the first division, embryonic cell size continuously reduces through successive cell divisions during early development (Menon et al., 2020). Cell size reduction continued at the developmental stage when we observed the gradual aggravation of the centrosome loss in haploid larvae."

*- Some correlations are used to draw conclusions: *

o Line 301-303. "The correlation between centrosome loss and spindle monopolarization indicates that haploid larval cells fail to form bipolar spindle because of the haploidy-linked centrosome loss."*. As stated by the authors, this is a correlation only. I agree it points in this direction. *

Response 1-10

We added a note to the corresponding sentence to draw readers' attention to the discussion on the limitation of the study with respect to the lack of centrosome rescue experiment (line 332, page 16).

O Line 305-308. "*Interestingly, centrosome loss occurred almost exclusively in haploid cells whose size became smaller than a certain border (Fig. 5), indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." This one is more problematic. There is no causal link established between cell size and centrosome number homeostasis. It could very well be that some unidentified problem induces both a reduction in cell size and the loss of centrioles. *

Response 1-11

To avoid an over-speculative description, we deleted the subsentence "indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." (line 336, page 17). We also added a new sentence, "Alternatively, it is also possible that other primary causes, such as the lack of second active allele producing sufficient protein pools induced cell size reduction and centrosome loss in parallel without causality between them." to discuss the possibility raised by the reviewer (line 348, page 17), in association with another comment from the reviewer #3 (see also Response 3-3).

• *

*I have concerns regarding the significance of the reported findings. Haploid zebrafish embryos show numerous developmental defects (some as early as gastrulation, as previously shown by the authors, Menon 2020), and they die by 4 dpf. That they experience massive apoptosis at day 3 does not seem very surprising, and that inhibiting p53 transiently improves the phenotype is not a big surprise. *

Response 1-12

Many reports have revealed tissue-level developmental abnormalities in haploid embryos since the discovery of haploid lethality in vertebrates more than 100 years ago. This has stimulated speculation of underlying causes of haploid intolerance for decades. However, there have been surprisingly few descriptions of cellular abnormalities underlying these tissue defects, precluding an evidence-based understanding of the principle that limits developmental ability in haploid embryos. Our findings of the haploidy-linked p53 upregulation and mitotic defects illustrate what happens in the dying haploid embryos at a cellular level. These findings would provide an evidence-based frame of reference for understanding why vertebrates cannot develop in the haploid state and also provide clues to controlling haploidy-linked embryonic defects in future studies. We added a new section in Discussion to discuss the importance of addressing the haploidy-linked defects at a cellular level (line 276, page 14).

*This reminds me of the non-specific effects of morpholino injection, which can be partially rescued by knocking down p53. *

Response 1-13

We believe the reviewer refers to the previous findings that different morpholinos generally have off-target effects activating p53-mediated apoptosis (e.g., Robu et al., 2007 PLoS Genet, PMID:17530925). However, p53 upregulation and apoptosis aggravation were also observed in uninjected haploid embryos free from morpholinos' artificial effects (Fig. 2, Fig. 3A, and B). To further address this issue, we plan to compare the frequency of cleavage caspase-3-positive cells between uninjected and control MO-injected haploids after revising the immunostaining of morphants in the original Fig. 3E-F (see Response 1-4 for details).

*The observation of mitotic arrest and mitotic defects and the observation that haploid cells often lack a centrosome is interesting. However, I felt that the manuscript suggested that these observations were novel and could explain the haploid syndrome specifically in non-mammalian embryos, when the authors reported the same observations in human haploid cells as well as in mouse haploid embryos (Yaguchi 2018). To me, this manuscript mainly confirms that their previous observation is not mammalian specific, but at least conserved in vertebrates. *

Response 1-14

As we originally wrote (line 341, page 17 in the original manuscript), we think these haploidy-linked cellular defects are conserved among mammalian and non-mammalian vertebrates. To improve the clarity of our interpretation, we rewrote a corresponding part of the manuscript (line 50, page 2).

*While I am no expert at centrosome duplication, I find the observation that haploidy leads to centrosome loss very intriguing, but have the impression that this manuscript falls short of improving our understanding of this phenomenon. *

Response 1-15

We express our gratitude to the reviewer for being interested in our findings. We hope the revisions made in the manuscript and the new results provided by the planned experiments will strengthen the contribution of this study to our understanding of haploidy-linked cellular defects.

• *

• *

Associated to Reviewer#2's comments

- Lack of proper controls in many experiments. For example, in the experiments where the authors treated haploids with reversine to suppress the SAC, there was no no-treatment control (Fig. 6A-C).

Response 2-1

We addressed the same point in__ Response 1-6__. In the original manuscript, we separately presented control and experimental conditions in the same experiment series in Fig. 4 and Fig. 6. We rejoined them in Fig. 4 as in the original experimental design. Please refer to __Response 1-6 __for further details.

• In Fig. 6D, when a DMSO control was included, the control fish were from 3 dpf while the reversine-treated fish were from 0.5-3 dpf. This is a big flaw in experimental design, especially considering the authors were looking at mitotic index, which is hugely impacted by developmental time. *

Response 2-2

In this experiment, we treated haploid larvae with either DMSO or reversine from 0.5 to 3 dpf, isolated cells from the larvae at 3 dpf, and subjected them to flow cytometry. Both DMSO- and reversine-treated larval cells were from 3-dpf larvae. Therefore, this experiment does not have the problem noted by the reviewer. To improve the clarity of the description of the experimental design, we rewrote the corresponding part of the figure legend (line 646, page 34).

- Subjective and inadequate data quantification. In the immunostaining experiments to detect caspase-3 and pH3, the authors either did not quantify at all and only showed single micrographs that might or might not be representative (for pH3), or only did very subjective and unconvincing quantification (for caspase-3). Objective measurements of fluorescence intensity could have been done, but the authors instead chose to categorize the staining into arbitrary categories with unclear standards. In example images they showed in the supplementary data, it is not obvious at all why some of the samples were classified as "mild" and others as "*severe" when their staining did not appear to be very different. *

Response 2-3

We apologize that we showed only "zoomed-out" images of the immunostained embryos in the original figures (Fig. 2A, 3E, and 6F), in which the distribution of individual cleaved caspase-3- or pH3-positive cells could not be clearly recognized. We added the enlarged view of identical immunostaining where these cells were clearly visualized in a countable manner (Fig. 2B, 3F, and 6D). Following the reviewer's suggestion, we newly conducted quantification by comparing the density of these cells within the right midbrain in haploids and diploids.

This new quantification demonstrated the haploidy-linked increase in cleaved caspase-3- or pH3-positive cells and a reversine-dependent decrease in pH3-positive cells. We added these new quantifications with statistical analyses to the revised manuscript (Fig. 2C and 6E). Accompanying these revisions, we omitted the categorization of the severeness of apoptosis, which was pointed out to be subjective. We rewrote the corresponding section of the manuscript to explain the new quantitative analyses (line 143, page 7; line 260, page 12).

While we also quantified cleaved caspase-3-positive cells in control and p53MO larvae in the original Fig. 3E, we realized that the staining quality of the inner larval layers of these morphants was relatively poor and could not apply the same standard of quantification as Fig. 2. Though we confirmed a statistically significant reduction in cleaved caspase-3-positive cells upon p53 depletion by quantified limited number of confocal sections (shown in Fig. 3G, please see also Response 1-4 for details), we decided to re-conduct this experiment for improving the staining quality to apply the same criteria of quantification for Fig 3 as Fig. 2 (Experimental plan is provided in Response 1-4).

Please note that we also tried to evaluate the extent of apoptosis and mitotic arrest based on the fluorescence intensity of organ areas. However, background staining outside the dead cell area precluded the precise quantification.

Additionally, the authors claimed that "*clusters of apoptotic cells" were only present in haploids but not diploids or p53 MO haploids, but they did not show any quantification. From the few example images (Fig.S3A), apoptotic clusters can be seen in p53 MO treated fish. Also, in some cases, the clusters were visible only because those fish were mounted in an incorrect orientation. For example, in Fig. S3A, control #2, that fish was visualized from its side, thus exposing areas around its eye that contained such clusters. These areas are not visible in other images where the fish were visualized from the top. *

__Response 2-4 __

We agree that the definition of "apoptotic clusters" was ambiguous in the original manuscript. We also agree that the visuals of the clusters could be affected by sample conditions, making them less reliable criteria for judging the severity of apoptotic upregulation in larvae. Following the reviewer's suggestion, we newly conducted apoptotic cell counting (Response 2-3), which recapitulated more reliably ploidy- or condition-dependent changes in the extent of apoptosis. Therefore, we decided to omit the description of the clusters in the new version of the manuscript.

*- Subpar data quality. Aside from issues with qualification, the IF data was not convincing as staining appeared to be inconsistent and uneven, with potential artefacts. *

Response 2-5

We apologize that the zoomed-out images in the original figures did not appropriately demonstrate the specific visualization of individual apoptotic or mitotic cells. As described in Response 2-3, we added enlarged views of the immunostaining to the revised manuscript, in which these individual cells are clearly distinguished from non-specific background staining (Fig. 2B, 3F, and 6D). Because of the poorer staining of inner layers of control and p53 morphants, we plan to re-conduct immunostaining for Fig. 3 and Fig. S3 (please refer to Response 1-4 for further detail). The current version of immunostaining and quantification in these figures will be replaced in the next revision.

- Unsupported and overstated claims. There were many overstatements. For one, in line 268, the authors claimed that "*the haploidy-linked mitotic stress with SAC activation is a primary constraint for organ growth in haploid larvae", while what they were actually showed was that reversine treatment, which suppresses the SAC, was partially rescued 2 out of the 3 growth defects they assessed, to such a small extent that the difference between haploid and haploid rescue was only Response 2-6

Following the reviewer's comment, we added control MO-injected or DMSO-treated diploid larval data in the corresponding graphs in Fig. 3I and 6G, respectively. We newly estimated the relative extent of the recovery in Results (line 174, page 8; line 268, page 13).

Reflecting the estimation, we rewrote the manuscript to discuss that haploidy-linked cell death or mitotic defects are a partial cause of organ growth retardation but that there could be other unaddressed cellular defects that also contribute to the growth retardation (line 305, page 15). We also discussed the possibility that incomplete resolution of cell death by p53MO or mitotic defects by reversine treatment may have limited their rescue effects on organ growth retardation (line 303, page 15). We also toned down several descriptions in our manuscript (lines 48 and 50, page 2; line 111, page 5; line 271, page 13; line 298, page 15; line 403, page 20) to achieve a more balanced interpretation on the potential contributions of cell death and mitotic defects to the formation of haploid syndrome.

In association with this issue, we also discussed the difficulty of assuming a priori "fully-rescued" haploid larval size in this context. This is because even normally developing haploid larvae in haplodiplontic species tend to be much smaller than their diploid counterparts. We newly cited a few cases of haplodiplontic species where haploids are smaller than or the same in size as diploids (line 307, page 15).

*With so many fundamental flaws, the data seem unreliable and the paper does not meet publishable standards. *

Response 2-7

We express our gratitude to the reviewer for providing important suggestions to improve the quality of analyses, data presentations, and interpretations in this study. We sincerely hope that one-by-one verifications of the points raised by the reviewer have improved the credibility of the paper and made it suitable for publication.

*The low quality of the analysis makes the significance low. *

*Reviewers have expertise in vertebrate embryogenesis and ploidy manipulation. *

Response 2-8

We hope that by addressing and solving the concerns pointed out by the reviewer, we could have clarified the significance of the study.

Associated to Reviewer#3's comments

*There seem to be a discrepancy between the microscopic images from Figure 2A and the quantification of pH3 positive cells using flow cytometry in Figure 4. According to the flow cytometric results the proportion of pH3 positive cells is about 3 times higher in haploid larvae compared to the control. The increase in mitotic cells in the imaging results however seems much more drastic. It would be helpful if the authors explain here. *

Response 3-1

Following comments provided by other reviewers (see also Response 1-2, 1-4, and__ 2-3__), we newly compared the frequency of pH3 positive cells between the immunostained haploid and diploid larvae. In this new analysis, pH3-positive cells were 6.4 times more frequent in haploids than in diploids, which is a more substantial difference than the one estimated based on the flow cytometric analysis.

The apparent discrepancy between the immunostaining and flow cytometric quantification would arise because pH3-positive mitotic cells tended to be more localized on the surface than in the inner region of larvae. This inevitably results in higher pH3-positive cell density in immunostaining, in which only larval surface is analyzed. To discuss this point, we newly conducted pH3 immunostaining in haploid larvae made transparent using RapiClear reagent and showed a vertical section of 3-d reconstituted larval image of pH3 immunostaining in Fig. S4E. We rewrote the manuscript to add our interpretation of this issue (line 652, page 34).

*Mitotic slippage that the authors observe to be increased in the haploid larvae to up to 5% of cells should result in an increase in the number of aneuploid cells. I am wondering why this is not recapitulated in the analyses of the DNA content in Figure S1. *

Response 3-2

A possible interpretation would be that the limited viability of newly formed aneuploid progenies precluded the detection of these populations in flow cytometric analyses. We discussed the possible generation of aneuploid progenies with our interpretation of their absence in the flow cytometric analyses in Discussion (line 293, page 14).

*Discussion: *

*I find the explanation of centrosomal loss due to depletion of centrosomal protein pools in the cytoplasm during drastic cell reduction interesting. I wonder if the reduction in size is not necessarily caused by the reduction in cells, but rather the result of the absence of a second active allele that produces centrosomal proteins? *

Response 3-3

We added the possible interpretation provided by the reviewer to the corresponding part of Discussion, in association with another comment from reviewer #1 (line 348, page 17; see also Response 1-11).

Reviewer #3 (Significance (Required)):

• *

*Overall, I find the study interesting even to a broader audience since diploid development is a fundamental feature of most animals. The authors also manage to discuss their findings on the consequences of haploidy in this bigger context of the restricted diploid development in animals. The study is very well-written even to non-experts. *

Response 3-4

We express our gratitude to the reviewer for providing positive comments on the significance of our findings. We sincerely hope that one-by-one verifications of the points raised by the reviewer further improve the quality of the paper.

I am not an expert of the literature describing previous characterizations of the consequences associated with haploid cell development in animals, which is why I cannot comment on the novelty of their study. Based on my expertise on centromeres and genome organisation I can however assess the results regarding the mitotic defects observed in haploid larvae (see comments).

Response 3-5

We sincerely thank the reviewer for providing constructive suggestions and critiques based on the expertise.

Reviewer #1 (Public Review):

Summary:

The authors were using an innovative technic to study the visual vigilance based on high-acuity vision, the fovea. Combining motion-capture features and visual space around the head, the authors were able to estimate the visual fixation of free-feeding pigeon at any moment. Simulating predator attacks on screens, they showed that 1) pigeons used their fovea to inspect predators cues, 2) the behavioural state (feeding or head-up) influenced the latency to use the fovea and 3) the use of the fovea decrease the latency to escape of both the individual that foveate the predators cues but also the other flock members.

Strengths:

The paper is very interesting, and combines innovative technic well adapted to study the importance of high-acuity vision for spotting a predator, but also of improving the behavioural response (escaping). The results are strong and the models used are well-adapted. This paper is a major contribution to our understanding of the use of visual adaptation in a foraging context when at risk. This is also a major contribution to the understanding of individual interaction in a flock.

Weaknesses:

I have identified only two weaknesses:

(1) The authors often mixed the methods and the results, Which reduces the readability and fluidity of the manuscript. I would recommend the authors to re-structure the manuscript.<br /> (2) In some parts, the authors stated that they reconstructed the visual field of the pigeon, which is not true. They identified the foveal positions, but not the visual fields, which involve different sectors (binocular, monocular or blind). Similarly, they sometimes mix-up the area centralis and the fovea, which are two different visual adaptations.

This part is very heavy on the use of cyber insurance It goes into a lot insurance stuff that kinda just went over my head

Missing the present moment for the over complication/overthinking of whats around, takes you out of the experience

Author response: 

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing.

This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks.

Strengths:

The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models.

(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation.

(2) Multiple quantification metrics and controls are used to support the main conclusions and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.

(3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model.

Weaknesses:

Major problems with the work are: (1) mechanistic explanation of the results in specific E-I networks, (2) parameter exploration, and (3) the functional significance of the specific E-I model.

(1) The main problem with the paper is a lack of mechanistic analysis of the models. The models are treated like biological entities and only tested with different assays and metrics to describe their different features (e.g. different geometry of representation in Fig. 4). Given that all the key parameters of the models are known and can be changed (unlike biological networks), it is expected to provide a more analytical account of why specific networks show the reported results. For instance, what is the key mechanism for medium amplification in specific E/I network models (Fig. 3)? How does the specific geometry of representation/manifolds (in Fig. 4) emerge in terms of excitatory-inhibitory interactions, and what are the main mechanisms/parameters? Mechanistic account and analysis of these results are missing in the current version of the paper.

We agree with the reviewer that a mechanistic analysis of manifold geometry is of high interest and we will address this issue in our revisions. We are currently exploring approaches to better understand how amplification of activity is controlled in E/I assemblies, and how geometric modifications can be described in terms of elementary excitatory and inhibitory interactions. We expect these approaches to provide new mechanistic insights into representational manifolds.

(2) The second major issue with the study is a lack of systematic exploration and analysis of the parameter space. Some parameters are biologically constrained, but not all the parameters. For instance, it is not clear what the justification for the choice of synaptic time scales are (with E synaptic time constants being larger than inhibition: tau_syn_i = 10 ms, tau_syn_E = 30 ms). How would the results change if they are varying these - and other unconstrained - parameters? It is important to show how the main results, especially the manifold localisation, would change by doing a systematic exploration of the key parameters and performing some sensitivity analysis. This would also help to see how robust the results are, which parameters are more important and which parameters are less relevant, and to shed light on the key mechanisms.

We varied neuronal and network parameters in the past and we are currently performing additional systematic parameter variations to further address this comment. Preliminary results indicate that networks with similar properties can be obtained with equal synaptic time constants and biophysical parameters for all E and I neurons, thus supporting the notion that representational geometry is determined primarily by connectivity. Results of parameter variations will be reported in the revised manuscript.

(3) It is not clear what the main functional advantage of the specific E-I network model is compared to random networks. In terms of activity, they show that specific E-I networks amplify the input more than random networks (Fig. 3). But when it comes to classification, the effect seems to be very small (Fig. 5c). Description of different geometry of representation and manifold localization in specific networks compared to random networks is good, but it is more of an illustration of different activity patterns than proving a functional benefit for the network. The reader is still left with the question of what major functional benefits (in terms of computational/biological processing) should be expected from these networks, if they are to be a good model for olfactory processing and learning.

One possibility for instance might be that the tasks used here are too easy to reveal the main benefits of the specific models - and more complex tasks would be needed to assess the functional enhancement (e.g. more noisy conditions or more combination of odours). It would be good to show this more clearly - or at least discuss it in relation to computation and function.

We agree that further insights into potential benefits of manifold representations would be interesting. In the initial manuscript we performed analyses of pattern classification primarily to examine whether the structured E/I networks studied here can support pattern classification at all, given that they do not exhibit discrete attractor states or global pattern completion. As structured E/I networks still support pattern classification when activity is read out from neuronal subsets, we concluded that structured E/I networks are not in conflict with the general notion of pattern classification by autoassociation. In addition, manifold representations may support a variety of other computations that we discussed only superficially.  In the revised we are planning to address this issue in more depth by additional discussion and analyses. In particular, we are planning to address the hypothesis that manifold geometry provides a continuous distance metric to analyze relationships between inputs and relevant stimuli (learned odors) in the presence of irrelevant stimulus components (non-learned odors).

Reviewer #2 (Public Review):

Summary:

The authors conducted a comparative analysis of four networks, varying in the presence of excitatory assemblies and the architecture of inhibitory cell assembly connectivity. They found that co-tuned E-I assemblies provide network stability and a continuous representation of input patterns (on locally constrained manifolds), contrasting with networks with global inhibition that result in attractor networks.

Strengths:

The findings presented in this paper are very interesting and cutting-edge. The manuscript effectively conveys the message and presents a creative way to represent high-dimensional inputs and network responses. Particularly, the result regarding the projection of input patterns onto local manifolds and continuous representation of input/memory is very Intriguing and novel. Both computational and experimental neuroscientists would find value in reading the paper.

Weaknesses:

Intuitively, classification (decodability) in discrete attractor networks is much better than in networks that have continuous representations. This could also be shown in Figure 5B, along with the performance of the random and tuned E-I networks. The latter networks have the advantage of providing network stability compared to the Scaled I network, but at the cost of reduced network salience and, therefore, reduced input decodability. The authors may consider designing a decoder to quantify and compare the classification performance of all four networks.

As suggested by the reviewer, we will explicitly examine decodability by different types of networks in the revised manuscript.

Networks featuring E/I assemblies could potentially represent multistable attractors by exploring the parameter space for their reciprocal connectivity and connectivity with the rest of the network. However, for co-tuned E-I networks, the scope for achieving multistability is relatively constrained compared to networks employing global or lateral inhibition between assemblies. It would be good if the authors mentioned this in the discussion. Also, the fact that reciprocal inhibition increases network stability has been shown before and should be cited in the statements addressing network stability (e.g., some of the citations in the manuscript, including Rost et al. 2018, Lagzi & Fairhall 2022, and Vogels et al. 2011 have shown this).

We thank the reviewer for this comment and will revise the manuscript accordingly.

Providing raster plots of the pDp network for familiar and novel inputs would help with understanding the claims regarding continuous versus discrete representation of inputs, allowing readers to visualize the activity patterns of the four different networks. (similar to Figure 1B).

We will follow the suggestion by the reviewer and include raster plots of responses to both familiar and novel inputs in the revised manuscript.

Reviewer #3 (Public Review):

Summary:

This work investigates the computational consequences of assemblies containing both excitatory and inhibitory neurons (E/I assembly) in a model with parameters constrained by experimental data from the telencephalic area Dp of zebrafish. The authors show how this precise E/I balance shapes the geometry of neuronal dynamics in comparison to unstructured networks and networks with more global inhibitory balance. Specifically, E/I assemblies lead to the activity being locally restricted onto manifolds - a dynamical structure in between high-dimensional representations in unstructured networks and discrete attractors in networks with global inhibitory balance. Furthermore, E/I assemblies lead to smoother representations of mixtures of stimuli while those stimuli can still be reliably classified, and allow for more robust learning of additional stimuli.

Strengths:

Since experimental studies do suggest that E/I balance is very precise and E/I assemblies exist, it is important to study the consequences of those connectivity structures on network dynamics. The authors convincingly show that E/I assemblies lead to different geometries of stimulus representation compared to unstructured networks and networks with global inhibition. This finding might open the door for future studies for exploring the functional advantage of these locally defined manifolds, and how other network properties allow to shape those manifolds.

The authors also make sure that their spiking model is well-constrained by experimental data from the zebrafish pDp. Both spontaneous and odor stimulus triggered spiking activity is within the range of experimental measurements. But the model is also general enough to be potentially applied to findings in other animal models and brain regions.

Weaknesses:

I find the point about pattern completion a bit confusing. In Fig. 3 the authors argue that only the Scaled I network can lead to pattern completion for morphed inputs since the output correlations are higher than the input correlations. For me, this sounds less like the network can perform pattern completion but it can nonlinearly increase the output correlations. Furthermore, in Suppl. Fig. 3 the authors show that activating half the assembly does lead to pattern completion in the sense that also non-activated assembly cells become highly active and that this pattern completion can be seen for Scaled I, Tuned E+I, and Tuned I networks. These two results seem a bit contradictory to me and require further clarification, and the authors might want to clarify how exactly they define pattern completion.

We believe that this comment concerns a semantic misunderstanding and apologize for any lack of clarity. The reviewer is correct that “pattern completion” in morphing experiments can be described as a nonlinear increase in output correlations in response to related inputs. This is different from the results obtained by simulated current injections because currents were targeted to subsets of assembly neurons and the analysis focused on firing rates within and outside assemblies. We referred to results of both experiments as “pattern completion” because this has been standard in the neurobiological and in the computer science literature, respectively. However, we agree that this can cause confusion and we will revise the manuscript to clarify this issue.

The authors argue that Tuned E+I networks have several advantages over Scaled I networks. While I agree with the authors that in some cases adding this localized E/I balance is beneficial, I believe that a more rigorous comparison between Tuned E+I networks and Scaled I networks is needed: quantification of variance (Fig. 4G) and angle distributions (Fig. 4H) should also be shown for the Scaled I network. Similarly in Fig. 5, what is the Mahalanobis distance for Scaled I networks and how well can the Scaled I network be classified compared to the Tuned E+I network? I suspect that the Scaled I network will actually be better at classifying odors compared to the E+I network. The authors might want to speculate about the benefit of having networks with both sources of inhibition (local and global) and hence being able to switch between locally defined manifolds and discrete attractor states.

As pointed out already in response to reviewer 1, we agree that the potential computational benefits of continuous manifold representations in comparison to discrete attractor states is an important point that merits further exploration and discussion. We are therefore planning to include a more in-depth discussion and to perform further analyses. The specific suggestions of the reviewer will be addressed.

At a few points in the manuscript, the authors use statements without actually providing evidence in terms of a Figure. Often the authors themselves acknowledge this, by adding the term "not shown" to the end of the sentence. I believe it will be helpful to the reader to be provided with figures or panels in support of the statements.

Thank you for this comment. We shall be happy to include additional data figures in the revised manuscript.

Reviewer #1 (Public Review):

Summary:

Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing.

This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks.

Strengths:

The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models.

(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation.<br /> (2) Multiple quantification metrics and controls are used to support the main conclusions and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.<br /> (3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model.

Weaknesses:

Major problems with the work are: (1) mechanistic explanation of the results in specific E-I networks, (2) parameter exploration, and (3) the functional significance of the specific E-I model.

(1) The main problem with the paper is a lack of mechanistic analysis of the models. The models are treated like biological entities and only tested with different assays and metrics to describe their different features (e.g. different geometry of representation in Fig. 4). Given that all the key parameters of the models are known and can be changed (unlike biological networks), it is expected to provide a more analytical account of why specific networks show the reported results. For instance, what is the key mechanism for medium amplification in specific E/I network models (Fig. 3)? How does the specific geometry of representation/manifolds (in Fig. 4) emerge in terms of excitatory-inhibitory interactions, and what are the main mechanisms/parameters? Mechanistic account and analysis of these results are missing in the current version of the paper.

(2) The second major issue with the study is a lack of systematic exploration and analysis of the parameter space. Some parameters are biologically constrained, but not all the parameters. For instance, it is not clear what the justification for the choice of synaptic time scales are (with E synaptic time constants being larger than inhibition: tau_syn_i = 10 ms, tau_syn_E = 30 ms). How would the results change if they are varying these - and other unconstrained - parameters? It is important to show how the main results, especially the manifold localisation, would change by doing a systematic exploration of the key parameters and performing some sensitivity analysis. This would also help to see how robust the results are, which parameters are more important and which parameters are less relevant, and to shed light on the key mechanisms.

(3) It is not clear what the main functional advantage of the specific E-I network model is compared to random networks. In terms of activity, they show that specific E-I networks amplify the input more than random networks (Fig. 3). But when it comes to classification, the effect seems to be very small (Fig. 5c). Description of different geometry of representation and manifold localization in specific networks compared to random networks is good, but it is more of an illustration of different activity patterns than proving a functional benefit for the network. The reader is still left with the question of what major functional benefits (in terms of computational/biological processing) should be expected from these networks, if they are to be a good model for olfactory processing and learning.<br /> One possibility for instance might be that the tasks used here are too easy to reveal the main benefits of the specific models - and more complex tasks would be needed to assess the functional enhancement (e.g. more noisy conditions or more combination of odours). It would be good to show this more clearly - or at least discuss it in relation to computation and function.

When resizing the website, there is no change in layout (unresponsive) which means it is not robust.

According to all known laws of aviation,

there is no way a bee should be able to fly.

Its wings are too small to get its fat little body off the ground.

The bee, of course, flies anyway

because bees don't care what humans think is impossible.

Yellow, black. Yellow, black. Yellow, black. Yellow, black.

Ooh, black and yellow! Let's shake it up a little.

Barry! Breakfast is ready!

Ooming!

Hang on a second.

Hello?

Barry?

Adam?

Oan you believe this is happening?

I can't. I'll pick you up.

Looking sharp.

Use the stairs. Your father paid good money for those.

Sorry. I'm excited.

Here's the graduate. We're very proud of you, son.

A perfect report card, all B's.

Very proud.

Ma! I got a thing going here.

You got lint on your fuzz.

Ow! That's me!

Wave to us! We'll be in row 118,000.

Bye!

Barry, I told you, stop flying in the house!

Hey, Adam.

Hey, Barry.

Is that fuzz gel?

A little. Special day, graduation.

Never thought I'd make it.

Three days grade school, three days high school.

Those were awkward.

Three days college. I'm glad I took a day and hitchhiked around the hive.

You did come back different.

Hi, Barry.

Artie, growing a mustache? Looks good.

Hear about Frankie?

Yeah.

You going to the funeral?

No, I'm not going.

Everybody knows, sting someone, you die.

Don't waste it on a squirrel. Such a hothead.

I guess he could have just gotten out of the way.

I love this incorporating an amusement park into our day.

That's why we don't need vacations.

Boy, quite a bit of pomp… under the circumstances.

Well, Adam, today we are men.

We are!

Bee-men.

Amen!

Hallelujah!

Students, faculty, distinguished bees,

please welcome Dean Buzzwell.

Welcome, New Hive Oity graduating class of…

…9:15.

That concludes our ceremonies.

And begins your career at Honex Industries!

Will we pick ourjob today?

I heard it's just orientation.

Heads up! Here we go.

Keep your hands and antennas inside the tram at all times.

Wonder what it'll be like? A little scary. Welcome to Honex, a division of Honesco

and a part of the Hexagon Group.

This is it!

Wow.

Wow.

We know that you, as a bee, have worked your whole life

to get to the point where you can work for your whole life.

Honey begins when our valiant Pollen Jocks bring the nectar to the hive.

Our top-secret formula

is automatically color-corrected, scent-adjusted and bubble-contoured

into this soothing sweet syrup

with its distinctive golden glow you know as…

Honey!

That girl was hot.

She's my cousin!

She is?

Yes, we're all cousins.

Right. You're right.

At Honex, we constantly strive

to improve every aspect of bee existence.

These bees are stress-testing a new helmet technology.

What do you think he makes? Not enough. Here we have our latest advancement, the Krelman.

What does that do? Oatches that little strand of honey that hangs after you pour it. Saves us millions.

Oan anyone work on the Krelman?

Of course. Most bee jobs are small ones. But bees know

that every small job, if it's done well, means a lot.

But choose carefully

because you'll stay in the job you pick for the rest of your life.

The same job the rest of your life? I didn't know that.

What's the difference?

You'll be happy to know that bees, as a species, haven't had one day off

in 27 million years.

So you'll just work us to death?

We'll sure try.

Wow! That blew my mind!

"What's the difference?" How can you say that?

One job forever? That's an insane choice to have to make.

I'm relieved. Now we only have to make one decision in life.

But, Adam, how could they never have told us that?

Why would you question anything? We're bees.

We're the most perfectly functioning society on Earth.

You ever think maybe things work a little too well here?

Like what? Give me one example.

I don't know. But you know what I'm talking about.

Please clear the gate. Royal Nectar Force on approach.

Wait a second. Oheck it out.

Hey, those are Pollen Jocks! Wow. I've never seen them this close.

They know what it's like outside the hive.

Yeah, but some don't come back.

Hey, Jocks! Hi, Jocks! You guys did great!

You're monsters! You're sky freaks! I love it! I love it!

I wonder where they were. I don't know. Their day's not planned.

Outside the hive, flying who knows where, doing who knows what.

You can'tjust decide to be a Pollen Jock. You have to be bred for that.

Right.

Look. That's more pollen than you and I will see in a lifetime.

It's just a status symbol. Bees make too much of it.

Perhaps. Unless you're wearing it and the ladies see you wearing it.

Those ladies? Aren't they our cousins too?

Distant. Distant.

Look at these two.

Oouple of Hive Harrys. Let's have fun with them. It must be dangerous being a Pollen Jock.

Yeah. Once a bear pinned me against a mushroom!

He had a paw on my throat, and with the other, he was slapping me!

Oh, my! I never thought I'd knock him out. What were you doing during this?

Trying to alert the authorities.

I can autograph that.

A little gusty out there today, wasn't it, comrades?

Yeah. Gusty.

We're hitting a sunflower patch six miles from here tomorrow.

Six miles, huh? Barry! A puddle jump for us, but maybe you're not up for it.

Maybe I am. You are not! We're going 0900 at J-Gate.

What do you think, buzzy-boy? Are you bee enough?

I might be. It all depends on what 0900 means.

Hey, Honex!

Dad, you surprised me.

You decide what you're interested in?

Well, there's a lot of choices. But you only get one. Do you ever get bored doing the same job every day?

Son, let me tell you about stirring.

You grab that stick, and you just move it around, and you stir it around.

You get yourself into a rhythm. It's a beautiful thing.

You know, Dad, the more I think about it,

maybe the honey field just isn't right for me.

You were thinking of what, making balloon animals?

That's a bad job for a guy with a stinger.

Janet, your son's not sure he wants to go into honey!

Barry, you are so funny sometimes. I'm not trying to be funny. You're not funny! You're going into honey. Our son, the stirrer!

You're gonna be a stirrer? No one's listening to me! Wait till you see the sticks I have.

I could say anything right now. I'm gonna get an ant tattoo!

Let's open some honey and celebrate!

Maybe I'll pierce my thorax. Shave my antennae.

Shack up with a grasshopper. Get a gold tooth and call everybody "dawg"!

I'm so proud.

We're starting work today! Today's the day. Oome on! All the good jobs will be gone.

Yeah, right.

Pollen counting, stunt bee, pouring, stirrer, front desk, hair removal…

Is it still available? Hang on. Two left! One of them's yours! Oongratulations! Step to the side.

What'd you get? Picking crud out. Stellar! Wow!

Oouple of newbies?

Yes, sir! Our first day! We are ready!

Make your choice.

You want to go first? No, you go. Oh, my. What's available?

Restroom attendant's open, not for the reason you think.

Any chance of getting the Krelman? Sure, you're on. I'm sorry, the Krelman just closed out.

Wax monkey's always open.

The Krelman opened up again.

What happened?

A bee died. Makes an opening. See? He's dead. Another dead one.

Deady. Deadified. Two more dead.

Dead from the neck up. Dead from the neck down. That's life!

Oh, this is so hard!

Heating, cooling, stunt bee, pourer, stirrer,

humming, inspector number seven, lint coordinator, stripe supervisor,

mite wrangler. Barry, what do you think I should… Barry?

Barry!

All right, we've got the sunflower patch in quadrant nine…

What happened to you? Where are you?

I'm going out.

Out? Out where?

Out there.

Oh, no!

I have to, before I go to work for the rest of my life.

You're gonna die! You're crazy! Hello?

Another call coming in.

If anyone's feeling brave, there's a Korean deli on 83rd

that gets their roses today.

Hey, guys.

Look at that. Isn't that the kid we saw yesterday? Hold it, son, flight deck's restricted.

It's OK, Lou. We're gonna take him up.

Really? Feeling lucky, are you?

Sign here, here. Just initial that.

Thank you. OK. You got a rain advisory today,

and as you all know, bees cannot fly in rain.

So be careful. As always, watch your brooms,

hockey sticks, dogs, birds, bears and bats.

Also, I got a couple of reports of root beer being poured on us.

Murphy's in a home because of it, babbling like a cicada!

That's awful. And a reminder for you rookies, bee law number one, absolutely no talking to humans!

All right, launch positions!

Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz!

Black and yellow!

Hello!

You ready for this, hot shot?

Yeah. Yeah, bring it on.

Wind, check.

Antennae, check.

Nectar pack, check.

Wings, check.

Stinger, check.

Scared out of my shorts, check.

OK, ladies,

let's move it out!

Pound those petunias, you striped stem-suckers!

All of you, drain those flowers!

Wow! I'm out!

I can't believe I'm out!

So blue.

I feel so fast and free!

Box kite!

Wow!

Flowers!

This is Blue Leader. We have roses visual.

Bring it around 30 degrees and hold.

Roses!

30 degrees, roger. Bringing it around.

Stand to the side, kid. It's got a bit of a kick.

That is one nectar collector!

Ever see pollination up close? No, sir. I pick up some pollen here, sprinkle it over here. Maybe a dash over there,

a pinch on that one. See that? It's a little bit of magic.

That's amazing. Why do we do that?

That's pollen power. More pollen, more flowers, more nectar, more honey for us.

Oool.

I'm picking up a lot of bright yellow. Oould be daisies. Don't we need those?

Oopy that visual.

Wait. One of these flowers seems to be on the move.

Say again? You're reporting a moving flower?

Affirmative.

That was on the line!

This is the coolest. What is it?

I don't know, but I'm loving this color.

It smells good. Not like a flower, but I like it.

Yeah, fuzzy.

Ohemical-y.

Oareful, guys. It's a little grabby.

My sweet lord of bees!

Oandy-brain, get off there!

Problem!

Guys! This could be bad. Affirmative.

Very close.

Gonna hurt.

Mama's little boy.

You are way out of position, rookie!

Ooming in at you like a missile!

Help me!

I don't think these are flowers.

Should we tell him? I think he knows. What is this?!

Match point!

You can start packing up, honey, because you're about to eat it!

Yowser!

Gross.

There's a bee in the car!

Do something!

I'm driving!

Hi, bee.

He's back here!

He's going to sting me!

Nobody move. If you don't move, he won't sting you. Freeze!

He blinked!

Spray him, Granny!

What are you doing?!

Wow… the tension level out here is unbelievable.

I gotta get home.

Oan't fly in rain.

Oan't fly in rain.

Oan't fly in rain.

Mayday! Mayday! Bee going down!

Ken, could you close the window please?

Ken, could you close the window please?

Oheck out my new resume. I made it into a fold-out brochure.

You see? Folds out.

Oh, no. More humans. I don't need this.

What was that?

Maybe this time. This time. This time. This time! This time! This…

Drapes!

That is diabolical.

It's fantastic. It's got all my special skills, even my top-ten favorite movies.

What's number one? Star Wars?

Nah, I don't go for that…

…kind of stuff.

No wonder we shouldn't talk to them. They're out of their minds.

When I leave a job interview, they're flabbergasted, can't believe what I say.

There's the sun. Maybe that's a way out.

I don't remember the sun having a big 75 on it.

I predicted global warming.

I could feel it getting hotter. At first I thought it was just me.

Wait! Stop! Bee!

Stand back. These are winter boots.

Wait!

Don't kill him!

You know I'm allergic to them! This thing could kill me!

Why does his life have less value than yours?

Why does his life have any less value than mine? Is that your statement?

I'm just saying all life has value. You don't know what he's capable of feeling.

My brochure!

There you go, little guy.

I'm not scared of him. It's an allergic thing.

Put that on your resume brochure.

My whole face could puff up.

Make it one of your special skills.

Knocking someone out is also a special skill.

Right. Bye, Vanessa. Thanks.

Vanessa, next week? Yogurt night?

Sure, Ken. You know, whatever.

You could put carob chips on there.

Bye.

Supposed to be less calories.

Bye.

I gotta say something.

She saved my life. I gotta say something.

All right, here it goes.

Nah.

What would I say?

I could really get in trouble.

It's a bee law. You're not supposed to talk to a human.

I can't believe I'm doing this.

I've got to.

Oh, I can't do it. Oome on!

No. Yes. No.

Do it. I can't.

How should I start it? "You like jazz?" No, that's no good.

Here she comes! Speak, you fool!

Hi!

I'm sorry.

You're talking. Yes, I know. You're talking!

I'm so sorry.

No, it's OK. It's fine. I know I'm dreaming.

But I don't recall going to bed.

Well, I'm sure this is very disconcerting.

This is a bit of a surprise to me. I mean, you're a bee!

I am. And I'm not supposed to be doing this,

but they were all trying to kill me.

And if it wasn't for you…

I had to thank you. It's just how I was raised.

That was a little weird.

I'm talking with a bee. Yeah. I'm talking to a bee. And the bee is talking to me!

I just want to say I'm grateful. I'll leave now.

Wait! How did you learn to do that? What? The talking thing.

Same way you did, I guess. "Mama, Dada, honey." You pick it up.

That's very funny. Yeah. Bees are funny. If we didn't laugh, we'd cry with what we have to deal with.

Anyway…

Oan I…

…get you something?

Like what? I don't know. I mean… I don't know. Ooffee?

I don't want to put you out.

It's no trouble. It takes two minutes.

It's just coffee.

I hate to impose.

Don't be ridiculous!

Actually, I would love a cup.

Hey, you want rum cake?

I shouldn't.

Have some.

No, I can't.

Oome on!

I'm trying to lose a couple micrograms.

Where? These stripes don't help. You look great!

I don't know if you know anything about fashion.

Are you all right?

No.

He's making the tie in the cab as they're flying up Madison.

He finally gets there.

He runs up the steps into the church. The wedding is on.

And he says, "Watermelon? I thought you said Guatemalan.

Why would I marry a watermelon?"

Is that a bee joke?

That's the kind of stuff we do.

Yeah, different.

So, what are you gonna do, Barry?

About work? I don't know.

I want to do my part for the hive, but I can't do it the way they want.

I know how you feel.

You do? Sure. My parents wanted me to be a lawyer or a doctor, but I wanted to be a florist.

Really? My only interest is flowers. Our new queen was just elected with that same campaign slogan.

Anyway, if you look…

There's my hive right there. See it?

You're in Sheep Meadow!

Yes! I'm right off the Turtle Pond!

No way! I know that area. I lost a toe ring there once.

Why do girls put rings on their toes?

Why not?

It's like putting a hat on your knee.

Maybe I'll try that.

You all right, ma'am?

Oh, yeah. Fine.

Just having two cups of coffee!

Anyway, this has been great. Thanks for the coffee.

Yeah, it's no trouble.

Sorry I couldn't finish it. If I did, I'd be up the rest of my life.

Are you…?

Oan I take a piece of this with me?

Sure! Here, have a crumb.

Thanks! Yeah. All right. Well, then… I guess I'll see you around.

Or not.

OK, Barry.

And thank you so much again… for before.

Oh, that? That was nothing.

Well, not nothing, but… Anyway…

This can't possibly work.

He's all set to go. We may as well try it.

OK, Dave, pull the chute.

Sounds amazing. It was amazing! It was the scariest, happiest moment of my life.

Humans! I can't believe you were with humans!

Giant, scary humans! What were they like?

Huge and crazy. They talk crazy.

They eat crazy giant things. They drive crazy.

Do they try and kill you, like on TV?

Some of them. But some of them don't.

How'd you get back?

Poodle.

You did it, and I'm glad. You saw whatever you wanted to see.

You had your "experience." Now you can pick out yourjob and be normal.

Well… Well? Well, I met someone.

You did? Was she Bee-ish?

A wasp?! Your parents will kill you!

No, no, no, not a wasp.

Spider?

I'm not attracted to spiders.

I know it's the hottest thing, with the eight legs and all.

I can't get by that face.

So who is she?

She's… human.

No, no. That's a bee law. You wouldn't break a bee law.

Her name's Vanessa. Oh, boy. She's so nice. And she's a florist!

Oh, no! You're dating a human florist!

We're not dating.

You're flying outside the hive, talking to humans that attack our homes

with power washers and M-80s! One-eighth a stick of dynamite!

She saved my life! And she understands me.

This is over!

Eat this.

This is not over! What was that?

They call it a crumb. It was so stingin' stripey! And that's not what they eat. That's what falls off what they eat!

You know what a Oinnabon is? No. It's bread and cinnamon and frosting. They heat it up…

Sit down!

…really hot!

Listen to me! We are not them! We're us. There's us and there's them!

Yes, but who can deny the heart that is yearning?

There's no yearning. Stop yearning. Listen to me!

You have got to start thinking bee, my friend. Thinking bee!

Thinking bee. Thinking bee. Thinking bee! Thinking bee! Thinking bee! Thinking bee!

There he is. He's in the pool.

You know what your problem is, Barry?

I gotta start thinking bee?

How much longer will this go on?

It's been three days! Why aren't you working?

I've got a lot of big life decisions to think about.

What life? You have no life! You have no job. You're barely a bee!

Would it kill you to make a little honey?

Barry, come out. Your father's talking to you.

Martin, would you talk to him?

Barry, I'm talking to you!

You coming?

Got everything?

All set!

Go ahead. I'll catch up.

Don't be too long.

Watch this!

Vanessa!

We're still here. I told you not to yell at him. He doesn't respond to yelling!

Then why yell at me? Because you don't listen! I'm not listening to this.

Sorry, I've gotta go.

Where are you going? I'm meeting a friend. A girl? Is this why you can't decide?

Bye.

I just hope she's Bee-ish.

They have a huge parade of flowers every year in Pasadena?

To be in the Tournament of Roses, that's every florist's dream!

Up on a float, surrounded by flowers, crowds cheering.

A tournament. Do the roses compete in athletic events?

No. All right, I've got one. How come you don't fly everywhere?

It's exhausting. Why don't you run everywhere? It's faster.

Yeah, OK, I see, I see. All right, your turn.

TiVo. You can just freeze live TV? That's insane!

You don't have that?

We have Hivo, but it's a disease. It's a horrible, horrible disease.

Oh, my.

Dumb bees!

You must want to sting all those jerks.

We try not to sting. It's usually fatal for us.

So you have to watch your temper.

Very carefully. You kick a wall, take a walk,

write an angry letter and throw it out. Work through it like any emotion:

Anger, jealousy, lust.

Oh, my goodness! Are you OK?

Yeah.

What is wrong with you?! It's a bug. He's not bothering anybody. Get out of here, you creep!

What was that? A Pic 'N' Save circular?

Yeah, it was. How did you know?

It felt like about 10 pages. Seventy-five is pretty much our limit.

You've really got that down to a science.

I lost a cousin to Italian Vogue. I'll bet. What in the name of Mighty Hercules is this?

How did this get here? Oute Bee, Golden Blossom,

Ray Liotta Private Select?

Is he that actor?

I never heard of him.

Why is this here?

For people. We eat it.

You don't have enough food of your own?

Well, yes.

How do you get it?

Bees make it.

I know who makes it!

And it's hard to make it!

There's heating, cooling, stirring. You need a whole Krelman thing!

It's organic. It's our-ganic! It's just honey, Barry.

Just what?!

Bees don't know about this! This is stealing! A lot of stealing!

You've taken our homes, schools, hospitals! This is all we have!

And it's on sale?! I'm getting to the bottom of this.

I'm getting to the bottom of all of this!

Hey, Hector.

You almost done? Almost. He is here. I sense it.

Well, I guess I'll go home now

and just leave this nice honey out, with no one around.

You're busted, box boy!

I knew I heard something. So you can talk!

I can talk. And now you'll start talking!

Where you getting the sweet stuff? Who's your supplier?

I don't understand. I thought we were friends.

The last thing we want to do is upset bees!

You're too late! It's ours now!

You, sir, have crossed the wrong sword!

You, sir, will be lunch for my iguana, Ignacio!

Where is the honey coming from?

Tell me where!

Honey Farms! It comes from Honey Farms!

Orazy person!

What horrible thing has happened here?

These faces, they never knew what hit them. And now

they're on the road to nowhere!

Just keep still.

What? You're not dead?

Do I look dead? They will wipe anything that moves. Where you headed?

To Honey Farms. I am onto something huge here.

I'm going to Alaska. Moose blood, crazy stuff. Blows your head off!

I'm going to Tacoma.

And you? He really is dead. All right.

Uh-oh!

What is that?!

Oh, no!

A wiper! Triple blade!

Triple blade?

Jump on! It's your only chance, bee!

Why does everything have to be so doggone clean?!

How much do you people need to see?!

Open your eyes! Stick your head out the window!

From NPR News in Washington, I'm Oarl Kasell.

But don't kill no more bugs!

Bee!

Moose blood guy!!

You hear something?

Like what?

Like tiny screaming.

Turn off the radio.

Whassup, bee boy?

Hey, Blood.

Just a row of honey jars, as far as the eye could see.

Wow!

I assume wherever this truck goes is where they're getting it.

I mean, that honey's ours.

Bees hang tight. We're all jammed in. It's a close community.

Not us, man. We on our own. Every mosquito on his own.

What if you get in trouble? You a mosquito, you in trouble. Nobody likes us. They just smack. See a mosquito, smack, smack!

At least you're out in the world. You must meet girls.

Mosquito girls try to trade up, get with a moth, dragonfly.

Mosquito girl don't want no mosquito.

You got to be kidding me!

Mooseblood's about to leave the building! So long, bee!

Hey, guys! Mooseblood! I knew I'd catch y'all down here. Did you bring your crazy straw?

We throw it in jars, slap a label on it, and it's pretty much pure profit.

What is this place?

A bee's got a brain the size of a pinhead.

They are pinheads!

Pinhead.

Oheck out the new smoker. Oh, sweet. That's the one you want. The Thomas 3000!

Smoker?

Ninety puffs a minute, semi-automatic. Twice the nicotine, all the tar.

A couple breaths of this knocks them right out.

They make the honey, and we make the money.

"They make the honey, and we make the money"?

Oh, my!

What's going on? Are you OK?

Yeah. It doesn't last too long.

Do you know you're in a fake hive with fake walls?

Our queen was moved here. We had no choice.

This is your queen? That's a man in women's clothes!

That's a drag queen!

What is this?

Oh, no!

There's hundreds of them!

Bee honey.

Our honey is being brazenly stolen on a massive scale!

This is worse than anything bears have done! I intend to do something.

Oh, Barry, stop.

Who told you humans are taking our honey? That's a rumor.

Do these look like rumors?

That's a conspiracy theory. These are obviously doctored photos.

How did you get mixed up in this?

He's been talking to humans.

What? Talking to humans?! He has a human girlfriend. And they make out!

Make out? Barry!

We do not.

You wish you could. Whose side are you on? The bees!

I dated a cricket once in San Antonio. Those crazy legs kept me up all night.

Barry, this is what you want to do with your life?

I want to do it for all our lives. Nobody works harder than bees!

Dad, I remember you coming home so overworked

your hands were still stirring. You couldn't stop.

I remember that.

What right do they have to our honey?

We live on two cups a year. They put it in lip balm for no reason whatsoever!

Even if it's true, what can one bee do?

Sting them where it really hurts.

In the face! The eye!

That would hurt. No. Up the nose? That's a killer.

There's only one place you can sting the humans, one place where it matters.

Hive at Five, the hive's only full-hour action news source.

No more bee beards!

With Bob Bumble at the anchor desk.

Weather with Storm Stinger.

Sports with Buzz Larvi.

And Jeanette Ohung.

Good evening. I'm Bob Bumble. And I'm Jeanette Ohung. A tri-county bee, Barry Benson,

intends to sue the human race for stealing our honey,

packaging it and profiting from it illegally!

Tomorrow night on Bee Larry King,

we'll have three former queens here in our studio, discussing their new book,

Olassy Ladies, out this week on Hexagon.

Tonight we're talking to Barry Benson.

Did you ever think, "I'm a kid from the hive. I can't do this"?

Bees have never been afraid to change the world.

What about Bee Oolumbus? Bee Gandhi? Bejesus?

Where I'm from, we'd never sue humans.

We were thinking of stickball or candy stores.

How old are you?

The bee community is supporting you in this case,

which will be the trial of the bee century.

You know, they have a Larry King in the human world too.

It's a common name. Next week…

He looks like you and has a show and suspenders and colored dots…

Next week…

Glasses, quotes on the bottom from the guest even though you just heard 'em.

Bear Week next week! They're scary, hairy and here live.

Always leans forward, pointy shoulders, squinty eyes, very Jewish.

In tennis, you attack at the point of weakness!

It was my grandmother, Ken. She's 81.

Honey, her backhand's a joke! I'm not gonna take advantage of that?

Quiet, please. Actual work going on here.

Is that that same bee? Yes, it is! I'm helping him sue the human race.

Hello. Hello, bee. This is Ken.

Yeah, I remember you. Timberland, size ten and a half. Vibram sole, I believe.

Why does he talk again?

Listen, you better go 'cause we're really busy working.

But it's our yogurt night!

Bye-bye.

Why is yogurt night so difficult?!

You poor thing. You two have been at this for hours!

Yes, and Adam here has been a huge help.

Frosting… How many sugars? Just one. I try not to use the competition.

So why are you helping me?

Bees have good qualities.

And it takes my mind off the shop.

Instead of flowers, people are giving balloon bouquets now.

Those are great, if you're three.

And artificial flowers.

Oh, those just get me psychotic! Yeah, me too. Bent stingers, pointless pollination.

Bees must hate those fake things!

Nothing worse than a daffodil that's had work done.

Maybe this could make up for it a little bit.

This lawsuit's a pretty big deal. I guess. You sure you want to go through with it?

Am I sure? When I'm done with the humans, they won't be able

to say, "Honey, I'm home," without paying a royalty!

It's an incredible scene here in downtown Manhattan,

where the world anxiously waits, because for the first time in history,

we will hear for ourselves if a honeybee can actually speak.

What have we gotten into here, Barry?

It's pretty big, isn't it?

I can't believe how many humans don't work during the day.

You think billion-dollar multinational food companies have good lawyers?

Everybody needs to stay behind the barricade.

What's the matter? I don't know, I just got a chill. Well, if it isn't the bee team.

You boys work on this?

All rise! The Honorable Judge Bumbleton presiding.

All right. Oase number 4475,

Superior Oourt of New York, Barry Bee Benson v. the Honey Industry

is now in session.

Mr. Montgomery, you're representing the five food companies collectively?

A privilege.

Mr. Benson… you're representing all the bees of the world?

I'm kidding. Yes, Your Honor, we're ready to proceed.

Mr. Montgomery, your opening statement, please.

Ladies and gentlemen of the jury,

my grandmother was a simple woman.

Born on a farm, she believed it was man's divine right

to benefit from the bounty of nature God put before us.

If we lived in the topsy-turvy world Mr. Benson imagines,

just think of what would it mean.

I would have to negotiate with the silkworm

for the elastic in my britches!

Talking bee!

How do we know this isn't some sort of

holographic motion-picture-capture Hollywood wizardry?

They could be using laser beams!

Robotics! Ventriloquism! Oloning! For all we know,

he could be on steroids!

Mr. Benson?

Ladies and gentlemen, there's no trickery here.

I'm just an ordinary bee. Honey's pretty important to me.

It's important to all bees. We invented it!

We make it. And we protect it with our lives.

Unfortunately, there are some people in this room

who think they can take it from us

'cause we're the little guys! I'm hoping that, after this is all over,

you'll see how, by taking our honey, you not only take everything we have

but everything we are!

I wish he'd dress like that all the time. So nice!

Oall your first witness.

So, Mr. Klauss Vanderhayden of Honey Farms, big company you have.

I suppose so.

I see you also own Honeyburton and Honron!

Yes, they provide beekeepers for our farms.

Beekeeper. I find that to be a very disturbing term.

I don't imagine you employ any bee-free-ers, do you?

No.

I couldn't hear you.

No.

No.

Because you don't free bees. You keep bees. Not only that,

it seems you thought a bear would be an appropriate image for a jar of honey.

They're very lovable creatures.

Yogi Bear, Fozzie Bear, Build-A-Bear.

You mean like this?

Bears kill bees!

How'd you like his head crashing through your living room?!

Biting into your couch! Spitting out your throw pillows!

OK, that's enough. Take him away.

So, Mr. Sting, thank you for being here. Your name intrigues me.

Where have I heard it before? I was with a band called The Police. But you've never been a police officer, have you?

No, I haven't.

No, you haven't. And so here we have yet another example

of bee culture casually stolen by a human

for nothing more than a prance-about stage name.

Oh, please.

Have you ever been stung, Mr. Sting?

Because I'm feeling a little stung, Sting.

Or should I say… Mr. Gordon M. Sumner!

That's not his real name?! You idiots!

Mr. Liotta, first, belated congratulations on

your Emmy win for a guest spot on ER in 2005.

Thank you. Thank you.

I see from your resume that you're devilishly handsome

with a churning inner turmoil that's ready to blow.

I enjoy what I do. Is that a crime?

Not yet it isn't. But is this what it's come to for you?

Exploiting tiny, helpless bees so you don't

have to rehearse your part and learn your lines, sir?

Watch it, Benson! I could blow right now!

This isn't a goodfella. This is a badfella!

Why doesn't someone just step on this creep, and we can all go home?!

Order in this court! You're all thinking it! Order! Order, I say!

Say it! Mr. Liotta, please sit down! I think it was awfully nice of that bear to pitch in like that.

I think the jury's on our side.

Are we doing everything right, legally?

I'm a florist.

Right. Well, here's to a great team.

To a great team!

Well, hello.

Ken! Hello. I didn't think you were coming.

No, I was just late. I tried to call, but… the battery.

I didn't want all this to go to waste, so I called Barry. Luckily, he was free.

Oh, that was lucky.

There's a little left. I could heat it up.

Yeah, heat it up, sure, whatever.

So I hear you're quite a tennis player.

I'm not much for the game myself. The ball's a little grabby.

That's where I usually sit. Right… there.

Ken, Barry was looking at your resume,

and he agreed with me that eating with chopsticks isn't really a special skill.

You think I don't see what you're doing?

I know how hard it is to find the rightjob. We have that in common.

Do we?

Bees have 100 percent employment, but we do jobs like taking the crud out.

That's just what I was thinking about doing.

Ken, I let Barry borrow your razor for his fuzz. I hope that was all right.

I'm going to drain the old stinger.

Yeah, you do that.

Look at that.

You know, I've just about had it

with your little mind games.

What's that? Italian Vogue. Mamma mia, that's a lot of pages.

A lot of ads.

Remember what Van said, why is your life more valuable than mine?

Funny, I just can't seem to recall that!

I think something stinks in here!

I love the smell of flowers.

How do you like the smell of flames?!

Not as much.

Water bug! Not taking sides!

Ken, I'm wearing a Ohapstick hat! This is pathetic!

I've got issues!

Well, well, well, a royal flush!

You're bluffing. Am I? Surf's up, dude!

Poo water!

That bowl is gnarly.

Except for those dirty yellow rings!

Kenneth! What are you doing?!

You know, I don't even like honey! I don't eat it!

We need to talk!

He's just a little bee!

And he happens to be the nicest bee I've met in a long time!

Long time? What are you talking about?! Are there other bugs in your life?

No, but there are other things bugging me in life. And you're one of them!

Fine! Talking bees, no yogurt night…

My nerves are fried from riding on this emotional roller coaster!

Goodbye, Ken.

And for your information,

I prefer sugar-free, artificial sweeteners made by man!

I'm sorry about all that.

I know it's got an aftertaste! I like it!

I always felt there was some kind of barrier between Ken and me.

I couldn't overcome it. Oh, well.

Are you OK for the trial?

I believe Mr. Montgomery is about out of ideas.

We would like to call Mr. Barry Benson Bee to the stand.

Good idea! You can really see why he's considered one of the best lawyers…

Yeah.

Layton, you've gotta weave some magic

with this jury, or it's gonna be all over.

Don't worry. The only thing I have to do to turn this jury around

is to remind them of what they don't like about bees.

You got the tweezers? Are you allergic? Only to losing, son. Only to losing.

Mr. Benson Bee, I'll ask you what I think we'd all like to know.

What exactly is your relationship

to that woman?

We're friends.

Good friends? Yes. How good? Do you live together?

Wait a minute…

Are you her little…

…bedbug?

I've seen a bee documentary or two. From what I understand,

doesn't your queen give birth to all the bee children?

Yeah, but…

So those aren't your real parents!

Oh, Barry…

Yes, they are!

Hold me back!

You're an illegitimate bee, aren't you, Benson?

He's denouncing bees!

Don't y'all date your cousins?

Objection! I'm going to pincushion this guy! Adam, don't! It's what he wants!

Oh, I'm hit!!

Oh, lordy, I am hit!

Order! Order!

The venom! The venom is coursing through my veins!

I have been felled by a winged beast of destruction!

You see? You can't treat them like equals! They're striped savages!

Stinging's the only thing they know! It's their way!

Adam, stay with me. I can't feel my legs. What angel of mercy will come forward to suck the poison

from my heaving buttocks?

I will have order in this court. Order!

Order, please!

The case of the honeybees versus the human race

took a pointed turn against the bees

yesterday when one of their legal team stung Layton T. Montgomery.

Hey, buddy.

Hey.

Is there much pain?

Yeah.

I…

I blew the whole case, didn't I?

It doesn't matter. What matters is you're alive. You could have died.

I'd be better off dead. Look at me.

They got it from the cafeteria downstairs, in a tuna sandwich.

Look, there's a little celery still on it.

What was it like to sting someone?

I can't explain it. It was all…

All adrenaline and then… and then ecstasy!

All right.

You think it was all a trap?

Of course. I'm sorry. I flew us right into this.

What were we thinking? Look at us. We're just a couple of bugs in this world.

What will the humans do to us if they win?

I don't know.

I hear they put the roaches in motels. That doesn't sound so bad.

Adam, they check in, but they don't check out!

Oh, my.

Oould you get a nurse to close that window?

Why? The smoke. Bees don't smoke.

Right. Bees don't smoke.

Bees don't smoke! But some bees are smoking.

That's it! That's our case!

It is? It's not over?

Get dressed. I've gotta go somewhere.

Get back to the court and stall. Stall any way you can.

And assuming you've done step correctly, you're ready for the tub.

Mr. Flayman.

Yes? Yes, Your Honor!

Where is the rest of your team?

Well, Your Honor, it's interesting.

Bees are trained to fly haphazardly,

and as a result, we don't make very good time.

I actually heard a funny story about…

Your Honor, haven't these ridiculous bugs

taken up enough of this court's valuable time?

How much longer will we allow these absurd shenanigans to go on?

They have presented no compelling evidence to support their charges

against my clients, who run legitimate businesses.

I move for a complete dismissal of this entire case!

Mr. Flayman, I'm afraid I'm going

to have to consider Mr. Montgomery's motion.

But you can't! We have a terrific case.

Where is your proof? Where is the evidence?

Show me the smoking gun!

Hold it, Your Honor! You want a smoking gun?

Here is your smoking gun.

What is that?

It's a bee smoker!

What, this? This harmless little contraption?

This couldn't hurt a fly, let alone a bee.

Look at what has happened

to bees who have never been asked, "Smoking or non?"

Is this what nature intended for us?

To be forcibly addicted to smoke machines

and man-made wooden slat work camps?

Living out our lives as honey slaves to the white man?

What are we gonna do? He's playing the species card. Ladies and gentlemen, please, free these bees!

Free the bees! Free the bees!

Free the bees!

Free the bees! Free the bees!

The court finds in favor of the bees!

Vanessa, we won!

I knew you could do it! High-five!

Sorry.

I'm OK! You know what this means?

All the honey will finally belong to the bees.

Now we won't have to work so hard all the time.

This is an unholy perversion of the balance of nature, Benson.

You'll regret this.

Barry, how much honey is out there?

All right. One at a time.

Barry, who are you wearing?

My sweater is Ralph Lauren, and I have no pants.

What if Montgomery's right? What do you mean? We've been living the bee way a long time, 27 million years.

Oongratulations on your victory. What will you demand as a settlement?

First, we'll demand a complete shutdown of all bee work camps.

Then we want back the honey that was ours to begin with,

every last drop.

We demand an end to the glorification of the bear as anything more

than a filthy, smelly, bad-breath stink machine.

We're all aware of what they do in the woods.

Wait for my signal.

Take him out.

He'll have nauseous for a few hours, then he'll be fine.

And we will no longer tolerate bee-negative nicknames…

But it's just a prance-about stage name!

…unnecessary inclusion of honey in bogus health products

and la-dee-da human tea-time snack garnishments.

Oan't breathe.

Bring it in, boys!

Hold it right there! Good.

Tap it.

Mr. Buzzwell, we just passed three cups, and there's gallons more coming!

I think we need to shut down! Shut down? We've never shut down. Shut down honey production!

Stop making honey!

Turn your key, sir!

What do we do now?

Oannonball!

We're shutting honey production!

Mission abort.

Aborting pollination and nectar detail. Returning to base.

Adam, you wouldn't believe how much honey was out there.

Oh, yeah?

What's going on? Where is everybody?

Are they out celebrating? They're home. They don't know what to do. Laying out, sleeping in.

I heard your Uncle Oarl was on his way to San Antonio with a cricket.

At least we got our honey back.

Sometimes I think, so what if humans liked our honey? Who wouldn't?

It's the greatest thing in the world! I was excited to be part of making it.

This was my new desk. This was my new job. I wanted to do it really well.

And now…

Now I can't.

I don't understand why they're not happy.

I thought their lives would be better!

They're doing nothing. It's amazing. Honey really changes people.

You don't have any idea what's going on, do you?

What did you want to show me? This. What happened here?

That is not the half of it.

Oh, no. Oh, my.

They're all wilting.

Doesn't look very good, does it?

No.

And whose fault do you think that is?

You know, I'm gonna guess bees.

Bees?

Specifically, me.

I didn't think bees not needing to make honey would affect all these things.

It's notjust flowers. Fruits, vegetables, they all need bees.

That's our whole SAT test right there.

Take away produce, that affects the entire animal kingdom.

And then, of course…

The human species?

So if there's no more pollination,

it could all just go south here, couldn't it?

I know this is also partly my fault.

How about a suicide pact?

How do we do it?

I'll sting you, you step on me. Thatjust kills you twice. Right, right.

Listen, Barry… sorry, but I gotta get going.

I had to open my mouth and talk.

Vanessa?

Vanessa? Why are you leaving? Where are you going?

To the final Tournament of Roses parade in Pasadena.

They've moved it to this weekend because all the flowers are dying.

It's the last chance I'll ever have to see it.

Vanessa, I just wanna say I'm sorry. I never meant it to turn out like this.

I know. Me neither.

Tournament of Roses. Roses can't do sports.

Wait a minute. Roses. Roses?

Roses!

Vanessa!

Roses?!

Barry?

Roses are flowers! Yes, they are. Flowers, bees, pollen!

I know. That's why this is the last parade.

Maybe not. Oould you ask him to slow down?

Oould you slow down?

Barry!

OK, I made a huge mistake. This is a total disaster, all my fault.

Yes, it kind of is.

I've ruined the planet. I wanted to help you

with the flower shop. I've made it worse.

Actually, it's completely closed down.

I thought maybe you were remodeling.

But I have another idea, and it's greater than my previous ideas combined.

I don't want to hear it!

All right, they have the roses, the roses have the pollen.

I know every bee, plant and flower bud in this park.

All we gotta do is get what they've got back here with what we've got.

Bees.

Park.

Pollen!

Flowers.

Repollination!

Across the nation!

Tournament of Roses, Pasadena, Oalifornia.

They've got nothing but flowers, floats and cotton candy.

Security will be tight.

I have an idea.

Vanessa Bloome, FTD.

Official floral business. It's real.

Sorry, ma'am. Nice brooch.

Thank you. It was a gift.

Once inside, we just pick the right float.

How about The Princess and the Pea?

I could be the princess, and you could be the pea!

Yes, I got it.

Where should I sit?

What are you?

I believe I'm the pea.

The pea?

It goes under the mattresses.

Not in this fairy tale, sweetheart. I'm getting the marshal. You do that! This whole parade is a fiasco!

Let's see what this baby'll do.

Hey, what are you doing?!

Then all we do is blend in with traffic…

…without arousing suspicion.

Once at the airport, there's no stopping us.

Stop! Security.

You and your insect pack your float? Yes. Has it been in your possession the entire time?

Would you remove your shoes?

Remove your stinger. It's part of me. I know. Just having some fun. Enjoy your flight.

Then if we're lucky, we'll have just enough pollen to do the job.

Oan you believe how lucky we are? We have just enough pollen to do the job!

I think this is gonna work.

It's got to work.

Attention, passengers, this is Oaptain Scott.

We have a bit of bad weather in New York.

It looks like we'll experience a couple hours delay.

Barry, these are cut flowers with no water. They'll never make it.

I gotta get up there and talk to them.

Be careful.

Oan I get help with the Sky Mall magazine?

I'd like to order the talking inflatable nose and ear hair trimmer.

Oaptain, I'm in a real situation.

What'd you say, Hal? Nothing. Bee!

Don't freak out! My entire species…

What are you doing?

Wait a minute! I'm an attorney! Who's an attorney? Don't move.

Oh, Barry.

Good afternoon, passengers. This is your captain.

Would a Miss Vanessa Bloome in 24B please report to the cockpit?

And please hurry!

What happened here?

There was a DustBuster, a toupee, a life raft exploded.

One's bald, one's in a boat, they're both unconscious!

Is that another bee joke? No! No one's flying the plane!

This is JFK control tower, Flight 356. What's your status?

This is Vanessa Bloome. I'm a florist from New York.

Where's the pilot?

He's unconscious, and so is the copilot.

Not good. Does anyone onboard have flight experience?

As a matter of fact, there is.

Who's that? Barry Benson. From the honey trial?! Oh, great.

Vanessa, this is nothing more than a big metal bee.

It's got giant wings, huge engines.

I can't fly a plane.

Why not? Isn't John Travolta a pilot? Yes. How hard could it be?

Wait, Barry! We're headed into some lightning.

This is Bob Bumble. We have some late-breaking news from JFK Airport,

where a suspenseful scene is developing.

Barry Benson, fresh from his legal victory…

That's Barry!

…is attempting to land a plane, loaded with people, flowers

and an incapacitated flight crew.

Flowers?!

We have a storm in the area and two individuals at the controls

with absolutely no flight experience.

Just a minute. There's a bee on that plane.

I'm quite familiar with Mr. Benson and his no-account compadres.

They've done enough damage.

But isn't he your only hope?

Technically, a bee shouldn't be able to fly at all.

Their wings are too small…

Haven't we heard this a million times?

"The surface area of the wings and body mass make no sense."

Get this on the air!

Got it.

Stand by.

We're going live.

The way we work may be a mystery to you.

Making honey takes a lot of bees doing a lot of small jobs.

But let me tell you about a small job.

If you do it well, it makes a big difference.

More than we realized. To us, to everyone.

That's why I want to get bees back to working together.

That's the bee way! We're not made of Jell-O.

We get behind a fellow.

Black and yellow! Hello! Left, right, down, hover.

Hover? Forget hover. This isn't so hard. Beep-beep! Beep-beep!

Barry, what happened?!

Wait, I think we were on autopilot the whole time.

That may have been helping me. And now we're not! So it turns out I cannot fly a plane.

All of you, let's get behind this fellow! Move it out!

Move out!

Our only chance is if I do what I'd do, you copy me with the wings of the plane!

Don't have to yell.

I'm not yelling! We're in a lot of trouble.

It's very hard to concentrate with that panicky tone in your voice!

It's not a tone. I'm panicking!

I can't do this!

Vanessa, pull yourself together. You have to snap out of it!

You snap out of it.

You snap out of it.

You snap out of it!

You snap out of it!

You snap out of it!

You snap out of it!

You snap out of it!

You snap out of it!

Hold it!

Why? Oome on, it's my turn.

How is the plane flying?

I don't know.

Hello?

Benson, got any flowers for a happy occasion in there?

The Pollen Jocks!

They do get behind a fellow.

Black and yellow. Hello. All right, let's drop this tin can on the blacktop.

Where? I can't see anything. Oan you?

No, nothing. It's all cloudy.

Oome on. You got to think bee, Barry.

Thinking bee. Thinking bee. Thinking bee! Thinking bee! Thinking bee!

Wait a minute. I think I'm feeling something.

What? I don't know. It's strong, pulling me. Like a 27-million-year-old instinct.

Bring the nose down.

Thinking bee! Thinking bee! Thinking bee!

What in the world is on the tarmac? Get some lights on that! Thinking bee! Thinking bee! Thinking bee!

Vanessa, aim for the flower. OK. Out the engines. We're going in on bee power. Ready, boys?

Affirmative!

Good. Good. Easy, now. That's it.

Land on that flower!

Ready? Full reverse!

Spin it around!

Not that flower! The other one!

Which one?

That flower.

I'm aiming at the flower!

That's a fat guy in a flowered shirt. I mean the giant pulsating flower

made of millions of bees!

Pull forward. Nose down. Tail up.

Rotate around it.

This is insane, Barry! This's the only way I know how to fly. Am I koo-koo-kachoo, or is this plane flying in an insect-like pattern?

Get your nose in there. Don't be afraid. Smell it. Full reverse!

Just drop it. Be a part of it.

Aim for the center!

Now drop it in! Drop it in, woman!

Oome on, already.

Barry, we did it! You taught me how to fly!

Yes. No high-five! Right. Barry, it worked! Did you see the giant flower?

What giant flower? Where? Of course I saw the flower! That was genius!

Thank you. But we're not done yet. Listen, everyone!

This runway is covered with the last pollen

from the last flowers available anywhere on Earth.

That means this is our last chance.

We're the only ones who make honey, pollinate flowers and dress like this.

If we're gonna survive as a species, this is our moment! What do you say?

Are we going to be bees, orjust Museum of Natural History keychains?

We're bees!

Keychain!

Then follow me! Except Keychain.

Hold on, Barry. Here.

You've earned this.

Yeah!

I'm a Pollen Jock! And it's a perfect fit. All I gotta do are the sleeves.

Oh, yeah.

That's our Barry.

Mom! The bees are back!

If anybody needs to make a call, now's the time.

I got a feeling we'll be working late tonight!

Here's your change. Have a great afternoon! Oan I help who's next?

Would you like some honey with that? It is bee-approved. Don't forget these.

Milk, cream, cheese, it's all me. And I don't see a nickel!

Sometimes I just feel like a piece of meat!

I had no idea.

Barry, I'm sorry. Have you got a moment?

Would you excuse me? My mosquito associate will help you.

Sorry I'm late.

He's a lawyer too?

I was already a blood-sucking parasite. All I needed was a briefcase.

Have a great afternoon!

Barry, I just got this huge tulip order, and I can't get them anywhere.

No problem, Vannie. Just leave it to me.

You're a lifesaver, Barry. Oan I help who's next?

All right, scramble, jocks! It's time to fly.

Thank you, Barry!

That bee is living my life!

Let it go, Kenny.

When will this nightmare end?!

Let it all go.

Beautiful day to fly.

Sure is.

Between you and me, I was dying to get out of that office.

You have got to start thinking bee, my friend.

Thinking bee! Me? Hold it. Let's just stop for a second. Hold it.

I'm sorry. I'm sorry, everyone. Oan we stop here?

I'm not making a major life decision during a production number!

All right. Take ten, everybody. Wrap it up, guys.

I had virtually no rehearsal for that.

Reviewer #2 (Public Review):

The manuscript investigates the function of basal forebrain cholinergic axons in mouse primary visual cortex (V1) during locomotion using two-photon calcium imaging in head-fixed mice. Cholinergic modulation has previously been proposed to mediate the effects of locomotion on V1 responses. The manuscript concludes that the activity of basal forebrain cholinergic axons in visual cortex provides a signal which is more correlated with binary locomotion state than locomotion velocity of the animal and finds no evidence for modulation of cholinergic axons by locomotion velocity. Cholinergic axons did not seem to respond to grating stimuli or visuomotor prediction error. Optogenetic stimulation of these axons increased the amplitude of responses to visual stimuli and decreased the response latency of layer 5 excitatory neurons, but not layer 2/3 neurons. Moreover, optogenetic or chemogenetic stimulation of cholinergic inputs reduced pairwise correlation of neuronal responses. These results provide insight into the role of cholinergic modulation to visual cortex and demonstrate that it affects different layers of visual cortex in a distinct manner. The experiments are well executed and the data appear to be of high quality. However, further analyses may be required to fully support some of the study's conclusions. Specifically, the analyses of the effects of locomotion and stimulation of cholinergic inputs present grand averages of responses across all neurons, and therefore may mask heterogeneity across layer 2/3 and layer 5 neurons.

〈下終南山過斛斯山人宿置酒〉 暮從碧山下，山月隨人歸。

I find social media to be kind of impartial to my mental health. I have loads of problems that go beyond how I perceive others and how others perceive me on social media. I will however say that social media is 100% addictive for me and I'm fully aware that it is. This is especially true when i'm infinite scrolling and I'm telling myself in my head that I need to put the phone down but I'm practically in a trance. I guess you could say that this would have a negative impact on my mental health, but for me, it's kind of normal. I experience a lot of joy from social media, as it gives me a platform to share my life but also to connect with others.

Taking a picture of this!!!!

UNETR là một kiến trúc mạng nơ-ron kết hợp giữa Vision Transformer (ViT) và 3D convolutions. Dưới đây là các điểm quan trọng về kiến trúc này: 1. Vision Transformer (ViT):

• UNETR là một phiên bản tổng quát của ViT cho 3D convolutions.
• Nó thay thế 3D convolutions trong phần mã hóa bằng multi-head self-attention.

1. Chuyển đổi dữ liệu đầu vào:

   • Dữ liệu đầu vào 3D được chia thành các patch không giao nhau với kích thước 16x16x16.
   • Sau đó, dữ liệu được chiếu vào không gian nhúng (768 chiều) bằng một lớp tuyến tính và kết hợp với positional embedding.
   • Dữ liệu sau đó được xử lý bởi một encoder multi-head self-attention.

I don't know how and why but this line made something in my head click and realise that the rule the main/starting/current class's name should be the exact same as the file name.

And that is to let the complier know which is the class that it should start executing from and which ones are the other classes that are to be used as supplementaries to the main class.

Wouldn't this fail if the remote has a different HEAD?

Ok

After taking psychology and reading about the recent global news on mental health I started thinking about the relationship between mental health and people with neurodiverse conditions.This article helped me learn that some mental health issues can be symptoms of neurodiversity in an individual. For example Arash E.Zaghi's, depression and anxiety contributed to his ADHD diagnosis. I chose to annotate this sentence because it suggests that even people with mental health issues or people with neurodiversity are smart people who are capable of achieving standards that neurotypical people can. This sentence goes to show that society has to come together to overcome stereotypes in order to represent the whole population of different types of people working in the stem field or in any field. I am interested in the healthcare field and it is important to represent all types of people and for people in healthcare to help neurodiverse people be confident in expressing their abilities.Furthermore it is important for healthcare providers to be aware of stereotypes and different biases that individuals encounter to ensure the best treatment and care for different types of patients.

the messenger explains how Samson did it and he did by breaking two pillars and making the theatre collapse on everyone. his strength was still prevalent

Samson is responding to harapha saying that his wife has dishonoroed and betrayed him and now he is blind bugt can still fight and wont need all of the armor and weaponry he said that they should fight in a "narrow place"(1037) that way it will give Samson the right advantage and make up for his blindness.. Samson will only need an "oak n staff"(1123).

dalila is approaching them crying with visible wetness from crying on her.

his pride has now brought him down--- he avoided alcohol but fell for the seductive dalila-- turned him into " a tame weather"(538) without his strength, he is useless

Samson recaps and tells them his problems and compares himself to a ship captain who crashed his ship that was "trusted to (him) from above"(199) all bc he is blind now "how counterfeit a coin...friends"(he is saying it is hard to find true friends like what he has now. they are all fake to him

the sight of samson is shocking and he cannot believe its him he thinks how could this be Samson if all he knows Samson to be is one who ripped apart lions and fdidnt use weapons

This thing brings me to ChatGPT. I once asked ChatGPT because I was worried about the theme of the essay, and what he presented to me was an idea that integrated many viewpoints into one idea. It makes me wonder if this idea is plagiarism. If it is not plagiarism, then this point of view also exists the idea that has been put forward before, if it is plagiarism, but this point of view has no direct article.

Author response:

Reviewer 1:

The paper “Quantifying gliding forces of filamentous cyanobacteria by self-buckling” combines experiments on freely gliding cyanobacteria, buckling experiments using two-dimensional V-shaped corners, and micropipette force measurements with theoretical models to study gliding forces in these organisms. The aim is to quantify these forces and use the results to perhaps discriminate between competing mechanisms by which these cells move. A large data set of possible collision events are analyzed, bucking events evaluated, and critical buckling lengths estimated. A line elasticity model is used to analyze the onset of buckling and estimate the effective (viscous type) friction/drag that controls the dynamics of the rotation that ensues post-buckling. This value of the friction/drag is compared to a second estimate obtained by consideration of the active forces and speeds in freely gliding filaments. The authors find that these two independent estimates of friction/drag correlate with each other and are comparable in magnitude. The experiments are conducted carefully, the device fabrication is novel, the data set is interesting, and the analysis is solid. The authors conclude that the experiments are consistent with the propulsion being generated by adhesion forces rather than slime extrusion. While consistent with the data, this conclusion is inferred.

We thank the reviewer for the positive evaluation of our work.

Summary:

The paper addresses important questions on the mechanisms driving the gliding motility of filamentous cyanobacteria. The authors aim to understand these by estimating the elastic properties of the filaments, and by comparing the resistance to gliding under a) freely gliding conditions, and b) in post-buckled rotational states. Experiments are used to estimate the propulsion force density on freely gliding filaments (assuming over-damped conditions). Experiments are combined with a theoretical model based on Euler beam theory to extract friction (viscous) coefficients for filaments that buckle and begin to rotate about the pinned end. The main results are estimates for the bending stiffness of the bacteria, the propulsive tangential force density, the buckling threshold in terms of the length, and estimates of the resistive friction (viscous drag) providing the dissipation in the system and balancing the active force. It is found that experiments on the two bacterial species yield nearly identical values of f (albeit with rather large variations). The authors conclude that the experiments are consistent with the propulsion being generated by adhesion forces rather than slime extrusion.

We appreciate this comprehensive summary of our work.

Strengths of the paper:

The strengths of the paper lie in the novel experimental setup and measurements that allow for the estimation of the propulsive force density, critical buckling length, and effective viscous drag forces for movement of the filament along its contour – the axial (parallel) drag coefficient, and the normal (perpendicular) drag coefficient (I assume this is the case, since the post-buckling analysis assumes the bent filament rotates at a constant frequency). These direct measurements are important for serious analysis and discrimination between motility mechanisms.

We thank the reviewer for this positive assessment of our work.

Weaknesses:

There are aspects of the analysis and discussion that may be improved. I suggest that the authors take the following comments into consideration while revising their manuscript.

The conclusion that adhesion via focal adhesions is the cause for propulsion rather than slime protrusion is consistent with the experimental results that the frictional drag correlates with propulsion force. At the same time, it is hard to rule out other factors that may result in this (friction) viscous drag - (active) force relationship while still being consistent with slime production. More detailed analysis aiming to discriminate between adhesion vs slime protrusion may be outside the scope of the study, but the authors may still want to elaborate on their inference. It would help if there was a detailed discussion on the differences in terms of the active force term for the focal adhesion-based motility vs the slime motility.

We appreciate this critical assessment of our conclusions. Of course we are aware that many different mechanisms may lead to similar force/friction characteristics, and that a definitive conclusion on the mechanism would require the combination of various techniques, which is beyond the scope of this work. Therefore, we were very careful in formulating the discussion of our findings, refraining, in particular, from a singular conclusion on the mechanism but instead indicating “support” for one hypothesis over another, and emphasizing “that many other possibilities exist”.

The most common concurrent hypotheses for bacterial gliding suggest that either slime extrusion at the junctional pore complex [A1], rhythmic contraction of fibrillar arrays at the cell wall [A2], focal adhesion sites connected to intracellular motor-microtubule complexes [A3], or modified type-IV pilus apparati [A4] provide the propulsion forces. For the slime extrusion hypothesis, which is still abundant today, one would rather expect an anticorrelation of force and friction: more slime extrusion would generate more force, but also enhance lubrication. The other hypotheses are more conformal to the trend we observed in our experiments, because both pili and focal adhesion require direct contact with a substrate. How contraction of fibrilar arrays would micromechanically couple to the environment is not clear to us, but direct contact might still facilitate force transduction. Please note that these hypotheses were all postulated without any mechanical measurements, solely based on ultra-structural electron microscopy and/or genetic or proteomic experiments. We see our work as complementary to that, providing a mechanical basis for evaluating these hypotheses.

We agree with the referee that narrowing down this discussion to focal adhesion should have been avoided. We rewrote the concluding paragraph (page 8):

“…it indicates that friction and propulsion forces, despite being quite vari able, correlate strongly. Thus, generating more force comes, inevitably, at the expense of added friction. For lubricated contacts, the friction coefficient is proportional to the thickness of the lubricating layer (Snoeijer et al., 2013 ), and we conjecture active force and drag both increase due to a more intimate contact with the substrate. This supports mechanisms like focal adhesion (Mignot et al., 2007 ) or a modified type-IV pilus (Khayatan et al., 2015 ), which generate forces through contact with extracellular surfaces, as the underlying mechanism of the gliding apparatus of filamentous cyanobacteria: more contacts generate more force, but also closer contact with the substrate, thereby increasing friction to the same extent. Force generation by slime extrusion (Hoiczyk and Baumeister, 1998 ), in contrast, would lead to the opposite behavior: More slime generates more propulsion, but also reduces friction. Besides fundamental fluid-mechanical considerations (Snoeijer et al., 2013 ), this is rationalized by two experimental observations: i. gliding velocity correlates positively with slime layer thickness (Dhahri et al., 2013 ) and ii. motility in slime-secretion deficient mutants is restored upon exogenous addition of polysaccharide slime. Still we emphasize that many other possibilities exist. One could, for instance, postulate a regulation of the generated forces to the experienced friction, to maintain some preferred or saturated velocity.”

Can the authors comment on possible mechanisms (perhaps from the literature) that indicate how isotropic friction may be generated in settings where focal adhesions drive motility? A key aspect here would probably be estimating the extent of this adhesion patch and comparing it to a characteristic contact area. Can lubrication theory be used to estimate characteristic areas of contact (knowing the radius of the filament, and assuming a height above the substrate)? If the focal adhesions typically cover areas smaller than this lubrication area, it may suggest the possibility that bacteria essentially present a flat surface insofar as adhesion is concerned, leading to a transversely isotropic response in terms of the drag. Of course, we will still require the effective propulsive force to act along the tangent.

We thank the referee for suggesting to estimate the dimensions of the contact region. Both pili and focal adhesion sites would be of sizes below one micron [A3, A4], much smaller than the typical contact region in the lubricated contact, which is on the order of the filament radius (few microns). So indeed, isotropic friction may be expected in this situation [A5] and is assumed frequently in theoretical work [A6–A8]. Anisotropy may then indeed be induced by active forces [A9], but we are not aware of measurements of the anisotropy of friction in bacterial gliding.

For a more precise estimate using lubrication theory, rheology and extrusion rate of the secreted polysaccharides would have to be known, but we are not aware of detailed experimental characterizations.

We extended the paragraph in the buckling theory on page 5 regarding the assumption of isotropic friction:

“We use classical Kirchhoff theory for a uniform beam of length L and bending modulus B, subject to a force density ⃗b = −f ⃗t− η ⃗v, with an effective active force density f along the tangent ⃗t, and an effective friction proportional to the local velocity ⃗v, analog to existing literature (Fily et al., 2020; Chelakkot et al., 2014; Sekimoto et al., 1995 ). Presumably, this friction is dominated by the lubrication drag from the contact with the substrate, filled by a thin layer of secreted polysaccharide slime which is much more viscous than the surrounding bulk fluid. Speculatively, the motility mechanism might also comprise adhering elements like pili (Khayatan et al., 2015 ) or foci (Mignot et al., 2007 ) that increase the overall friction (Pompe et al., 2015 ). Thus, the drag due to the surrounding bulk fluid can be neglected (Man and Kanso, 2019 ), and friction is assumed to be isotropic, a common assumption in motility models (Fei et al., 2020; Tchoufag et al., 2019; Wada et al., 2013 ). We assume…”

We also extended the discussion regarding the outcome of isotropic friction (page 7):

“…Thus we plot f/v over η in Figure 4 D, finding nearly identical values over about two decades. Since f and η are not correlated with v0, this is due to a correlation between f and η. This relation is remarkable in two aspects: On the one hand, it indicates that friction is mainly isotropic. This suggests that friction is governed by an isotropic process like bond friction or lubrication from the slime layer in the contact with the substrate, the latter being consistent with the observation that mutations deficient of slime secretion do not glide but exogenous addition of slime restores motility (Khayatan et al., 2015 ). In contrast, hydrodynamic drag from the surrounding bulk fluid (Man and Kanso, 2019 ), or the internal friction of the gliding apparatus would be expected to generate strongly anisotropic friction. If the latter was dominant, a snapping-like transition into the buckling state would be expected, rather than the continuously growing amplitude that is observed in experiments. On the other hand, it indicates that friction and propulsion forces…”

I am not sure why the authors mention that the power of the gliding apparatus is not rate-limiting. The only way to verify this would be to put these in highly viscous fluids where the drag of the external fluid comes into the picture as well (if focal adhesions are on the substrate-facing side, and the upper side is subject to ambient fluid drag). Also, the friction referred to here has the form of a viscous drag (no memory effect, and thus not viscoelastic or gel-like), and it is not clear if forces generated by adhesion involve other forms of drag such as chemical friction via temporary bonds forming and breaking. In quasi-static settings and under certain conditions such as the separation of chemical and elastic time scales, bond friction may yield overall force proportional to local sliding velocities.

We agree with the referee that the origin of the friction is not easily resolved. Lubrication yields an isotropic force density that is proportional to the velocity, and the same could be generated by bond friction. Importantly, both types of friction would be assumed to be predominantly isotropic. We explicitly referred to lubrication drag because it has been shown that mutations deficient of slime extrusion do not glide [A4].

Assuming, in contrast, that in free gliding, friction with the environment is not rate limiting, but rather the internal friction of the gliding apparatus, i.e., the available power, we would expect a rather different behavior during early-buckling evolution. During early buckling, the tangential motion is stalled, and the dynamics is dominated by the growing buckling amplitude of filament regions near the front end, which move mainly transversely. For geometric reasons, in this stage the (transverse) buckling amplitude grows much faster than the rear part of the filament advances longitudinally. Thus that motion should not be impeded much by the internal friction of the gliding apparatus, but by external friction between the buckling parts of the filament and the ambient. The rate at which the buckling amplitude initially grows should be limited by the accumulated compressive stress in the filament and the transverse friction with the substrate. If the latter were much smaller than the (logitudinal) internal friction of the gliding apparatus, we would expect a snapping-like transition into the buckled state, which we did not observe.

In our paper, we do not intend to evaluate the exact origin of the friction, quantifying the gliding force is the main objective. A linear force-velocity relation agrees with our observations. A detailed analysis of friction in cyanobacterial gliding would be an interesting direction for future work.

To make these considerations more clear, we rephrased the corresponding paragraph on page 7 & 8:

“…Thus we plot f/v over η in Figure 4 D, finding nearly identical values over about two decades. Since f and η are not correlated with v0, this is due to a correlation between f and η. This relation is remarkable in two aspects: On the one hand, it indicates that friction is mainly isotropic. This suggests that friction is governed by an isotropic process like bond friction or lubrication from the slime layer in the contact with the substrate, the latter being consistent with the observation that mutations deficient of slime secretion do not glide but exogenous addition of slime restores motility (Khayatan et al., 2015 ). In contrast, hydrodynamic drag from the surrounding bulk fluid (Man and Kanso, 2019 ), or the internal friction of the gliding apparatus would be expected to generate strongly anisotropic friction. If the latter was dominant, a snapping-like transition into the buckling state would be expected, rather than the continuously growing amplitude that is observed in experiments. On the other hand, it indicates that friction and propulsion forces…”

For readers from a non-fluids background, some additional discussion of the drag forces, and the forms of friction would help. For a freely gliding filament if f is the force density (per unit length), then steady gliding with a viscous frictional drag would suggest (as mentioned in the paper) f ∼ v! L η||. The critical buckling length is then dependent on f and on B the bending modulus. Here the effective drag is defined per length. I can see from this that if the active force is fixed, and the viscous component resulting from the frictional mechanism is fixed, the critical buckling length will not depend on the velocity (unless I am missing something in their argument), since the velocity is not a primitive variable, and is itself an emergent quantity.

We are not sure what “f ∼ v! L η||” means, possibly the spelling was corrupted in the forwarding of the comments.

We assumed an overdamped motion in which the friction force density ff (per unit length of the filament) is proportional to the velocity v0, i.e. ff ∼ η v0, with a friction coefficient η. Overdamped means that the friction force density is equal and opposite to the propulsion force density, so the propulsion force density is f ∼ ff ∼ η v0. The total friction and propulsion forces can be obtained by multiplication with the filament length

L, which is not required here. In this picture, v0 is an emergent quantity and f and η are assumed as given and constant. Thus, by observing v0, f can be inferred up to the friction coefficient η. Therefore, by using two descriptive variables, L and v0, with known B, the primitive variable η can be inferred by logistic regression, and f then follows from the overdamped equation of motion.

To clarify this, we revised the corresponding section on page 5 of the paper:

“The substrate contact requires lubrication from polysaccharide slime to enable bacteria to glide (Khayatan et al., 2015 ). Thus we assume an over- damped motion with co-linear friction, for which the propulsion force f and the free gliding velocity v0 of a filament are related by f = η v0, with a friction coefficient η. In this scenario, f can be inferred both from the observed Lc ∼ (f/B)−1/3 and, up to the proportionality coefficient η, from the observed free gliding velocity. Thus, by combining the two relations, one may expect also a strong correlation between Lc and v0. In order to test this relation for consistency with our data, we include v0 as a second regressor, by setting x = (L−Lc(v0))/∆Lc in Equation 1, with Lc(v0) = (η v0/(30.5722 B))−1/3, to reflect our expectation from theory (see below). Now, η rather than f is the only unknown, and its ensemble distribution will be determined in the regression. Figure 3 E,F show the buckling behavior…”

Reviewer 2:

In the presented manuscript, the authors first use structured microfluidic devices with gliding filamentous cyanobacteria inside in combination with micropipette force measurements to measure the bending rigidity of the filaments.

Next, they use triangular structures to trap the bacteria with the front against an obstacle. Depending on the length and rigidity, the filaments buckle under the propulsive force of the cells. The authors use theoretical expressions for the buckling threshold to infer propulsive force, given the measured length and stiffnesses. They find nearly identical values for both species, f ∼ (1.0 ± 0.6) nN/µm, nearly independent of the velocity.

Finally, they measure the shape of the filament dynamically to infer friction coefficients via Kirchhoff theory. This last part seems a bit inconsistent with the previous inference of propulsive force. Before, they assumed the same propulsive force for all bacteria and showed only a very weak correlation between buckling and propulsive velocity. In this section, they report a strong correlation with velocity, and report propulsive forces that vary over two orders of magnitude. I might be misunderstanding something, but I think this discrepancy should have been discussed or explained.

We regret the misunderstanding of the reviewer regarding the velocity dependence, which indicates that the manuscript should be improved to convey these relations correctly.

First, in the Buckling Measurements section, we did not assume the same propulsion force for all bacteria. The logistic regression yields an ensemble median for Lc (and thus an ensemble median for f ), along with the width ∆Lc of the distribution (and thus also the width of the distribution of f ). Our result f ∼ (1.0 ± 0.6) nN/µm indicates the median and the width of the distribution of the propulsion force densities across the ensemble of several hundred filaments used in the buckling measurements. The large variability of the forces found in the second part is consistently reflected by this very wide distribution of active forces detected in the logistic regression in the first part.

We did small modifications to the buckling theory paragraph to clarify that in the first part, a distribution of forces rather than a constant value is inferred (page 6)

“Inserting the population median and quartiles of the distributions of bending modulus and critical length, we can now quantify the distribution of the active force density for the filaments in the ensemble from the buckling measurements. We obtain nearly identical values for both species, f ∼ (1.0±0.6) nN/µm, where the uncertainty represents a wide distribution of f across the ensemble rather than a measurement error.”

The same holds, of course, when inferring the distribution of the friction coefficients (page 5):

“The substrate contact requires lubrication from polysaccharide slime to enable bacteria to glide (Khayatan et al., 2015 ). Thus we assume an over- damped motion with co-linear friction, for which the propulsion force f and the free gliding velocity v0 of a filament are related by f = η v0, with a friction coefficient η. In this scenario, f can be inferred both from the observed Lc ∼ (f/B)−1/3 and, up to the proportionality coefficient η, from the observed free gliding velocity. Thus, by combining the two relations, one may expect also a strong correlation between Lc and v0. In order to test this relation for consistency with our data, we include v0 as a second regressor, by setting x = (L−Lc(v0))/∆Lc in Equation 1, with Lc(v0) = (η v0/(30.5722 B))−1/3, to reflect our expectation from theory (see below). Now, η rather than f is the only unknown, and its ensemble distribution will be determined in the regression. Figure 3 E,F show the buckling behavior…”

The (naturally) wide distribution of force (and friction) leads to a distribution of Lc as well. However, due to the small exponent of 1/3 in the buckling threshold Lc ∼ f 1/3, the distribution of Lc is not as wide as the distributions of the individually inferred f or η. This is visualized in panel G of Figure 3, plotting Lc as a function of v0 (v0 is equivalent to f , up to a proportionality coefficient η). The natural length distribution, in contrast, is very wide. Therefore, the buckling propensity of a filament is most strongly characterized by its length, while force variability, which alters Lc of the individual, plays a secondary role.

In order to clarify this, we edited the last paragraph of the Buckling Measurements section on page 5 of the manuscript:

“…Within the characteristic range of observed velocities (1 − 3 µm/s), the median Lc depends only mildly on v0, as compared to its rather broad distribution, indicated by the bands in Figure 3 G. Thus a possible correlation between f and v0 would only mildly alter Lc. The natural length distribution (cf. Appendix 1—figure 1 ), however, is very broad, and we conclude that growth rather than velocity or force distributions most strongly impacts the buckling propensity of cyanobacterial colonies. Also, we hardly observed short and fast filaments of K. animale, which might be caused by physiological limitations (Burkholder, 1934 ).”

Second, in the Profile analysis section, we did not report a correlation between force and velocity. As can be seen in Figure 4—figure Supplement 1, neither the active force nor the friction coefficient, as determined from the analysis of individual filaments, show any significant correlation with the velocity. This is also written in the discussion (page 7):

We see no significant correlation between L or v0 and f or η, but the observed values of f and η cover a wide range (Figure 4 B, C and Figure 4—figure Supplement 1 ).

Note that this is indeed consistent with the logistic regression: Using v0 as a second regressor did not significantly reduce the width of the distribution of Lc as compared to the simple logistic regression, indicating that force and velocity are not strongly correlated.

In order to clarify this in the manuscript, we modified that part (page 7):

“…We see no significant correlation between L or v0 and f or η, but the observed values of f and η cover a wide range (Figure 4 B,C and Figure 4— figure Supplement 1 ). This is consistent with the logistic regression, where using v0 as a second regressor did not significantly reduce the width of the distribution of critical lengths or active forces. The two estimates of the friction coefficient, from logistic regression and individual profile fits, are measured in (predominantly) orthogonal directions: tangentially for the logistic regression where the free gliding velocity was used, and transversely for the evolution of the buckling profiles. Thus we plot f/v over η in Figure 4 D, finding nearly identical values over about two decades. Since f and η are not correlated with v0, this is due to a correlation between f and η. This relation is remarkable in two aspects: On the one hand, it indicates that friction is mainly isotropic…”

From a theoretical perspective, not many new results are presented. The authors repeat the well-known calculation for filaments buckling under propulsive load and arrive at the literature result of buckling when the dimensionless number (f L3/B) is larger than 30.6 as previously derived by Sekimoto et al in 1995 [1] (see [2] for a clamped boundary condition and simulations). Other theoretical predictions for pushed semi-flexible filaments [1–4] are not discussed or compared with the experiments. Finally, the Authors use molecular dynamics type simulations similar to [2–4] to reproduce the buckling dynamics from the experiments. Unfortunately, no systematic comparison is performed.

[1]        Ken Sekimoto, Naoki Mori, Katsuhisa Tawada, and Yoko Y Toyoshima. Symmetry breaking instabilities of an in vitro biological system. Physical review letters, 75(1):172, 1995.

[2]       Raghunath Chelakkot, Arvind Gopinath, Lakshminarayanan Mahadevan, and Michael F Hagan. Flagellar dynamics of a connected chain of active, polar, brownian particles. Journal of The Royal Society Interface, 11(92):20130884, 2014.

[3]       Rolf E Isele-Holder, Jens Elgeti, and Gerhard Gompper. Self-propelled worm-like filaments: spontaneous spiral formation, structure, and dynamics. Soft matter, 11(36):7181–7190, 2015.

[4]       Rolf E Isele-Holder, Julia J¨ager, Guglielmo Saggiorato, Jens Elgeti, and Gerhard Gompper. Dynamics of self-propelled filaments pushing a load. Soft Matter, 12(41):8495–8505, 2016.

We thank the reviewer for pointing us to these publications, in particular the work by Sekimoto we were not aware of. We agree with the referee that the calculation is straight forward (basically known since Euler, up to modified boundary conditions). Our paper focuses on experimental work, the molecular dynamics simulations were included mainly as a consistency check and not intended to generate the beautiful post-buckling patterns observed in references [2-4]. However, such shapes do emerge in filamentous cyanobacteria, and with the data provided in our manuscript, simulations can be quantitatively matched to our experiments, which will be covered by future work.

We included the references in the revision of our manuscript, and a statement that we do not claim priority on these classical theoretical results.

Introduction, page 2:

“…Self-Buckling is an important instability for self-propelling rod-like micro-organisms to change the orientation of their motion, enabling aggregation or the escape from traps (Fily et al., 2020; Man and Kanso, 2019; Isele-Holder et al., 2015; Isele-Holder et al., 2016 ). The notion of self-buckling goes back to work of Leonhard Euler in 1780, who described elastic columns subject to gravity (Elishakoff, 2000 ). Here, the principle is adapted to the self-propelling, flexible filaments (Fily et al., 2020; Man and Kanso, 2019; Sekimoto et al., 1995 ) that glide onto an obstacle. Filaments buckle if they exceed a certain critical length Lc ∼ (B/f)1/3, where B is the bending modulus and f the propulsion force density…”

Buckling theory, page 5:

“…The buckling of gliding filaments differs in two aspects: the propulsion forces are oriented tangentially instead of vertically, and the front end is supported instead of clamped. Therefore, with L < Lc all initial orientations are indifferently stable, while for L > Lc, buckling induces curvature and a resultant torque on the head, leading to rotation (Fily et al., 2020; Chelakkot et al., 2014; Sekimoto et al., 1995 ). Buckling under concentrated tangential end-loads has also been investigated in literature (de Canio et al., 2017; Wolgemuth et al., 2005 ), but leads to substantially different shapes of buckled filaments. We use classical Kirchhoff theory for a uniform beam of length L and bending modulus B, subject to a force density ⃗b = −f ⃗t − η ⃗v, with an effective active force density f along the tangent ⃗t, and an effective friction proportional to the local velocity ⃗v, analog to existing literature (Fily et al., 2020; Chelakkot et al., 2014; Sekimoto et al., 1995 )…”

Further on page 6:

“To derive the critical self-buckling length, Equation 5 can be linearized for two scenarios that lead to the same Lc: early-time small amplitude buckling and late-time stationary rotation at small and constant curvature (Fily et al., 2020; Chelakkot et al., 2014 ; Sekimoto et al., 1995 ). […] Thus, in physical units, the critical length is given by Lc = (30.5722 B/f)1/3, which is reproduced in particle based simulations (Appendix Figure 2 ) analogous to those in Isele-Holder et al. (2015, 2016).”

Discussion, page 7 & 8:

“…This, in turn, has dramatic consequences on the exploration behavior and the emerging patterns (Isele-Holder et al., 2015, 2016; Abbaspour et al., 2021; Duman et al., 2018; Prathyusha et al., 2018; Jung et al., 2020 ): (L/Lc)3 is, up to a numerical prefactor, identical to the flexure number (Isele-Holder et al., 2015, 2016; Duman et al., 2018; Winkler et al., 2017 ), the ratio of the Peclet number and the persistence length of active polymer melts. Thus, the ample variety of non-equilibrium phases in such materials (Isele-Holder et al., 2015, 2016; Prathyusha et al., 2018; Abbaspour et al., 2021 ) may well have contributed to the evolutionary success of filamentous cyanobacteria.”

Reviewer 3:

Summary:

This paper presents novel and innovative force measurements of the biophysics of gliding cyanobacteria filaments. These measurements allow for estimates of the resistive force between the cell and substrate and provide potential insight into the motility mechanism of these cells, which remains unknown.

We thank the reviewer for the positive evaluation of our work. We have revised the manuscript according to their comments and detail our replies and modifications next to the individual points below.

Strengths:

The authors used well-designed microfabricated devices to measure the bending modulus of these cells and to determine the critical length at which the cells buckle. I especially appreciated the way the authors constructed an array of pillars and used it to do 3-point bending measurements and the arrangement the authors used to direct cells into a V-shaped corner in order to examine at what length the cells buckled at. By examining the gliding speed of the cells before buckling events, the authors were able to determine how strongly the buckling length depends on the gliding speed, which could be an indicator of how the force exerted by the cells depends on cell length; however, the authors did not comment on this directly.

We thank the referee for the positive assessment of our work. Importantly, we do not see a significant correlation between buckling length and gliding speeds, and we also do not see a correlation with filament length, consistent with the assumption of a propulsion force density that is more or less homogeneously distributed along the filament. Note that each filament consists of many metabolically independent cells, which renders cyanobacterial gliding a collective effort of many cells, in contrast to gliding of, e.g., myxobacteria.

In response also to the other referees’ comments, we modified the manuscript to reflect more on the absence of a strong correlation between velocity and force/critical length. We modified the Buckling measurements section on page 5 of the paper:

“The substrate contact requires lubrication from polysaccharide slime to enable bacteria to glide (Khayatan et al., 2015 ). Thus we assume an over-damped motion with co-linear friction, for which the propulsion force f and the free gliding velocity v0 of a filament are related by f = η v0, with a friction coefficient η. In this scenario, f can be inferred both from the observed Lc ∼ (f/B)−1/3 and, up to the proportionality coefficient η, from the observed free gliding velocity. Thus, by combining the two relations, one may expect also a strong correlation between Lc and v0. In order to test this relation for consistency with our data, we include v0 as a second regressor, by setting x = (L−Lc(v0))/∆Lc in Equation 1, with Lc(v0) = (η v0/(30.5722 B))−1/3, to reflect our expectation from theory (see below). Now, η rather than f is the only unknown, and its ensemble distribution will be determined in the regression. Figure 3 E, F show the buckling behavior…”

Further, we edited the last paragraph of the Buckling measurements section on page 5 of the manuscript:

“Within the characteristic range of observed velocities (1 − 3 µm/s), the median Lc depends only mildly on v0, as compared to its rather broad distribution, indicated by the bands in Figure 3 G. Thus a possible correlation between f and v0 would only mildly alter Lc. The natural length distribution (cf. Appendix 1—figure 1 ), however, is very broad, and we conclude that growth rather than velocity or force distributions most strongly impacts the buckling propensity of cyanobacterial colonies. Also, we hardly observed short and fast filaments of K. animale, which might be caused by physiological limitations (Burkholder, 1934 ).”

We also rephrased the corresponding discussion paragraph on page 7:

“…Thus we plot f/v over η in Figure 4 D, finding nearly identical values over about two decades. Since f and η are not correlated with v0, this is due to a correlation between f and η. This relation is remarkable in two aspects: On the one hand, it indicates that friction is mainly isotropic. This suggests that friction is governed by an isotropic process like bond friction or lubrication from the slime layer in the contact with the substrate, the latter being consistent with the observation that mutations deficient of slime secretion do not glide but exogenous addition of slime restores motility (Khayatan et al., 2015 ). In contrast, hydrodynamic drag from the surrounding bulk fluid (Man and Kanso, 2019 ), or the internal friction of the gliding apparatus would be expected to generate strongly anisotropic friction. If the latter was dominant, a snapping-like transition into the buckling state would be expected, rather than the continuously growing amplitude that is observed in experiments. On the other hand, it indicates that friction and propulsion forces…”

Weaknesses:

There were two minor weaknesses in the paper.

First, the authors investigate the buckling of these gliding cells using an Euler beam model. A similar mathematical analysis was used to estimate the bending modulus and gliding force for Myxobacteria (C.W. Wolgemuth, Biophys. J. 89: 945-950 (2005)). A similar mathematical model was also examined in G. De Canio, E. Lauga, and R.E Goldstein, J. Roy. Soc. Interface, 14: 20170491 (2017). The authors should have cited these previous works and pointed out any differences between what they did and what was done before.

We thank the reviewer for pointing us to these references. The paper by Wolgemuth is theoretical work, describing A-motility in myxobacteria by a concentrated propulsion force at the rear end of the bacterium, possibly stemming from slime extrusion. This model was a little later refuted by [A3], who demonstrated that focal adhesion along the bacterial body and thus a distributed force powers A-motility, a mechanism that has by now been investigated in great detail (see [A10]). The paper by Canio et al. contains a thorough theoretical analysis of a filament that is clamped at one end and subject to a concentrated tangential load on the other. Since both models comprise a concentrated end-load rather than a distributed propulsion force density, they describe a substantially different motility mechanism, leading also to substantially different buckling profiles. Consequentially, these models cannot be applied to cyanobacterial gliding.

We included both citations in the revision and pointed out the differences to our work in the introduction (page 2):

“…A few species appear to employ a type-IV-pilus related mechanism (Khayatan et al., 2015; Wilde and Mullineaux, 2015 ), similar to the better- studied myxobacteria (Godwin et al., 1989; Mignot et al., 2007; Nan et al., 2014; Copenhagen et al., 2021; Godwin et al., 1989 ), which are short, rod-shaped single cells that exhibit two types of motility: S (social) motility based on pilus extension and retraction, and A (adventurous) motility based on focal adhesion (Chen and Nan, 2022 ) for which also slime extrusion at the trailing cell pole was earlier postulated as mechanism (Wolgemuth et al., 2005 ). Yet, most gliding filamentous cyanobacteria do not exhibit pili and their gliding mechanism appears to be distinct from myxobacteria (Khayatan et al., 2015 ).”

And in Buckling theory, page 5:

“….The buckling of gliding filaments differs in two aspects: the propulsion forces are oriented tangentially instead of vertically, and the front end is supported instead of clamped. Therefore, with L < Lc all initial orientations are indifferently stable, while for L > Lc, buckling induces curvature and a resultant torque on the head, leading to rotation (Fily et al., 2020; Chelakkot et al., 2014; Sekimoto et al., 1995 ). Buckling under concentrated tangential end-loads has also been investigated in literature (de Canio et al., 2017; Wolgemuth et al., 2005 ), but leads to substantially different shapes of buckled filaments.”

The second weakness is that the authors claim that their results favor a focal adhesion-based mechanism for cyanobacterial gliding motility. This is based on their result that friction and adhesion forces correlate strongly. They then conjecture that this is due to more intimate contact with the surface, with more contacts producing more force and pulling the filaments closer to the substrate, which produces more friction. They then claim that a slime-extrusion mechanism would necessarily involve more force and lower friction. Is it necessarily true that this latter statement is correct? (I admit that it could be, but is it a requirement?)

We thank the referee for raising this interesting question. Our claim regarding slime extrusion is based on three facts: i. mutations deficient of slime extrusion do not glide, but start gliding as soon as slime is provided externally [A4]. ii. A positive correlation between speed and slime layer thickness was observed in Nostoc [A11]. iii. The fluid mechanics of lubricated sliding contacts is very well understood and predicts a decreasing resistance with increasing layer thickness.

We included these considerations in the revision of our manuscript (page 8):

“…it indicates that friction and propulsion forces, despite being quite variable, correlate strongly. Thus, generating more force comes, inevitably, at the expense of added friction. For lubricated contacts, the friction coefficient is proportional to the thickness of the lubricating layer (Snoeijer et al., 2013 ), and we conjecture active force and drag both increase due to a more intimate contact with the substrate. This supports mechanisms like focal adhesion (Mignot et al., 2007 ) or a modified type-IV pilus (Khayatan et al., 2015 ), which generate forces through contact with extracellular surfaces, as the underlying mechanism of the gliding apparatus of filamentous cyanobacteria: more contacts generate more force, but also closer contact with the substrate, thereby increasing friction to the same extent. Force generation by slime extrusion (Hoiczyk and Baumeister, 1998 ), in contrast, would lead to the opposite behavior: More slime generates more propulsion, but also reduces friction. Besides fundamental fluid-mechanical considerations (Snoeijer et al., 2013 ), this is rationalized by two experimental observations: i. gliding velocity correlates positively with slime layer thickness (Dhahri et al., 2013 ) and ii. motility in slime-secretion deficient mutants is restored upon exogenous addition of polysaccharide slime. Still we emphasize that many other possibilities exist. One could, for instance, postulate a regulation of the generated forces to the experienced friction, to maintain some preferred or saturated velocity.”

Related to this, the authors use a model with isotropic friction. They claim that this is justified because they are able to fit the cell shapes well with this assumption. How would assuming a non-isotropic drag coefficient affect the shapes? It may be that it does equally well, in which case, the quality of the fits would not be informative about whether or not the drag was isotropic or not.

The referee raises another very interesting point. Given the typical variability and uncertainty in experimental measurements (cf. error Figure 4 A), a model with a sightly anisotropic friction could be fitted to the observed buckling profiles as well, without significant increase of the mismatch. Yet, strongly anisotropic friction would not be consistent with our observations.

Importantly, however, we did not conclude on isotropic friction based on the fit quality, but based on a comparison between free gliding and early buckling (Figure 4 D). In early buckling, the dominant motion is in transverse direction, while longitudinal motion is insignificant, due to geometric reasons. Thus, independent of the underlying model, mostly the transverse friction coefficiont is inferred. In contrast, free gliding is a purely longitudinal motion, and thus only the friction coefficient for longitudinal motion can be inferred. These two friction coefficients are compared in Figure 4 D. Still, the scatter of that data would allow to fit a certain anisotropy within the error margins. What we can exclude based on out observation is the case of a strongly anisotropic friction. If there is no ab-initio reason for anisotropy, nor a measurement that indicates it, we prefer to stick with the simplest

assumption. We carefully chose our wording in the Discussion as “mainly isotropic” rather

than “isotropic” or “fully isotropic”.

We added a small statement to the Discussion on page 7 & 8:

“... Thus we plot f/v over η in Figure 4 D, finding nearly identical values over about two decades. Since f and η are not correlated with v0, this is due to a correlation between f and η. This relation is remarkable in two aspects: On the one hand, it indicates that friction is mainly isotropic. This suggests that friction is governed by an isotropic process like bond friction or lubrication from the slime layer in the contact with the substrate, the latter being consistent with the observation that mutations deficient of slime secretion do not glide but exogenous addition of slime restores motility (Khayatan et al., 2015 ). In contrast, hydrodynamic drag from the surrounding bulk fluid (Man and Kanso, 2019 ), or the internal friction of the gliding apparatus would be expected to generate strongly anisotropic friction. If the latter was dominant, a snapping-like transition into the buckling state would be expected, rather than the continuously growing amplitude that is observed in experiments. On the other hand, it indicates that friction and propulsion forces ...”

Recommendations for the authors

The discussion regarding how the findings of this paper imply that cyanobacteria filaments are propelled by adhesion forces rather than slime extrusion should be improved, as this conclusion seems questionable. There appears to be an inconsistency with a buckling force said to be only weakly dependent on the gliding velocity, while its ratio with the velocity correlates with a friction coefficient. Finally, data and source code should be made publicly available.

In the revised version, we have modified the discussion of the force generating mechanism according to the reviewer suggestions. The perception of inconsistency in the velocity dependence of the buckling force was based on a misunderstanding, as we detailed in our reply to the referee. We revised the corresponding section to make it more clear. Data and source code have been uploaded to a public data repository.

Reviewer #2 (recommendations for the authors)

Despite eLife policy, the authors do not provide a Data Availability Statement. For the presented manuscript, data and source code should be provided “via trusted institutional or third-party repositories that adhere to policies that make data discoverable, accessible and usable.” https://elifesciences.org/inside-elife/51839f0a/for-authors-updates- to-elife-s-data-sharing-policies

Most of the issues in this reviewer’s public review should be easy to correct, so I would strongly support the authors to provide an amended manuscript.

We added the Data Availability Statement in the amended manuscript.

References

[A1] E. Hoiczyk and W. Baumeister. “The junctional pore complex, a prokaryotic secretion organelle, is the molecular motor underlying gliding motility in cyanobacteria”. In: Curr. Biol. 8.21 (1998), pp. 1161–1168. doi: 10.1016/s0960-9822(07)00487-3.

[A2] N. Read, S. Connell, and D. G. Adams. “Nanoscale Visualization of a Fibrillar Array in the Cell Wall of Filamentous Cyanobacteria and Its Implications for Gliding Motility”. In: J. Bacteriol. 189.20 (2007), pp. 7361–7366. doi: 10.1128/jb.00706- 07.

[A3] T. Mignot, J. W. Shaevitz, P. L. Hartzell, and D. R. Zusman. “Evidence That Focal Adhesion Complexes Power Bacterial Gliding Motility”. In: Science 315.5813 (2007), pp. 853–856. doi: 10.1126/science.1137223.

[A4] Behzad Khayatan, John C. Meeks, and Douglas D. Risser. “Evidence that a modified type IV pilus-like system powers gliding motility and polysaccharide secretion in filamentous cyanobacteria”. In: Mol. Microbiol. 98.6 (2015), pp. 1021–1036. doi: 10.1111/mmi.13205.

[A5] Tilo Pompe, Martin Kaufmann, Maria Kasimir, Stephanie Johne, Stefan Glorius, Lars Renner, Manfred Bobeth, Wolfgang Pompe, and Carsten Werner. “Friction- controlled traction force in cell adhesion”. In: Biophysical journal 101.8 (2011), pp. 1863–1870.

[A6] Hirofumi Wada, Daisuke Nakane, and Hsuan-Yi Chen. “Bidirectional bacterial gliding motility powered by the collective transport of cell surface proteins”. In: Physical Review Letters 111.24 (2013), p. 248102.

[A7] Jo¨el Tchoufag, Pushpita Ghosh, Connor B Pogue, Beiyan Nan, and Kranthi K Mandadapu. “Mechanisms for bacterial gliding motility on soft substrates”. In: Proceedings of the National Academy of Sciences 116.50 (2019), pp. 25087–25096.

[A8] Chenyi Fei, Sheng Mao, Jing Yan, Ricard Alert, Howard A Stone, Bonnie L Bassler, Ned S Wingreen, and Andrej Kosmrlj. “Nonuniform growth and surface friction determine bacterial biofilm morphology on soft substrates”. In: Proceedings of the National Academy of Sciences 117.14 (2020), pp. 7622–7632.

[A9] Arja Ray, Oscar Lee, Zaw Win, Rachel M Edwards, Patrick W Alford, Deok-Ho Kim, and Paolo P Provenzano. “Anisotropic forces from spatially constrained focal adhesions mediate contact guidance directed cell migration”. In: Nature communications 8.1 (2017), p. 14923.

[A10] Jing Chen and Beiyan Nan. “Flagellar motor transformed: biophysical perspectives of the Myxococcus xanthus gliding mechanism”. In: Frontiers in Microbiology 13 (2022), p. 891694.

[A11] Samia Dhahri, Michel Ramonda, and Christian Marliere. “In-situ determination of the mechanical properties of gliding or non-motile bacteria by atomic force microscopy under physiological conditions without immobilization”. In: PLoS One 8.4 (2013), e61663.

Replacing the key cap [as a means of switching from QWERTZ to QWERTY] isn't going to help at all, it's just a label. You'd have to swap out internal parts too. Depending on the model, you'd either have to remove and swap typebars or remove the head off the typebar and resolder it onto the appropriate alternate (and ensure that it's properly aligned, not an easy task). Then you'd have to swap the key caps (labels). It's definitely a mechanically doable process, but it's probably almost never done in practice. Doing it as a newbie probably isn't recommendable; you're better off having a repair shop do it for you if you decide to go this route. Depending on the keyboard/model, you'd also have to deal with accents, umlauts, etc.

Given the difficulty (or cost) of the process and the potential end results, you're assuredly better off locating a QWERTY machine and paying a bit more for shipping to your area if necessary.

Your mileage may vary depending on model.

reply to u/imprisoningmymemory at https://www.reddit.com/r/typewriters/comments/1cg1avp/replacing_keys/

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

General response of the authors to the editor and the reviewers:

We thank the reviewers for their feedback, input and questions as these have helped us to (hopefully) improve the manuscript. We have rewritten several sections of the manuscript, moved methodological descriptions from the Results to the Methods section, and added imaging data for two cytoskeletal proteins, Shot and Cofilin/Twinstar, which confirm the predicted differential DV expression. Because the changes to the text were extensive, we did not mark them by track changes (the manuscript would have been illegible), but would be happy to provide an additional version that includes the tracked changes.

We provide below the point-by-point response to each question and comment made by the reviewers. Our text is in blue.

__Reviewer #1 __

__Evidence, reproducibility and clarity __

__Summary __

This manuscript investigated changes in the proteome and phosphoproteome during dorsovental axis specification in the Drosophila embryo. To model the three regions in the embryo that are relevant for DV axis development, the authors used specific mutations to enrich for a single type of cells (ventral, lateral, or dorsal). The detected proteins and phosphopeptides were clustered according to the region of expression. There were differences between the protein and corresponding phosphopeptide abundance, suggesting that phosphorylation is a regulatory modification in DV axis establishment. Two different mutations that both result in a ventralized phenotype were found to change marker protein expression in different ways. Using inhibition of microtubule polymerization, this study also investigated the role of microtubules in epithelial folding.

__Major comments __

1. Generally, there is a lack of significance testing throughout the manuscript. Simply reporting fold changes can be misleading, if these changes are not significant. Examples:

2. Rigor of the proteomics evidence showing changes for the expected markers is insufficient because no statistical evaluation is provided. Specifically, in Fig. 1D and Suppl Fig 2: are the fold changes statistically significant?

3. Data in Fig. 4F, 5F need to be assessed for significance. There are other instances in the manuscript where significance should be tested.

We did ANOVA testing for all proteome and phosphoproteome data, and the outcome of these analyses is reported in Supplementary Tables 2 and 3. We have added references to significance throughout, wherever possible and relevant and have included a table that summarizes all p values for all comparisons in all of the figures (Supplementary Table 2). However, note that we do our clustering independent of statistical significance, i.e., we include all values, as we explain in the manuscript.

It is difficult to see the value of the obtained dataset for the community, in part because the data are analyzed by a linear model and cluster assignment developed by the authors, which is a somewhat arbitrary representation. Perhaps the authors could explain how their data could be used by other researchers, and maybe even develop an accessible portal for interacting with the data.

We do provide the entire set of data in a formatted Excel Table as Supplementary Tables 3 and 4, which contain common pairwise comparisons and ANOVA tests that allow a researcher without a strong proteomics background to explore the data, and we also provide the raw proteomics datasets deposited in PRIDE, so any interested colleague can re-analyse them in the manner that suits their purposes best.

We analysed the data in the way we did because it takes account of the knowledge from genetics that we have of all these cell populations. This also allowed us to include the important set of proteins and phosphosites that are completely absent from all but one mutant genotype, and would therefore have dropped out of the statistical analyses.

For example, what does it mean biologically that a protein is a member of a specific cluster shown in Fig. 3C? Is there a predictive value in such an assignment, and how does it relate to the main question of DV axis regulation? An example of a novel insight obtained for specific protein(s) would be useful to illustrate the utility of this analysis.

The clusters represent groups of proteins that are present at higher or lower abundance in subsets of cell populations. So, for example, being present in cluster 5 means (Fig. 3C) that this protein is predicted to be more abundant in the mesoderm than elsewhere (which includes being detected ONLY in the mesoderm, like Snail). This clustering therefore is the way for us to find new proteins that conform to these groups.

We provide here the immunostainings of two cytoskeleton-associated proteins that our proteomic analyses predicted to be more abundant in the ectoderm (Cluster 6: dorsal+lateral):

• The actin-microtubule crosslinker Short-stop (Shot), which is seen to be reduced in the mesoderm.
• The actin-severing protein Cofilin/Twinstar, which was also found downregulated in the mesoderm in the work cited in Ref.:10 Gong L. et al., Development (2004). The staining shows that cofilin-GFP is abundant in the entire subapical region of ectodermal cells, but strongly reduced in ventral furrow cells, where it is only retained in a few apical membrane blebs. These proteins are targets for functional analyses in follow-up work.

[Imaging Data for Reviewers]

Figure: Physical cross-sections of fixed embryos showing the enrichment of proteins in the ectoderm (cluster 6: DL). Dorsal is top, ventral is bottom. Scale bar: 50 um Top panel: Staining for short-stop (shot; cyan / grayscale) and snail (yellow) in embryos expressing gap43-mCherry. Bottom panel: staining for discs large (dlg, magenta) and GFP (green / grayscale) in embryos expressing cofilin-GFP (Kyoto protein trap for Cofilin/Twinstar).

Overall, at present the study appears to have limited novelty and mechanistic insight. The data generally align with prior expectations, but it is unclear how this work advances the field.

We were reassured that the data align with previous studies, but as we state in the text, they go well beyond these valuable and important studies in several dimensions. We had made the following assumptions:

1. DV patterning mutants recapitulate biological qualities of DV cell populations and the differential expression of DV fate determinants, as confirmed in Fig. 1 and Fig. 3D.
2. The differential regulation of the proteomes and phosphoproteomes across DV patterning mutants recapitulates the abundances of proteins and phosphosites within DV cell populations of a wildtype embryo. We confirmed this in Fig. 3A and Fig. 5C with the implementation of a linear model for the abundances of detected proteins and phosphosites. The resulting analysis revealed new avenues for future functional studies, as intended. Most of the work on cell shape regulation at the gastrulation stage has focused on actomyosin and a subset of cell adhesion molecules. We have identified networks of proteins and phosphoproteins that may also control gastrulation (Fig. 6 and Supplementary Fig. 5), including microtubules, which were significantly enriched in networks of phosphoproteins (Fig. 7 and Supplementary Fig. 6).

For example, the observed differences between marker proteins in Toll10B vs. spn27A data seem to confirm previous suggestions that spn27A has a stronger ventralizing effect.

This suggestion was made by colleagues who had unpublished observations on a limited number of gene expression patterns that supported their contention. A correlation analysis (see figure below) of our results now shows that proteins with a restricted dorso-ventral pattern change more in spn27Aex mutants than in Toll10B. If we look at the known mesodermal genes such as Snail, Twist, Mdr49 and CG4500 we find them at higher abundance in spn27Aex than Toll10B , while the ectodermal genes Egr, Zen, Dtg, Tsg, Bsk, and Ptr are reduced more strongly in spn27Aex than in Toll10B. This takes the prior observation of a stronger ventralization of spn27Aex from an anecdotal to a systematic analysis.

[Correlation analyses available for reviewers]

Cross-correlation between the fold changes (FCs) in Toll10B/WT vs. spn27Aex/WT for all proteins detected in wildtype, Toll10B and spn27Aex. Each dot is a protein. The green line is the 'identity' function (slope = 1) that would be expected if the FCs for each protein in both ventralized mutants were exactly the same. A set of proteins with restricted dorso-ventral distribution are highlighted in yellow: mesodermal (ventral) and blue: ectodermal (dorsal).

The role of microtubules in epithelial folding in the embryo has also been demonstrated before.

   The role of microtubules in epithelial folding in the *Drosophila *embryo has indeed been examined in three previous studies that studied dorsal fold formation (Ref.: 35, Takeda et al. NCB 2018), ventral furrow formation (VFF, Ref.: 36, Ko et al. JCB 2019), and salivary gland invagination (Booth et al. Dev Cell 2014). These data reveal diverse and non-conservative functional requirements, ranging from acto-myosin contractility during apical constriction (Booth et al. 2014), force transmission and repair of the supracellular contractile network (but not apical constriction per se, Ko et al 2019), to the generation of expansile forces during cell shape homeostasis (Takeda et al 2018). In light of this potentially broad functional spectrum, we sought to compare three epithelial folds that form within the context of gastrulation: ventral furrow, cephalic furrow and dorsal folds. We confirmed that the initiation of VFF was normal, but the final invagination failed, as per Ko et al. 2019, while dorsal fold initiation did not occur (extending conclusions from Takeda et al 2018). In contrast, cephalic furrow formation, though delayed, did not require microtubules. We also revealed a novel commonality of MT function. Specifically, prior to the initiation of all three epithelial folds, proper nuclear positioning requires MTs. We additionally discovered novel membrane abnormalities in two distinct types of blebs during ventral furrow and dorsal fold formation, respectively. Thus, our data provide insights into the roles of microtubules during epithelial folding that go beyond prior work.

The shown phosphorylation changes (if they are significant) for Toll and Cactus are difficult to explain. In Suppl Fig 2B, E: why is Toll more phosphorylated in the lateralized than in ventralized embryos? (the provided reference 20 does not seem to clarify this).

   These changes are indeed significant (Toll-S871: Vtl vs. WT p = 0.01 , Vsp vs. WT p = 0.002; Cactus-S463: Vsp vs WT p = 0.03); see Supplementary Figure 2B and Supplementary Table 2).

   We have corrected Ref. 20 (Shen B. and Manley J.L., Development 1998). Ref. 20 only shows that Tl is phosphorylated by Pelle (Ref 20: Fig. 6A), although neither the exact position of Tl phosphosite(s) nor the function of Tl phosphorylation were explored in this article. A hallmark of Toll Like Receptor (TLR) regulation is these receptors are subject to tyrosine phosphorylation, which has been widely connected to the regulation of the binding of adaptor proteins to the cytoplasmic tail of TLRs. Both our finding of Serine phosphorylation in Tl, and the differential phosphorylation across cell populations is new, but since we do not know what this particular Serine phosphorylation site does in TLRs in general, we cannot speculate on the meaning of it occurring more in lateral than in ventral cells. In Ref. 20, the authors speculate that Tl phosphorylation by Pelle regulates the association between Tl and Pelle, which then enables Dorsal translocation to the nucleus. It might also be part of a feedback regulation loop, but this is entirely speculative.

Also, certain Cactus phosphorylations appear higher in dorsalized and ventralized embryos, but not in lateralized embryos. Are such changes expected and do they make sense biologically? It is unclear why these phosphorylation data are used to validate the success of the approach.

   The three Cactus phosphosites S463, S467 and S468 were identified and characterised in the work cited in Ref. 19 (Liu Z.P. et al., Genes and Development, 1997), and we used these sites to validate that our approach was sensitive enough to detect known phosphosites in proteins that act on the dorso-ventral patterning pathway specifically at the point of gastrulation (Stage 6 of embryonic development). We also reported in this manuscript the detection of known phosphosites within the Rho-pathway (Fig. 5E,F, Myosin Light Chain: T21, S22; Cofilin: S3).

   Liu Z.P. et al. reported that these three sites map to the Cactus PEST domain, which is required for Cactus degradation in the mesoderm (Belvin M. et al, Genes and Development 1995).  Liu Z.P. et al. also showed that mutating these phosphosites impairs Cactus turnover without affecting the ability of Cactus to bind Dorsal. We can only speculate that the differential phosphorylation across dorso-ventral embryonic cell populations is associated with the regulation of Cactus turnover. Consistent with this, we find Cactus downregulated 1.5 log2 fold in ventralized embryos derived from *spn27Aex/def* mothers. Furthermore, there are a number of signalling pathways that act both in the dorsal and the ventral-lateral domain (e.g., rhomboid/EGF), so it is not surprising to find modifications that are shared by these regions.

The rationale to use a diffusion algorithm for data analysis is not clear. How would the analysis differ if diffusion was not used?

Phosphoproteomics data are often sparse and noisy for a number of reasons (technical; low abundance of phosphorylated peptides compared to other peptides in the cell; biological: not all phosphosites are functional). Network diffusion is a common way used for various data types to boost the signal-to-noise ratio. For example, if from a list of 10 phosphosites, 5 all fall in the same network region or process, and the rest are randomly distributed in the network, chances are that the first region is more representative of the regulated process in that dataset. Using network propagation, the signal coming from the first 5 phosphosites would give a higher score to that network region, marking it as the predominant signal. Our specific implementation, which uses the semantic similarity between nodes to model the edges in the network, further boosts the functional signal by preferentially including nodes that have a higher functional similarity to the initial phosphosites. Our approach therefore allows us to identify the processes that are predominantly ‘active’ in our dataset. We refer the reviewer to our recent preprint for more evidence that this strategy boosts the signal-to-noise ratio in phosphoproteomic datasets and further prioritises more functional phosphosites (https://www.biorxiv.org/content/10.1101/2023.08.07.552249v1). If this approach was not used and we based the identification of relevant processes only on the list of phosphosites, we would have acquired more spurious terms in our functional enrichment analysis. The above preprint also shows that different methods such as the Prize Collecting Steiner Forest algorithm perform worse for phosphoproteomics data.

Generally, the discussion of enriched GO categories presented in Fig. 6 is not rigorous, and it is unclear what biological insight is provided by this figure, probably because the categories are extremely diverse and not clustered in a meaningful way. Despite stating that the work on microtubules came out as a result of proteomic analysis, there is no connection between proteomic data (e.g., data shown in Fig. 6) and microtubule analysis in Fig. 7.

   The connection is between the __phosphoproteomic__ data and the microtubules. The reviewer is correct about the fact there is little connection at the proteomic level with microtubules. Only the diffused network analyses performed on the phosphoproteomic data pointed in this direction. We have improved the writing about this point.

The Discussion section touches on areas of differential protein degradation and mRNA regulation; however, these data are not presented in Results or Figures and so it is difficult to assess the relevance of this analysis.

     We present these data in Figure 6A,B. The network analyses of the clusters showed significant enrichment of cellular component terms that are connected with protein turnover and mRNA regulation. We have added a reference to figure 6 in the Discussion for clarity.

There is insufficient citation of prior literature throughout the manuscript: many statements are lacking proper references.

We have corrected the mistakes and added missing references.

Proteomics data should be deposited into a standard repository that is a member of ProtomeXchange Consortium, such as PRIDE, etc.

All proteomics and phosphoproteomics data have been uploaded to PRIDE:

The raw files for the proteomics and phosphoproteomics experiments were deposited in PRIDE under separate identifiers:

Proteome: Identifier PXD046050 (Reviewer account details: reviewer_pxd046050@ebi.ac.uk, pw: coJ9otiX).

Phosphoproteome: Identifier PXD046192 (Reviewer account details: reviewer_pxd046192@ebi.ac.uk, pw: nvkbwClp).

We have included a statement of raw data availability in the revised version of the manuscript with the PRIDE access information.

__Minor comments __

The text has several typos and should be proof-read, and references to figures and tables should be checked, as some of these are not correct.

We have corrected typos, references to figures and tables in the revised version of the manuscript.

The genotypes for the mutations used in this study should be accompanied by citation describing identification of these mutations and the resulting phenotypes. It would also be helpful to describe the nature of these alleles (molecular lesion, gain vs loss of function, etc.). Some of this information is included in the Discussion, but it would be useful for the reader to learn this early on, when the chosen genotypes are presented.

All this information is and was provided in the methods section and in Table 1, including stock numbers and sources of the stocks. Please see 'Methods, Drosophila genetics and embryo collections'.

2G,H - the X axis should be clearly labeled as logarithmic.

We introduced the log2 label in the X-axis of Fig. 2G,H and any other panel in which this was not expressly made clear.

In Fig. 2G the locations of lines showing fold changes for Twist and Snail seem incorrect. In Fig. 2H the dotted line does not appear to correspond to 50% of the number of phosphosites.

We apologise for these errors, both have been corrected in the revised version of the manuscript.

5D can be improved by adding letters for the coloured clusters.

We have labelled the clusters in Fig. 3B and Fig. 5D. to ease the identification of biologically relevant clusters.

It is unclear if any specific additional insight was obtained using SILAC, the authors may want to discuss this approach and outcomes more.

SILAC has been widely used to deal with the inherent variability of proteomic analyses by introducing a standard that is metabolically labelled, in our case, w1118 flies fed with SILAC yeast were used as the standard. Because the inherent variability is larger in phosphoproteomic experiments (because protein identification is based on phosphorylated peptides only, see Methods), we used SILAC labelling only in the phosphoproteomic experiment.

__Reviewer #2 __

Evidence, reproducibility and clarity

The present article by Gomez et al describes a deep proteomics analysis of the proteome and phosphoproteome of embryos mutated for key genes involved in the dorso-ventral axis in Drosophila melanogaster. Overall, this is a nice article showing new insight in this development process. The results are mainly descriptive, yet identifies potential new players in the definition of the dorso-ventral axis.

The generation of mutants for genes found up- or down-regulated in each mutant strain would be a significant addition to this manuscript. But I think in its current form the data brings enough new information on this particular developmental step and would be of interest for the fly community.

My main concern is that the manuscript can be difficult to read and overly convoluted at times even for experts in the field. I would suggest the author move some methodological explanations from the results to the methods section to further detail the goals of some results sections.

We have followed these suggestions and hope we have made the manuscript more easily readable.

As an example, the goal of part 3) « A linear model for quantitative interpretation of the proteomes » is not clear to me. Are the authors comparing the abundance of a protein in the WT versus a theoretical WT in order to determine which fractions of mesoderm, lateral ectoderm and dorsal region are actually present in the WT? (...)

Yes, in part, but the main purpose was to compare how well the theoretical WT, as ‘reconstituted’ from the mutants, corresponds to the observed actual WT (for which we have at least approximate values).

The question that we faced when we started these calculations was: what is the ‘correct’ fraction (or proportion) we should use to weight each protein (or phosphosite) measurement in the mutants. Theoretically, these values should be those that result in the best match between the theoretical WT and the measured WT abundance of each protein (or phosphosite). We knew from actual measurements only the mesodermal fraction, which was determined to be ~20% of the cross-sectional area (Ref. 21: Rahimi, N., et al Dev. Cell. 2016). The neuroectoderm and ectoderm fractions were estimated to be approx. 40% each (Ref.: 22, Jazwinska, A et al. Development 1999), but we lacked an exact number. The systematic exploration of these proportions led us to conclude that indeed both the neuroectoderm and ectoderm fractions should be around 40% each, provided the mesoderm is fixed at 20%. Thus, we used these fractions: D: 0.4 L: 0.4 V: 0.2 for our follow-up analyses.

(...) Or are they using it as a reference to obtain a fold change for the different proteins quantified (in this case why not use the WT?)?

yes, again, in part: as a reference for the EXPECTED fold changes, as would be predicted from the WT.

Since we have moved some of the details of this approach from the main text to the methods section, we have also revised the remaining text and hope it is now clearer.

The proteomics data must be deposited in a public repository. I did not see it stated in the methods section.

All proteomics and phosphoproteomics data have been uploaded to PRIDE; see further comments above in response 13.

The version of the uniprot database is quite old (2016) so is the version of MaxQuant used in this study. Any reasons for that (other than that the analysis was performed in 2016)?

That is indeed the reason.

The data were run on different MS platforms, how did the authors account for the variability in MS signals? What samples were run on which MS platform? Were the WT embryos ran on both?

We measured three replicates, and all five genotypes (four mutant genotypes plus wildtype) for each of the replicates were measured on the same instrument. Specifically, for the whole proteome analyses, replicate one and three of all genotypes were measured on the QExactive Plus instrument and replicate 2 of all genotypes were measured on a QExactive HF-x instrument, as were the phosphoproteomes. So, indeed, the wildtype was measured on both instruments. We thus did not observe instrument-specific bias in the PCA analysis for the proteome data.

We have added this in more detail to the method section:

“Samples of replicate one and three were measured on the QE-Plus system and replicate two was measured on the QE-HF-x system.

For phosphoproteome analysis, (…) Samples of all three replicates were measured on the QEx-HFx system. We added trial samples measured on the QEx-Plus system to increase the phosphosite coverage using the match between runs algorithm.”

In the methods section the authors mention that a high-pH reverse phase fractionation was performed? How many fractions of High-pH reverse phase separation were injected per sample? Was this separation performed for all the samples?

We have adjusted the Methods section regarding the high-pH fractionation by adding the following sentence: “Fractions were collected every 60s in a 96 well plate over 60 min gradient time collecting a total number of 8 fractions per sample.“

Why did the authors used label-free (proteome) and SILAC (phosphoproteome) quantification methods?

See our response to reviewer #1, point 19.

Why is the threshold based on the Q3 of the standard deviation (if I got it right) ? Couldn't they be calculated directly on the distribution of the ratio?

We could also have done it that way.

However, we had wanted also to take into account the variation between the replicates, i.e., the quality of the individual measurements, and we therefore devised the procedure we used, by which the standard deviation of the individual technical replicates enters the calculation with the ratio of the averages, the variability between replicates would have been ignored and we considered it more appropriate to take the more conservative approach. But as it turns out, the cut-off would have ended up being very similar had we calculated it the way the referee suggests,

Page 6: The supplementary figure 2E refers to the protein Cactus and the text to CKII, please modify one or the other to avoid any confusion. Page 7: A dot is missing at the end of the following sentence « if used with the assumed weightings for the populations »

We have corrected these sentences.

Page 19: Replace SppedVac by SpeedVac

We have corrected the error in the manuscript and thank the reviewer for the detailed inspection.

Page 8: why not using a z-score with thresholds directly instead of a -1/+1/0 system and then using the z-score?

Because we wanted to compare the relative changes over wt between mutants (i.e. the similarity between 1 0 0 and 0 -1 -1) rather than the relationship of their absolute values to the wt, and to assign proteins with similar relationships into the same dorso-ventral regulation categories.

The text states this (previously in main text, now in methods):

“The reason for this is that this method takes into account that value sets that represent similar relative differences between the mutants (for example, 0 -1 -1 vs. 1 -1 -1 or 1, 0, 0) are biologically more similar to each other than the raw values indicate. The z-scores for all of these cases would be 1.1547 -0.5774 -0.5774.”

In the abstract it is mentioned that 3,399 proteins are differentially regulated at the proteome level versus 1,699 significantly deregulated at a 10 % FDR in the main text (page 5). Is there a reason for this discrepancy? Same comment for the phosphopeptides.

But we now also see the need to better clarify this point, and we have edited the text accordingly.

The second number refers to those proteins that show statistically significant changes based on ANOVA (1699 proteins).

The first number (3398; note that the number 3399 in the abstract was a typo, now corrected) includes all proteins that were detected in at least 1 replicate in the wildtype (5883/6111) minus those that do not change between the genotypes (2156/6111) and minus all those that change in the same direction in all mutants (329).

This includes proteins that are automatically excluded from ANOVA, i.e., those that are detected only in the wildtype (35/6111 proteins) or in two or more genotypes but only in 1 technical replicate ANOVA negative ones.

As we stated, we did this because it “allows us to include the important group of proteins that show a ‘perfect’ behaviour, like dMyc and WntD, in that they are undetectable in the mutants that correspond to the regions in the normal embryo where these genes are not expressed.”. This 'regulated' set consists of those proteins that exceed the |0.5| fold threshold.

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

This review is a list of many individual critiques. It is unclear what the expertise of the reviewer is (they do not provide the answer to that question in the review form, unlike the other referees), but several of the criticisms are unfounded. Three of the PIs of this work are researchers with extensive experience in Drosophila genetics and early development but are nevertheless confounded by some of the comments made by this referee.

The mutants do not completely "flatten" the embryos.

We do not claim that they do. Nor are the ventral, lateral and dorsal regions in the normal embryo completely ‘flat’ or homogeneous. But the mutants are good representations of the major fates in these regions, as a wealth of published literature from the last 30 years indicates.

For instance, Tl10B broadly expresses snail but also expresses sog in the head. (i.e. Fig 1B - sog and sna expression in Figure 1B mutant backgrounds looks odd.) The sog expression likely relates to a deficiency specific effect.

This ‘sensitive’ area is well known also from other genetic conditions – e.g. partial loss of dorsal and indeed in Spn27A mutants. It is therefore not specific to the Tl10B deficiency but says something about gene interactions in this region. Thus, this cannot be a deficiency-specific effect.

Is sog seen in a Toll10B/+ mutant background?

Yes, it is, and more frequently than in Toll10B/Def.

The deficiency used for the Toll10B experiment is Df(3R)ro80b which is quite large and deletes 14+ genes.

True. However, this does not matter: the mothers are heterozygous, so the genes are not missing, they are present in one wildtype copy! And these mothers are then mated with wildtype fathers, so if expression of these genes were needed in the embryo, then there would be another full wt copy of each. We appreciate that maternal effect genetics can be difficult to follow, but this is all work that has been done a long time ago, and is not the point of this paper at all.

The deficiency used for the spn27A experiment is Df(2L)BSC7 and removes 4+ genes.

Again, this would only matter if these were maternal effect genes that were needed for the establishment of the dorso-ventral axis, and they are not.

Furthermore, the gd9 allele may not be a complete loss of function.

It may not be – but what matters is the well characterized phenotype which has been shown to represent dorsal cell types.

It is possible that the Toll10B allele picked up an accessory dominant mutation.

This again would only matter if it was a dominant AND maternal effect mutation that affects the DV axis in the embryo – and there are very few of these known. And nothing in our analysis of these embryos, with which we have been working on and off over 3 decades and therefore know very well, indicates that our current stock is any different from those we have seen in the past.

Unfortunately, these mutant phenotypes that affect DV and AP patterning mean that conclusions cannot be made that changes in protein relate to DV patterning.

We simply do not understand this statement.

Why do the mutant phenotypes (gene expression patterns and cell morphologies representative of the ventral, lateral and dorsal cell populations) not mean that the proteins downstream of the fate changes correspond to the cell fates?

To get a better view of the ventralized phenotype, the authors should repeat the analysis by ectopically expressing Toll10B using the Gal4-UAS system; UAS-activate Toll transgenes are available.

All Gal4-UAS maternal drivers, even the best and the strongest, result in mosaic expression. Our lab has extensive experience with this system and we know that, for example, the homogeneous, high levels of twist or snail expression that we see in spn or Tl10B embryos cannot be achieved with GAL4.

Fig 1C-F - due to combined AP and DV effects seen with ventralizing mutants, it is important that the authors confirm that cross-section views relate to the middle to posterior of the embryo.

We confirm this.

Costaining with anti-Kr or -Caudal would help to ensure they are assaying the correct AP domain for pure DV effects.

In our view, this is an unnecessary experiment. I know where the middle of the embryo is. If the reviewer does not believe when we say we are showing a section from the middle, they can see that the sections are not from the end region by, for example, the cell number, and the section angles.

The authors refer to reference [60] for stages but there is no information regarding morphological criteria used under the microscope to stage the embryos.

We have now added more detail in the methods section:

Briefly. using a Zeiss binocular, the embryos were individually hand-selected on wet agar which made the embryos semi-transparent, allowing the assessment of a range of morphological features, of which at least some are visible in each of the mutants:

• Yolk distance to embryonic surface: distinguishes between early (stage 5a) and late cellularisation (stage 5b).
• Yolk distribution within the embryo: identification of large embryonic movements of the germ band (e.g.: Initiation of germ band extension, marking the initiation of stage 7). In DV patterning mutants this is seen as twisting of the embryo.
• Change in the outline of the dorsal-posterior region: polar cell movement from the posterior most region of the embryo (stage 5a/b) to stage 6a/b.
• Formation of the cephalic and dorsal folds: identification of stage 6 (initiation of cephalic fold) and stage 7 (dorsal folds). The combined use of these morphological criteria, together with the synchronised egg collections allows accurate staging of wild type and mutant embryos.

Furthermore, what is stage 6a,b? Stage 6 is not typically divided in two stages nor is it clear what a,b relate to.

We used a generally accepted standard for staging embryos: Campos-Ortega J.A. and Hartenstein V. ‘The embryonic development of Drosophila melanogaster’ book (ref. Nº 60). In this book, they describe the morphological criteria that can be followed in living embryos for proper staging. These stages, with these exact names, are shown on pages 11 and 12 of the 1997 edition (2nd edition).

According to the published timetable of Drosophila development by Foe et al. 1993 (not cited), gastrulating embryos are 200 min or 3 hr 20'. It's unclear if this is the stage that was assayed.

Foe is a beautiful paper, but we did not cite it because the commonly used nomenclature predates it (Campos-Ortega and Hartenstein 1985).

In addition, timing depends on temperature whereas morphological criteria do not.

The mutant embryos likely develop at different rates relative to wildtype. It seems important to provide details about the staging of embryos. If the mutant embryos take longer to gastrulate, for instance, might that also be a factor that impacts the proteome.

As described above, we used a combination of criteria to accurately judge staging. DV patterning embryos could in principle develop faster or slower than wildtype. We performed synchronised egg collections (Methods: Embryo collections) for 15’. Therefore, any developmental timing defect would have become evident based on a difference in the number of embryos entering stage 6 and 7 at the point of visual inspection of the collections. This was not the case.

How many replicates for each genotype? In the text it states, "replicates from the same genotype clustered together (Fig. 2E)....." Similar vague reference for phosphoproteome follows (Fig 2F). It is then stated that it was impossible to determine the experimental source for this variation. Could it relate to differences in timing of samples?

We had given the numbers of replicates in the figure legend but have now also included them in the methods section for more clarity. We did 3 replicates for each genotype in each experiment, with the exception of gd9 and spn27aex mutants, for which we did 2 biological replicates each with 3 replicates, making a total of 6 replicates for these genotypes in the proteomic experiment. We have included an additional clarification in figure legend 2. The number of replicates per genotype per experiment can also be seen from the correlation matrices shown Fig. 2E and 2F, in which the replicates are shown individually. The measurements for each replicate for each genotype within each experiment were reported in Supplementary Tables 2 and 3, 'description' tabs of the worksheets.

The lengthy discussion of ratio estimation on page 7 should be streamlined and made more clear. Are the authors throwing out data and only keeping samples that support their model? This seems like overfitting - if I am understanding correctly, you are selecting the samples that support the "majority of proteins fit the linear model" but this isn't necessarily the case.

No, this is a misunderstanding. We do not select data.

We have rephrased this section, but to explain here briefly: We do not select any samples, we state that the majority of proteins fit the theoretical model (and that is not even surprising, because any protein that does not change across the populations will automatically fit the model). We then discuss why some might NOT fit the model. The model doesn’t need to be supported, it simply is a calculation that allows us to stratify the data.

They call this the 'correct' manner (see section 4 page 7) but it seems like a working model and presumptuous to imply that it is the correct way.

We explained in the text why we refer to this as ‘correct’. It is a matter or definition, not presumption, and we even used quotes to be clear about this. ’Correct’ indicates a combination of values that is consistent with the biological model that the DV mutants are good representations of the corresponding embryonic cell populations in a wild type embryo. We do not in any way ‘throw out’ other data, we just note they don’t fit that model. Clarifications on the concept for the model have been added in various places in the text

Figure 3C - it is confusing to use a circular diagram to show DV inferred position of the 14 clusters as their position on the circle does not correspond to where they are expressed on the embryos. Perhaps a stacked bar graph for 6 different domains would be better.

This figure does not show positions of clusters. It is simply a pie chart, as is stated in the figure legend and as can be seen by the numbers and the corresponding sizes of the sectors. We have tried a stacked representation (shown below), but find it no clearer and have therefore stuck with this very common way of representing quantities, and in particular, proportions. We use the same representation with the same colour schemes in all subsequent figures, so proportions can be compared across figures.

It is very hard to follow the text on page 9.

We have rephrased this section

It is very hard to see the gene expression patterns shown in Fig 4A with the color scheme/scale used.

We appreciate this colour scheme does not correspond to the commonly used dark colour on a light background which would mimic histochemistry to show gene expression. The ‘inferno’ colour scheme was used because it allows better quantitative comparisons between subtly different patterns. However, to make these figures more similar to the types of in situ hybridisations that embryologists are used to seeing, we now use a different representation.

In general, Figure 4 is uninterpretable - in particular, what do the numbers mean on the greyscale circle plots in panel D?

We apologize for having failed to explicitly include the explanation for this in the figure legend. The reader will notice that these numbers add up to the number in the circle to the left, and the numbers indicate the number of proteins showing perfect matches (white), partial overlaps (grey) and mismatches (black). We have improved the graphic representation and added an explanation in the figure legend.

Figure 5A. Why wasn't protein abundance and phosphosites identified from an individual, identical sample?

This was because of the way the project developed over the course of the research, and the protein part was originally intended only as a proof of concept, with the intended focus being the phosphoproteome. We later decided to include a full analysis of the proteome, but did not consider it worthwhile and necessary to repeat the entire laborious and expensive experiment with both analyses being done from the same samples.

How can one be sure that the phosphosites were correctly assigned if the proteins were not detected in the proteome but they were only identified in the phosphosite analysis?

We are not sure we understand this question. The phosphoproteomic analysis identifies phosphopeptides of proteins that in turn allow one to identify the protein itself and the amino acid in that peptide that is phosphorylated. So the identification is done only WITHIN the phosphoproteomic analysis and does not relate directly to the proteomic analysis. This explains why we found some phosphopeptides for which we did not detect the full host protein in the proteomic analysis.

Thus, if a protein was detected only in either of the experiments, this fact doesn’t modify the validity of the result, because the identification was done individually for each experiment.

Page 16 - much discussion about the difference between Spn27A and Toll10b/def mutant background. One has half as much Toll receptor. The phenotype of Toll10b/+ should be examined.

Both genotypes have been extensively examined in the past. Tl10B/def has only one copy of the gene from the mother, and the mutant protein is constitutively active. By putting it over a deficiency, we (and others in the past) made sure that the exclusive source for Tl signalling is from this gain of function Tl allele, and that the wildtype receptor, which would still be activated by the natural ligand in a graded pattern along the DV axis, does not confound the result.

The Tl10B/+ combination creates a less ventralized phenotype which is not more similar to that of spn27Aex/def but in fact less similar.

Page 12 - hard to follow the discussion of modeling (?) presented in Figure 6. The results (bottom of page 12 - #1 "most networks are enriched for cellular components associated with regulation of gene expression" and page 13 #2 - "cytoskleeton emerges as a major target of regulation") seem vague and unsubstantiated. Rhabdomere, P granule, micropyle, autophagosome?

We agree with the reviewer that there are many cellular components that are enriched in the diffused network analyses, many of them unrelated to morphogenesis. We had highlighted this finding on page 12, paragraph 3. Nevertheless, we have rephrased the statements as ‘the heat maps illustrate that most of the enriched cellular components in both experiments were highly enriched with cellular components associated with DNA and RNA metabolism or the regulation of gene expression.’ and have now included numbers.

We think ‘a major target’ for phosphorylation does in fact apply to the cytoskeleton, and we had already supplied the number to substantiate this in the manuscript (14/62).

Readers will be able to evaluate these network analyses based on their own fields of interest or particular questions they may wish to address. We haven’t excluded any cellular component terms.

Figure 7 seems like a separate study.

Why were the phosphopeptides investigated to determine if they relate to phosphorylated proteins? Phosphoantibodies could have been generated for a subset. Instead the manuscript pivots to analysis of microtubules.

We are reporting here one example of a proof-of-concept study that we carried out, chosen based on our own research interests and on available tools and reagents. There are clearly many other avenues that could have been explored and that others may want to explore, but that go well beyond this report. We have made this more explicit in the text.

Page 14 - discussion first paragraph. Please cite ref[10] when discussing the "previous study" otherwise the reader will not understand which study you are referring to until the next paragraph.

We have moved the reference from its current position to the one suggested by the reviewer.

• In general, the study would benefit from more attention to references and citations of prior work. A comparison of this work to the Gong et al. Development 2004 study should be made earlier. This work is cited very early on, namely in the introduction.

• The authors start off saying that no other study has looked at proteins from a spatial perspective. We are unsure what the reviewer refers to. We say precisely the opposite: we indicate that studies have been performed to look at differences in cell populations, including that by the lab of Jon Minden (Gong et al), a highly respected former co-author of one of the current authors (ML). We do state that the technologies at the time did not allow the same depth and temporal resolution as the methods that are available nowadays. For instance, Gong et al. used an excellent and original approach at the time, which however did not detect Snail and Twist in the ventralized mutants.

The only time we say ‘no other study’ is about ‘region-specific post-translational regulation of proteins’ - though we do state in the discussion that Gong et al would have detected some of these cases because they used 2D gels.

• Along these lines, there is another more recent proteomic study from Beati et al. Fly 2020 using similarly staged embryos. How do these other experiments compare to the current ones? As they apparently analyzed proteome and phosphopeptides from an identical sample, are the authors' new data using separate samples consistent? This study is actually about a later stage (stage 8 embryos, post-gastrulation). Again, an excellent study, but not directly relevant to our current analysis. It validates the use of SILAC in Drosophila, although it is not the first study to do this. Furthermore, it looks at a different question and biological process using a mutant, htl, to understand the effect of FGF signalling.

• Furthermore, Adam Martin's lab has been studying microtubule action along the dorsoventral axis (Denk-Lobnig et al 2021) and this work is not cited. Denk-Lobnig et al 2021 is about spatial patterns of myosin and actin and how that is governed genetically on the ventral side of the embryo, pertaining primarily to ventral furrow formation. It does not analyse microtubules nor dorsal-ventral cell populations.

It is possible there may be some confusion with another excellent study from Adam Martin’s lab, in which the role of microtubules is analysed. But this is exclusively in the ventral furrow, and the study did not look at the effect of microtubule depolymerisation on nuclear positioning nor membrane behaviour. We cite this work extensively (Ref.: 36, Ko et al. JCB 2019) and we compare our results to that paper. However, our work here goes beyond this study in that it looks at all cells along the DV axis.

General comments:

Typos throughout. For example, page .4 section heading "dorso-ventral cell..."

We have scanned the entire document for typos.

Font size extremely small - for example see Figure 1A gene names, and 1F magnified view.

We have adjusted the fonts in the main figures.

Scale bars not shown when showing magnified views. For example, see Fig 1E,

We have added these.

Reviewer #3 (Significance (Required)): This study by Gomez et al. uses a proteomic-centered approach to study proteomes associated with cell populations in the embryo that they argue relate to different positions along the dorso-ventral axis. They generate a proteomic resource, though it was unclear how anyone could use the data they produce. There is no searchable database and we have to trust that the authors will ultimately provide such a resource to the community.

All proteomics and phosphoproteomics data have been uploaded to PRIDE. Also see responses to the other referees’ queries about this point.

There is the potential for interesting insights but the work is not presented in a way that is accessible or useful. The presentation needs significant improvement.

We have improved the presentation and way the results are presented as per the suggestion of all reviewers.

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 mutants do not completely "flatten" the embryos. For instance, Tl10B broadly expresses snail but also expresses sog in the head. (i.e. Fig 1B - sog and sna expression in Figure 1B mutant backgrounds looks odd.) The sog expression likely relates to a deficiency specific effect. Is sog seen in a Toll10B/+ mutant background? The deficiency used for the Toll10B experiment is Df(3R)ro80b which is quite large and deletes 14+ genes. The deficiency used for the spn27A experiment is Df(2L)BSC7 and removes 4+ genes. Furthermore, the gd9 allele may not be a complete loss of function. It is possible that the Toll10B allele picked up an accessory dominant mutation. Unfortunately, these mutant phenotypes that affect DV and AP patterning mean that conclusions cannot be made that changes in protein relate to DV patterning. To get a better view of the ventralized phenotype, the authors should repeat the analysis by ectopically expressing Toll10B using the Gal4-UAS system; UAS-activate Toll transgenes are available.

• Fig 1C-F - due to combined AP and DV effects seen with ventralizing mutants, it is important that the authors confirm that cross-section views relate to the middle to posterior of the embryo. Costaining with anti-Kr or -Caudal would help to ensure they are assaying the correct AP domain for pure DV effects.

• The authors refer to reference [60] for stages but there is no information regarding morphological criteria used under the microscope to stage the embryos. Furthermore, what is stage 6a,b? Stage 6 is not typically divided in two stages nor is it clear what a,b relate to. According to the published timetable of Drosophila development by Foe et al. 1993 (not cited), gastrulating embryos are 200 min or 3 hr 20'. It's unclear if this is the stage that was assayed.

• The mutant embryos likely develop at different rates relative to wildtype. It seems important to provide details about the staging of embryos. If the mutant embryos take longer to gastrulate, for instance, might that also be a factor that impacts the proteome.

• How many replicates for each genotype? In the text it states, "replicates from the same genotype clustered together (Fig. 2E)....." Similar vague reference for phosphoproteome follows (Fig 2F). It is then stated that it was impossible to determine the experimental source for this variation. Could it relate to differences in timing of samples?

• The lengthy discussion of ratio estimation on page 7 should be streamlined and made more clear. Are the authors throwing out data and only keeping samples that support their model? This seems like overfitting - if I am understanding correctly, you are selecting the samples that support the "majority of proteins fit the linear model" but this isn't necessarily the case. They call this the 'correct' manner (see section 4 page 7) but it seems like a working model and presumptuous to imply that it is the correct way.

• Figure 3C - it is confusing to use a circular diagram to show DV inferred position of the 14 clusters as their position on the circle does not correspond to where they are expressed on the embryos. Perhaps a stacked bar graph for 6 different domains would be better.

• It is very hard to follow the text on page 9.

• It is very hard to see the gene expression patterns shown in Fig 4A with the color scheme/scale used.

• In general, Figure 4 is uninterpretable - in particular, what do the numbers mean on the greyscale circle plots in panel D?

• Figure 5A. Why wasn't protein abundance and phosphosites identified from an individual, identical sample? How can one be sure that the phosphosites were correctly assigned if the proteins were not detected in the proteome but they were only identified in the phosphosite analysis?

• Page 16 - much discussion about the difference between Spn27A and Toll10b/def mutant background. One has half as much Toll receptor. The phenotype of Toll10b/+ should be examined.

• Page 12 - hard to follow the discussion of modeling (?) presented in Figure 6. The results (bottom of page 12 - #1 "most networks are enriched for cellular components associated with regulation of gene expression" and page 13 #2 - "cytoskleeton emerges as a major target of regulation" ) seem vague and unsubstantiated. Rhabdomere, P granule, micropyle, autophagosome?

• Figure 7 seems like a separate study. Why were the phosphopeptides investigated to determine if they relate to phosphorylated proteins? Phosphoantibodies could have been generated for a subset. Instead the manuscript pivots to analysis of microtubules.

• Page 14 - discussion first paragraph. Please cite ref[10] when discussing the "previous study" otherwise the reader will not understand which study you are referring to until the next paragraph. In general, the study would benefit from more attention to references and citations of prior work. A comparison of this work to the Gong et al. Development 2004 study should be made earlier. The authors start off saying that no other study has looked at proteins from a spatial perspective - but this other study from 2004 did just that. They compared ventralized to lateralized embryos. Along these lines, there is another more recent proteomic study from Beati et al. Fly 2020 using similarly staged embryos. How do these other experiments compare to the current ones? As they apparently analyzed proteome and phosphopeptides from an identical sample, are the authors' new data using separate samples consistent?

General comments:

1. Typos throughout. For example, page .4 section heading "dorso-ventral cell..."

2. Font size extremely small - for example see Figure 1A gene names, and 1F magnified view.

3. Scale bars not shown when showing magnified views. For example, see Fig 1E,F

Significance

This study by Gomez et al. uses a proteomic-centered approach to study proteomes associated with cell populations in the embryo that they argue relate to different positions along the dorso-ventral axis. They generate a proteomic resource, though it was unclear how anyone could use the data they produce. There is no searchable database and we have to trust that the authors will ultimately provide such a resource to the community. Furthermore, Adam Martin's lab has been studying microtubule action along the dorsoventral axis (Denk-Lobnig et al 2021) and this work is not cited. There is the potential for interesting insights but the work is not presented in a way that is accessible or useful. The presentation needs significant improvement.

author mentions this as the locking in of rotting google search.

The start of Google search decreasing effectiveness

OK WHATS WITH THE ORANGE BLOSSOMS

oo cassandra fits this

urghh please don't betray her

NAH WHAT

NAH WHAT

Do me a favor and read this book: https://www.penguinrandomhouse.com/books/237251/what-we-see-when-we-read-by-peter-mendelsund/

Author response:

Reviewer #1 (Public Review):

Given that this is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C, the manuscript would benefit from placing these findings in the context of what has been reported in other populations, for example, how decay rates of intact and defective genomes compare with that of other subtypes where known.

Most published studies are from men living with HIV-1 subtype B and the studies are not from the hyperacute infection phase and therefore a direct head-to-head comparison with the FRESH study is difficult. However, we can cite/highlight and contrast our study with a few examples from other acute infection studies as follows.

(1) Peluso et. al., JCI, 2020, showed that in Caucasian men (SCOPE study), with subtype B infection, initiating ART during chronic infection virus intact genomes decayed at a rate of 15.7% per year, while defective genomes decayed at a rate of 4% per year. In our study we showed that in chronic treated participants genomes decreased at a rate of 25% (intact) and 3% (defective) per month for the first 6 months of treatment.

(2) White et. al., PNAS, 2021, demonstrated that in a cohort of African, white and mixed-race American men treated during acute infection, the rate of decay of intact viral genomes in the first phase of decay was <0.3 logs copies in the first 2-3 weeks following ART initiation. In the FRESH cohort our data from acute treated participants shows a comparable decay rate of 0.31 log copies per month for virus intact genomes.

(3) A study in Thailand (Leyre et. al., 2020, Science Translational Medicine), of predominantly HIV-1 CRF01-AE subtype compared HIV-reservoir levels in participants starting ART at the earliest stages of acute HIV infection (in the RV254/SEARCH 010 cohort) and participants initiating ART during chronic infection (in SEARCH 011 and RV304/SEARCH 013 cohorts). In keeping with our study, they showed that the frequency of infected cells with integrated HIV DNA remained stable in participants who initiated ART during chronic infection, while there was a sharp decay in these infected cells in all acutely treated individuals during the first 12 weeks of therapy. Rates of decay were not provided and therefore a direct comparison with our data from the FRESH cohort is not possible.

(4) A study by Bruner et. al., Nat. Med. 2016, described the composition of proviral populations in acute treated (within 100 days) and chronic treated (>180 days), predominantly male subtype B cohort. In comparison to the FRESH chronic treated group, they showed that in chronic treated infection 98% (87% in FRESH) of viral genomes were defective, 80% (60% in FRESH) had large internal deletions and 14% (31% in FRESH) were hypermutated. In acute treated 93% (48% in FRESH) were defective and 35% (7%) in FRESH were hypermutated. The differences frequency of hypermutations could be explained by the differences in timing of infection specifically in the acute treated groups were FRESH participants initiate ART at a median of 1 day after infection. It is also possible that sex- or race-based differences in immunological factors that impact the reservoir may play a role.

This study also showed that large deletions are non-random and occur at hotspots in the HIV-1 genome. The design of the subtype B IPDA assay (Bruner et. al., Nature, 2019) is based on optimal discrimination between intact and deleted sequences - obtained with a 5′ amplicon in the Ψ region and a 3′ amplicon in Envelope. This suggest that Envelope is a hotspot for large while deletions in Ψ is the site of frequent small deletions and is included in larger 5′ deletions. In the FRESH cohort of HIV-1 subtype C, genome deletions were most frequently observed between Integrase and Envelope relative to Gag (p<0.0001–0.001).

(5) In 2017, Heiner et. al., in Cell Rep, also described genetic characteristics of the latent HIV-1 reservoir in 3 acute treated and 3 chronic treated male study participants with subtype B HIV. Their data was similar to Bruner et. al. above showing proportions of intact proviruses in participants who initiated therapy during acute/early infection at 6% (94% defective) and chronic infection at 3% (97% defective). In contrast the frequencies in FRESH in acute treated were 52% intact and 48% defective and in chronic infection were 13% intact and 87% defective. These differences could be attributed to the timing of treatment initiation where in the aforementioned study early treatment ranged from 0.6-3.4 months after infection.

Indeed, in the abstract, the authors indicate that treatment was initiated before the peak. The use of the term 'peak' viremia in the hyperacute-treated group could perhaps be replaced with 'highest recorded viral load'. The statistical comparison of this measure in the two groups is perhaps more relevant with regards to viral burden over time or area under the curve viral load as these are previously reported as correlates of reservoir size.

We will edit the manuscript text to describe the term peak viraemia in hyperacute treated participants more clearly. We will perform an analysis of area under the curve to compare viral burden in the two study groups.

Reviewer #2 (Public Review):

Other factors also deserve consideration and include age, and environment (e.g. other comorbidities and coinfections.)

We agree that these factors could play a role however participants in this study were of similar age (18-23), and information on co-morbidities and coinfections are not known.

Reviewer #3 (Public Review):

The word reservoir should not be used to describe proviral DNA soon after ART initiation. It is generally agreed upon that there is still HIV DNA from actively infected cells (phase 1 & 2 decay of RNA) during the first 6-12 months of ART. Only after a full year of uninterrupted ART is it really safe to label intact proviral HIV DNA as an approximation of the reservoir. This should be amended throughout.

We agree and will amend the use of the word reservoir to only refer to the proviral DNA load after full viral suppression, i.e., during undetectable viral load.

All raw, individualized data should be made available for modelers and statisticians. It would be very nice to see the RNA and DNA data presented in a supplementary figure by an individual to get a better grasp of intra-host kinetics.

We will make all relevant data available and accessible to interested parties.

The legend of Supplementary Figure 2 should list when samples were taken.

The data in this figure represents an overall analysis of all sequences available for each participant at all time points. This will be explained more clearly in the manuscript and added to the figure legend.

At this line, Eurydice drops her head, turns around, and leaves the room. She moves slowly, almost wandering out of the room. The chorus watches her with a confused face as the messenger finishes his monologue. He does not notice her leaving until he hears the door bang shut.

The welfare policies were built to support men and center around men. It seems as if women can never in the eyes of the law be the head of a household without a man, reinforcing the need to marry in order to be economically independent while caring for children.

Apple supposedly slashed production for their 3k5 USD ski goggles, a sign they may soon be joining the long list at https://en.wikipedia.org/wiki/List_of_virtual_reality_headsets of attempts that all don't solve the key thing: adding 1kg of ski goggles to your head and shove it between yourself and the world is only acceptable in a very limited set of contexts and uses, and not a mainstream thing.

eLife assessment

The authors perform voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. They suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, namely theta and ripples. However, evidence for the potentially useful findings is currently incomplete due to major weaknesses in the experimental and analytical approach.

Reviewer #2 (Public Review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased-locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single-cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population-level activity in CA1.

Weaknesses:

The evidence provided is weak, with the authors making surprising population-level claims based on a very sparse data set (5 data sets, each with less than 20 neurons simultaneously recorded) acquired with exciting, but less tested technology. Further, while the authors link these observations to the novelty of the context, both in the title and text, they do not include data from subsequent visits to support this. Detailed comments are below:

(1) My first question for the authors, which is not addressed in the discussion, is why these events have not been observed in the countless extracellular recording experiments conducted in rodent CA1 during the exploration of novel environments. Those data sets often have 10x the neurons simultaneously recording compared to these present data, thus the highly synchronous firing should be very hard to miss. Ideally, the authors could confirm their claims via the analysis of publicly available electrophysiology data sets. Further, the claim of high extra-SWR synchrony is complicated by the observation that their recorded neurons fail to spike during the limited number of SWRs recorded during behavior- again, not agreeing with much of the previous electrophysiological recordings.

(2) The authors posit that these events are linked to the novelty of the context, both in the text, as well as in the title and abstract. However, they do not include any imaging data from subsequent days to demonstrate the failure to see this synchrony in a familiar environment. If these data are available it would strengthen the proposed link to novelty if they were included.

(3) In the discussion the authors begin by speculating the theta present during these synchronous events may be slower type II or attentional theta. This can be supported by demonstrating a frequency shift in the theta recording during these events/immobility versus the theta recording during movement.

(4) The authors mention in the discussion that they image deep-layer PCs in CA1, however, this is not mentioned in the text or methods. They should include data, such as imaging of a slice of a brain post-recording with immunohistochemistry for a layer-specific gene to support this.

Reviewer #3 (Public Review):

Summary:

In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected in the other side of the brain, and the investigation is flawed due to multiple problems with the point process analyses. The synchrony terminology refers to dozens of milliseconds as opposed to the millisecond timescale referred to in prior work, and the interpretations do not take into account theta phase locking as a simple alternative explanation.

Weaknesses:

The two main messages of the manuscript indicated in the title are not supported by the data. The title gives two messages that relate to CA1 pyramidal neurons in behaving head-fixed mice: (1) synchronous ensembles are associated with theta (2) synchronous ensembles are not associated with ripples.

There are two main methodological problems with the work: (1) experimentally, the theta and ripple signals were recorded using electrophysiology from the opposite hemisphere to the one in which the spiking was monitored. However, both signals exhibit profound differences as a function of location: theta phase changes with the precise location along the proximo-distal and dorso-ventral axes, and importantly, even reverses with depth. And ripples are often a local phenomenon - independent ripples occur within a fraction of a millimeter within the same hemisphere, let alone different hemispheres. Ripples are very sensitive to the precise depth - 100 micrometers up or down, and only a positive deflection/sharp wave is evident. (2) The analysis of the point process data (spike trains) is entirely flawed. There are many technical issues: complex spikes ("bursts") are not accounted for; differences in spike counts between the various conditions ("locomotion" and "immobility") are not accounted for; the pooling of multiple CCGs assumes independence, whereas even conditional independence cannot be assumed; etc.

Beyond those methodological issues, there are two main interpretational problems: (1) the "synchronous ensembles" may be completely consistent with phase locking to the intracellular theta (as even shown by the authors themselves in some of the supplementary figures). (2) The definition of "synchrony" in the present work is very loose and refers to timescales of 20-30 ms. In previous literature that relates to synchrony of point processes, the timescales discussed are 1-2 ms, and longer timescales are referred to as the "baseline" which is actually removed (using smoothing, jittering, etc.).

An instance of where Gordo is assimilating to his family’s culture of calling people by their nicknames instead of their actual name. On page 62 where Gordo’s father gave the nickname “Head and Shoulders” to Primi. Gordo wanting to fit in not only with his family but also with his father, on the next page we see Gordo calling Primi, “Head and Shoulders”.

新的Tokio调度器只需要为每个生成的任务分配一次内存，而旧版本需要两次。以前，任务结构（Task struct）看起来像这样：

```rust struct Task { /// 管理任务所需的所有状态 state: TaskState,

/// 以未来特性对象表示的运行逻辑
future: Box<dyn Future<Output = ()>>,

} `` 然后，Task 结构也会被分配在一个 Box 中。这一直是一个问题，我长期以来一直想解决（我最初在2014年尝试解决这个问题）。自旧的Tokio调度器以来，有两个变化。首先，std::alloc` 稳定了。其次，Future任务系统转向了显式虚拟表（vtable）策略。这是最终消除每个任务双重分配低效的两个缺失部分。

现在，任务结构表示为：

rust struct Task<T> { header: Header, future: T, trailer: Trailer, } Header 和 Trailer 都是推动任务所需的状态，但它们被分为“热”数据（header）和“冷”数据（trailer），即大致上是经常访问的数据和很少使用的数据。热数据被放在结构的头部，并尽可能保持小尺寸。当 CPU 解引用任务指针时，它将一次性加载一个缓存行大小的数据（在64到128字节之间）。我们希望这些数据尽可能相关。

这样的设计减少了内存分配的次数，同时优化了数据的局部性，提高了缓存效率，从而提升了任务的处理性能。这种结构也更加灵活，允许在不牺牲性能的前提下，更自由地定义任务的存储和管理方式。

这里的 mask 实际在现在的代码里已经被移除了，因为 cap 是固定的常数，直接通过 cap 就能计算出 ringbuffer 溢出位的位移量。也就是 mask = cap - 1 = 255

mask 在 ringbuffer 的实现中很常见，当我们即将写入数组的最后一个元素时，下一次写入应该在数组前开始。如果每次都通过条件语句来判断就会不够高效，引入 mask 后每次push 尾idx + 1，每次 pop 头 idx +1，实际访问数组时将当前 idx 和 mask 做 mod 即可得到合法的 idx 位置。 这里用与运算代替 mod 也是常见做法，因为 mod 值的二机制位全为 1（mask = 255）。

Too many historical inaccuracies here... universities always had an economic function. Generalisations doing my head in a bit...

Author response:

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

We thank the reviewers for their thoughtful comments. We were pleased that they thought our study was "well crafted and written", "important", and that it provides a "valuable resource for researchers studying color vision". They also expressed several constructive criticisms, concerning – among other things – the lack of details regarding experimental procedures and analysis, the challenge in relating retinal data to cortical recordings, and consistency of results across animals. In response to the reviewers’ comments and following their suggestions, we performed additional analyses, and substantially revised the paper:

We added a section in the Discussion about "Limitations of the stimulus paradigm". In addition, we added a new Suppl. Figure that illustrates the effect of deconvolution of calcium traces on our results and clarified in the text why we use deconvolved signals for all analyses. The new Suppl. Figure also shows an additional analysis with a more conservative threshold of neuron exclusion.

We now clarify how retinal signaling relates to our cortical results and rewrote the text to be more conservative regarding our conclusions.

In addition, we added a new Suppl. Figure showing the key analyses from Figures 2 and 4 separately for each animal. We now mention consistency across animals in the Results section and clearly state which analyses were performed an data pooled across animals.

We are positive that these additions address the issues raised by the reviewers. Please find our point-by-point replies to all comments below.

eLife assessment

Franke et al. explore and characterize the color response properties in the mouse primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The data is solid; however, the evidence supporting some conclusions and details about some procedures are incomplete. In its current form, the paper makes a useful contribution to how color is coded in mouse V1. Significance would be enhanced with some additional analyses and resolution of some technical issues.

We thank the reviewers for appreciating our manuscript and their thoughtful comments.

Referee 1 (Remarks to the Author):

Summary:

In this study, Franke et al. explore and characterize the color response properties across the primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The authors use awake-behaving 2P imaging to define the spectral response properties of visual interneurons in Layer 2/3. They find that opponent responses are more prominent at photopic light levels, and diversity in color opponent responses exists across the visual science, with green ON/ UV OFF responses being stronger represented in the upper visual field. This is argued to be relevant for detecting certain features that are more salient when the chromatic space is used, possibly due to noise reductions.

Strengths:

The work is well crafted and written and provides a thorough characterization that reveals an uncharacterized diversity of visual properties in V1. I find this characterization important because it reveals how strongly chromatic information can modulate the response properties in V1. In the upper visual field, 25% of the cells differentially relay chromatic information, and one may wonder how this information will be integrated and subsequently used to aid vision beyond the detection of color per see. I personally like the last paragraph of the discussion that highlights this fact.

We thank the reviewer for appreciating our manuscript.

Weaknesses: One major point highlighted in this paper is the fact that Green ON/UV OFF responses are not generated in the retina. But glancing through the literature, I saw this is not necessarily true. Fig 1. of Joesch and Meister, a paper cited, shows this can be the case. Thus, I would not emphasize that this wasn’t present in the retina. This is a minor point, but even if the retina could not generate these signals, I would be surprised if the diversity of responses would only arise through feed-forward excitation, given the intricacies of cortical connectivity. Thus, I would argue that the argument holds for most of the responses seen in V1; they need to be further processed by cortical circuitries.

We thank the reviewer for this comment. When analyzing available data from the retina using a similar center-surround color flicker stimulus (Szatko et al. 2020), we found that Green On/UV Off color opponency is very rare in the RF center of retinal ganglion cells (Suppl. Fig. 5). This suggests that center Green On/UV Off color opponency in V1 neurons is not inherited by the RF center of retinal neurons. However, we agree with the reviewer that retinal neurons might still contribute to V1 color opponency, for example by being center-surround color opponent (e.g. Joesch et al. 2016 and Szatko et al. 2020). We rephrased the text to acknowledge this fact.

This takes me to my second point, defining center and surround. The center spot is 37.5 deg of visual angle, more than 1 mm of the retinal surface. That means that all retinal cells, at least half and most likely all of their surrounds will also be activated. Although 37.5 deg is roughly the receptive field size previously determined for V1 neurons, the one-to-one comparison with retinal recording, particularly with their center/surround properties, is difficult. This should be discussed. I assume that the authors tried a similar approach with sparse or dense checker white noise stimuli. If so, it would be interesting if there were better ways of defining the properties of V1 neurons on their complex/simple receptive field properties to define how much of their responses are due to an activation of the true "center" or a coactivation of the surround. Interestingly, at least some of the cells (Fig. 1d, cells 2 and 5) don’t have a surround. Could it be that in these cases, the "center" and "surround" are being excited together? How different would the overall statistics change if one used a full-filed flicker stimulus instead of a center/surround stimulus? How stable are the results if the center/surround flicker stimulus is shifted? These results won’t change the fact that chromatic coding is present in the VC and that there are clear differences depending on their position, but it might change the interpretation. Thus, I would encourage you to test these differences and discuss them.

Thanks for this comment. We agree with the reviewer that a one-to-one comparison of retina and V1 data is challenging, due to differences in both RF and stimulus size. We rephrased the Results text to clarify this point and now also mention it in the Discussion.

To be able to record from many V1 neurons simultaneously, we used a stimulus size of 37.5 degree visual angle in diameter, which is slightly larger than center RFs of single V1 neurons. As the reviewer mentions, the disadvantage of this approach is that the stimulus is only roughly centered on the neurons’ center RFs. To reduce the impact of potential stimulus misalignment on our results, we used the following steps:

For each recording, we positioned the monitor such that the mean RF across all neurons lies within the center of the stimulus field of view.

We confirmed that this procedure results in good stimulus alignment for the large majority of recorded neurons within individual recording fields by using a sparse noise stimulus (Suppl. Fig. 1a-c). Specifically, we found that for 83% of tested neurons, more than two thirds of their center RF, determined by the sparse noise stimulus, overlapped with the center spot of the color noise stimulus.

For analysis, we excluded neurons without a significant center STA, which may be caused by misalignment of the stimulus.

Together, we believe these points strongly suggest that the center spot and the surround annulus of the noise stimulus predominantly drive center (i.e. classical RF) and surround (i.e. extraclassical RF), respectively, of the recorded V1 neurons. This is further supported by the fact that color response types identified using an automated clustering method were robust across mice (Suppl. Fig. 6c), indicating consistent stimulus centering.

Nevertheless, we cannot exclude that the stimulus was misaligned for a subset of the recorded neurons used for analysis. We agree with the reviewer that such misalignment might have contributed to cells not having surround STAs, due to simultaneous activation of antagonistic center and surround RF components by the surround stimulus. While a full-field stimulus would get rid of the misalignment problem, it would not allow to study color tuning in center and surround RF components separately. Instead, one could compare the results of our approach with an approach that centers the stimulus on individual neurons. However, we believe that performing these additional experiments is out of the scope of the current study.

To acknowledge the experimental limitations of our study and the concerns brought up by the reviewer, we now explicitly mention the steps we perform to reduce the effects of stimulus misalignment in the Results section and discuss the problem of stimulus alignment in the Discussion. We believe these changes will help the reader to interpret our results.

Referee 2 (Remarks to the Author):

Summary:

Franke et al. characterize the representation of color in the primary visual cortex of mice and how it changes across the visual field, with a particular focus on how this may influence the ability to detect aerial predators. Using calcium imaging in awake, head-fixed mice, they characterize the properties of V1 neurons (layer 2/3) using a large center-surround stimulation where green and ultra-violet were presented in random combinations. Using a clustering approach, a set of functional cell-types were identified based on their preference to different combinations of green and UV in their center and surround. These functional types were demonstrated to have varying spatial distributions in V1, including one neuronal type (Green-ON/UV-OFF) that was much more prominent in the posterior V1 (i.e. upper visual field). Modelling work suggests that these neurons likely support the detection of predator-like objects in the sky.

Strengths:

The large-scale single-cell resolution imaging used in this work allows the authors to map the responses of individual neurons across large regions of the visual cortex. Combining this large dataset with clustering analysis enabled the authors to group V1 neurons into distinct functional cell types and demonstrate their relative distribution in the upper and lower visual fields. Modelling work demonstrated the different capacity of each functional type to detect objects in the sky, providing insight into the ethological relevance of color opponent neurons in V1.

We thank the reviewer for appreciating our manuscript.

Weaknesses:

While the study presents solid evidence a few weaknesses exist, including the size of the dataset, clarity regarding details of data included in each step of the analysis and discussion of caveats of the work. The results presented here are based on recordings of 3 mice. While the number of neurons recorded is reasonably large (n > 3000) an analysis that tests for consistency across animals is missing. Related to this, it is unclear how many neurons at each stage of the analysis come from the 3 different mice (except for Suppl. Fig 4).

Thank you for this comment. We apologize that the original manuscript did not clearly indicate the consistency of our results across animals. We have revised the manuscript in the following ways:

We have added an additional Suppl. Figure, which shows the variability of the data within and across animals (Suppl. Fig. 4). Specifically, we show the distribution of color and luminance selectivity for (i) center and surround components of V1 RFs and (ii) for upper and lower visual field. This data is used for all analyses shown in Figures 2-4. The figure legend of this figure also states the number of neurons per animal.

We now clearly state in the Results section that all analyses in the main figures were performed by pooling data across animals, and refer to the Suppl. Figures for consistency across animals.

We believe these changes help the reader to interpret our results.

Finally, the paper would greatly benefit from a more in depth discussion of the caveats related to the conclusion drawn at each stage of the analysis. This is particularly relevant regarding the caveats related to using spike triggered averages to assess the response preferences of ON-OFF neurons, and the conclusions drawn about the contribution of retinal color opponency.

Thanks. We substantially revised the text to discuss caveats and limitations of the approach. For example, we added a section into the Discussion called "Limitations of the stimulus paradigm". In addition, we clarified how retinal signals relate to cortical ones and phrased our conclusions more conservatively.

The authors provide solid evidence to support an asymmetric distribution of color opponent cells in V1 and a reduced color contrast representation in lower light levels. Some statements would benefit from more direct evidence such as the integration of upstream visual signals for color opponency in V1.

Based on the comments from Reviewer 1, we have rephrased the statements regarding the integration of upstream visual signals for color opponency in V1. We think these revisions increase the clarity of the results and help the reader with interpretation.

Overall, this study will be a valuable resource for researchers studying color vision, cortical processing, and the processing of ethologically relevant information. It provides a useful basis for future work on the origin of color opponency in V1 and its ethological relevance.

Thanks! We thank the reviewer again for the helpful comments.

Referee 3 (Remarks to the Author):

This paper studies chromatic coding in mouse primary visual cortex. Calcium responses of a large collection of cells are measured in response to a simple spot stimulus. These responses are used to estimate chromatic tuning properties - specifically sensitivity to UV and green stimuli presented in a large central spot or a larger still surrounding region. Cells are divided based on their responses to these stimuli into luminance or chromatic sensitive groups. Several technical concerns limit how clearly the data support the conclusions. If these issues can be fixed, the paper would make a valuable contribution to how color is coded in mouse V1.

We thank the reviewer for the helpful comments.

Analysis: The central tool used to analyze the data is a "spike triggered average" of the responses to randomly varying stimuli. There are several steps in this analysis that are not documented, and hence evaluating how well it works is difficult. Central to this is that the paper does not measure spikes. Instead, measured calcium traces are converted to estimated spike rates, which are then used to estimate STAs. There are no raw calcium traces shown, and the approach to estimate spike rates is not described in any detail. Confirming the accuracy of these steps is essential for a reader to be able to evaluate the paper. Further, it is not clear why the linear filters connecting the recorded calcium traces and the stimulus cannot be estimated directly, without the intermediate step of estimating spike rates.

Thank you for this comment. We have used the genetically encoded calcium sensor GCaMP6s in our recordings. This sensor is a very sensitive GCaMP6 variant, but also one with slow kinetics. To remove the effect of the slow sensor kinetics from recorded calcium responses, the recorded traces are commonly deconvolved with the impulse function of the sensor to obtain the deconvolved calcium traces. We now include this reasoning in the Results section. To illustrate the effect of the deconvolution, we added a new Suppl. Figure (Suppl. Fig. 2) showing raw calcium and deconvolved traces, and the STAs estimated from both types of traces. This illustrates that the results regarding neuronal color preferences are consistent across raw and deconvolved calcium traces.

We agree with the reviewer that the term STA might be confusing. We have replaced it with the term "even-triggered-average" (ETA). In addition, we have replaced the phrase "estimated spike rate" with "deconvolved calcium trace" throughout the manuscript because the unit of the deconvolved traces is not interpretable, like spike rate would be (spikes per second). In the revised version, we now clarify in the Methods section that we estimate the ETAs based on deconvolved calcium traces, which is correlated with and an approximation for spike rate.

A further issue about the STAs is that the inclusion criterion (correlation of predicted vs measured responses of 0.25) is pretty forgiving. It would be helpful to see a distribution of those correlation values, and some control analyses to check whether the STA is providing a sufficiently accurate measure to support the results (e.g. do the central results hold for the cells with the highest correlations).

We thank the reviewer for this comment. To exclude noisy neurons from analysis, we used the following procedure:

For each of the four stimulus conditions (center and surround for green and UV stimuli), kernel quality was measured by comparing the variance of the STA with the variance of the baseline, defined as the first 500 ms of the STA. Only cells with at least 10-times more variance of the kernel compared to baseline for UV or green center STA were considered for further analysis.

We have added the distribution of quality values to a new Suppl. Figure (Suppl. Fig. 2d,e). We now also show the percentage of neurons above threshold, given different quality thresholds. Finally, we have repeated the analysis shown in Figure 2 for a much more conservative threshold, including only the top 25% of neurons (Suppl. Fig. 2e,f). We now mention this new analysis in the Methods and Results section.

Limitations of stimulus choice: The paper relies on responses to a large (37.5 degree diameter) modulated spot and surrounding region. This spot is considerably larger than the receptive fields of both V1 cells and retinal ganglion cells. As a result, the spot itself is very likely to strongly activate both center and surround mechanisms, and responses of cells are likely to depend on where the receptive fields are located within the spot (and, e.g., how much of the true neural surround samples the center spot vs the surround region). The impact of these issues on the conclusions is considered briefly at the start of the results but needs to be evaluated in considerably more detail. This is particularly true for retinal ganglion cells given the size of their receptive fields (see also next point).

We agree with the reviewer that the centering of the stimulus is critical and apologize if this point was not discussed sufficiently. To be able to record from many V1 neurons simultaneously, we used a stimulus size of 37.5 degree visual angle in diameter, which is slightly larger than center RFs of single V1 neurons. As the reviewer mentions, the disadvantage of this approach is that the stimulus is only roughly centered on the neurons’ center RFs. To reduce the impact of potential stimulus misalignment on our results, we have used different experimental and analysis steps and controls (see also second comment of Reviewer 1):

For each recording, we positioned the monitor such that the mean RF across all neurons lies within the center of the stimulus field of view.

We confirmed that this procedure results in good stimulus alignment for the large majority of recorded neurons within individual recording fields by using a sparse noise stimulus (Suppl. Fig. 1a-c). Specifically, we found that for 83% of tested neurons, more than two thirds of their center RF, determined by the sparse noise stimulus, overlapped with the center spot of the color noise stimulus.

For analysis, we excluded neurons without a significant center STA, which may be caused by misalignment of the stimulus.

We now mention those clearly in the Results section and added the limitations of our approach to the Discussion section.

Comparison with retina: A key conclusion of the paper is that the chromatic tuning in V1 is not inherited from retinal ganglion cells. This conclusion comes from comparing chromatic tuning in a previously-collected data set from retina with the present results. But the retina recordings were made using a considerably smaller spot, and hence it is not clear that the comparison made in the paper is accurate. This issue may be handled by the analysis presented in the paper, but if so it needs to be described more clearly. The paper from which the retina data is taken argues that rod-cone chromatic opponency originates largely in the outer retina. This mechanism would be expected to be shared across retinal outputs. Thus it is not clear how the Green-On/UV-Off vs Green-Off/UV-On asymmetry could originate. This should be discussed.

We agree with the reviewer that a one-to-one comparison of retina and V1 data is challenging, due to differences in both RF and stimulus size. We rephrased the Results text to clarify this point and now also mention it in the Discussion.

When analyzing available data from the retina using a similar center-surround color flicker stimulus (Szatko et al. 2020), we found that Green On/UV Off color opponency is very rare in the RF center of retinal ganglion cells (Suppl. Fig. 5). This suggests that center Green On/UV Off color opponency in V1 neurons is not inherited by the RF center of retinal neurons. However, we agree with the reviewer that retinal neurons might still contribute to V1 color opponency, for example by being center-surround color opponent (e.g. Joesch et al. 2016 and Szatko et al. 2020). We rephrased the text to acknowledge this fact.

Residual chromatic cells at low mesopic light levels The presence of chromatically tuned cells at the lowest light level probed is surprising. The authors describe these conditions as rod-dominated, in which case chromatic tuning should not be possible. This again is discussed only briefly. It either reflects the presence of an unexpected pathway that amplifies weak cone signals under low mesopic conditions such that they can create spectral opponency or something amiss in the calibrations or analysis. Data collected at still lower light levels would help resolve this.

Thank you for this comment. We call the lowest light level "low mesopic" and "rod-dominated" because the spectral contrast of V1 center responses in posterior recording fields is green-shifted for this light level (Fig. 3a). This is only expected if responses in the UV-cone dominant ventral retina are predominantly driven by rod photoreceptors. We now explain this rationale in the Results section. In addition, we mention in the Discussion that future studies are required to test whether cone signals need to be amplified for low light levels. While we agree with the reviewer that it would be exciting to use even lower light levels during recordings, we believe this is out of the scope of the current study due to the technical challenges involved in achieving scotopic stimulation.

Reviewer #2 (Public Review):

Summary:

Franke et al. characterize the representation of color in the primary visual cortex of mice, highlighting how this changes across the visual field. Using calcium imaging in awake, head-fixed mice, they characterize the properties of V1 neurons (layer 2/3) using a large center-surround stimulation where green and ultra-violet colors were presented in random combinations. Clustering of responses revealed a set of functional cell-types based on their preference to different combinations of green and UV in their center and surround. These functional types were demonstrated to have different spatial distributions across V1, including one neuronal type (Green-ON/UV-OFF) that was much more prominent in the posterior V1 (i.e. upper visual field). Modelling work suggests that these neurons likely support the detection of predator-like objects in the sky.

Strengths:

The large-scale single-cell resolution imaging used in this work allows the authors to map the responses of individual neurons across large regions of the visual cortex. Combining this large dataset with clustering analysis enabled the authors to group V1 neurons into distinct functional cell types and demonstrate their relative distribution in the upper and lower visual fields. Modelling work demonstrated the different capacity of each functional type to detect objects in the sky, providing insight into the ethological relevance of color opponent neurons in V1.

Weaknesses:

While the study presents convincing evidence about the asymmetric distribution of color-opponent neurons in V1, the paper would greatly benefit from a more in-depth discussion of the caveats related to the conclusions drawn about their origin. This is particularly relevant regarding the conclusion drawn about the contribution of color opponent neurons in the retina. The mismatch between retinal color opponency and V1 color opponency could imply that this feature is not solely inherited from the retina, however, there are other plausible explanations that are not discussed here. Direct evidence for this statement remains weak.

In addition, the paper would benefit from adding explicit neuron counts or percentages to the quadrants of each of the density plots in Figures 2-5. The variance explained by the principal components does not capture the percentage of color opponent cells. Additionally, there appear to be some remaining errors in the figure legend and labels that have not been addressed (e.g. '??' in Fig 2 legend).

Overall, this study will be a valuable resource for researchers studying color vision, cortical processing, and the processing of ethologically relevant information. It provides a useful basis for future work on the origin of color opponency in V1 and its ethological relevance.

Its so true!

Very invocative wording

Reviewer #1 (Public Review):

Summary:

In this manuscript by Thronlow Lamson et al., the authors develop a "beads-on-a-string" or BOAS strategy to link diverse hemagglutinin head domains, to elicit broadly protective antibody responses. The authors are able to generate varying formulations and lengths of the BOAS and immunization of mice shows induction of antibodies against a broad range of influenza subtypes. However, several major concerns are raised, including the stability of the BOAS, that only 3 mice were used for most immunization experiments, and that important controls and analyses related to how the BOAS alone, and not the inclusion of diverse heads, impacts humoral immunity.

Strengths:

Vaccine strategy is new and exciting.

Analyses were performed to support conclusions and improve paper quality.

Weaknesses:

Controls for how different hemagglutinin heads impact immunity versus the multivalency of the BOAS.

Only 3 mice were used for most experiments.

There were limited details on size exclusion data.

Reviewer #2 (Public Review):

Summary:

The authors describe a "beads-on-a-string" (BOAS) immunogen, where they link, using a non-flexible glycine linker, up to eight distinct hemagglutinin (HA) head domains from circulating and non-circulating influenzas and assess their immunogenicity. They also display some of their immunogens on ferritin NP and compare the immunogenicity. They conclude that this new platform can be useful to elicit robust immune responses to multiple influenza subtypes using one immunogen and that it can also be used for other viral proteins.

Strengths:

The paper is clearly written. While the use of flexible linkers has been used many times, this particular approach (linking different HA subtypes in the same construct resembling adding beads on a string, as the authors describe their display platform) is novel and could be of interest.

Weaknesses:

The authors did not compare to individuals HA ionized as cocktails and did not compare to other mosaic NP published earlier. It is thus difficult to assess how their BOAS compare.

Other weaknesses include the rationale as to why these subtypes were chosen and also an explanation of why there are different sizes of the HA1 construct (apart from expression). Have the authors tried other lengths? Have they expressed all of them as FL HA1?

This relates directly back to ECE expulsion rates for (especially) young Black boys. And how expensive expansive programs can cost - children of color with disabilities are also much more likely to be low-income and so a class analysis of this is also important when we think about increasing access. Head Start programs can be WONDERFUL resources for families - but looking at the Creative Curriculum, the level of burnout and turnover amongst staff, they are so much less able to truly be inquiry-based and emergent (not to mention the waitlists...)

Good remark of syntactic vs semantic intent preservation.

Semantics are in the head of a person, that conveys them as syntactic ops. I.e., semantics get specified down to ops.

Merging syntactically may not always preserve semantics. I.e., one wants to "make defs easier to read by converting them to CamelCase", another wants the same but via snake-case. Having merged them syntactically, we get Camel-Snake-Case-Hybrid, which does not preserve any semantic intent. The semantics intent here are not conflict-free in the first case, though.

Make defs readable | | as CamelCase as Snake Case | | modify to CC modify to SC They diverged at this point, even before getting to syntactic changes.

The best solution would be to solve original problem in a different way - let defs be user-specific. But that's blue sky thinking. Although done in Unison, we do have syntactic systems around.

So staying in a syntactic land, the best we could do is to capture the original intent: "Make defs readable".

Then we need a smart agent, human or an AI, specify it further.

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

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Reply to the reviewers

Revision Plan

Manuscript number: RC-2024-02385

Corresponding author(s): Jennifer R. Kowalski

1. General Statements [optional]

Our manuscript describes a novel role for the conserved glycoprotein hormone receptor, FSHR-1, in regulating C. elegans neuromuscular function through an inter-tissue, gut-brain signaling pathway. FSHR-1 is the sole C. elegans homolog of a family of vertebrate glycoprotein receptors that includes FSHR, TSHR, and LHR, and has previously been shown to regulate body size, germline differentiation, lipid homeostasis, and various stress responses in the worm (Kenis at al 2023; Cho et al 2007; Torzone et al 2023; Powell et al 2009; Miller et al 2015; Robinson and Powell 2016; Wei and Kowalski, 2018; Kim and Sieburth 2020; Wang et al 2023) but its role in neuromuscular regulation, although identified in a 2005 RNA interference screen (Sieburth et al 2005), has not been previously explored. Here, through a combination of genetic, behavioral, and fluorescence imaging approaches, we demonstrate that FSHR-1 is both necessary and sufficient in the intestine of the worm (and may also act in several other distal tissues, including glia and head neurons) to promote muscle excitation through effects on active zone protein localization and synaptic vesicle release from cholinergic motor neurons. Additionally, we identify the FSHR-1 ligands, glycoproteins GPLA-1 and GPLB-1, as well as several known downstream effectors of FSHR-1 in other contexts, GSA-1/GalphaS, ACY-1/adenylyl cyclase, and the lipid kinase SPHK-1, as interactors in the FSHR-1 pathway for neuromuscular control. This work represents a detailed description of the ability of this conserved and multi-functional receptor in inter-tissue coordination that may ultimately be connected to its functions in other physiological processes, such as the stress response, and may also prove relevant for understanding roles for FSHR-1 homologs in humans.

We greatly appreciate the thoughtful and constructive feedback provided by each of the three reviewers of this manuscript. We are pleased that all three reviewers noted the novelty of the mechanisms of cross-tissue regulation of neuromuscular function by FSHR-1 that we uncovered. Reviewer 1 comments, "They demonstrate a novel phenomenon of cross-tissue regulation by restoring FSHR-1 in neurons, intestines, or glia to restore NMJ function." Reviewer 2 echoes this sentiment, also noting, "The data is well presented, compelling and the conclusions are well supported by the data. . .. [T]his study provides a solid foundation to address many interesting questions regarding the role of fshr-1 signaling in regulating neuronal function." Reviewer 3 adds "This is a highly worthy contribution to the field of cell non-autonomous signaling and neuromodulation, and specifically synaptic transmission modulation. The study deepens and enhances the understanding of fshr-1 function within the C. elegans intestine and adds in several molecular components into the signaling pathway, acting both upstream and downstream. . . While this work relies on an invertebrate system of C. elegans, all components have vertebrate counterparts, so findings are likely of broader interest."

As described below, we are working to address many of the comments made by the reviewers and have already made some of the suggested minor changes to the manuscript. We are hopeful that, given the reviewers' excitement about this work, the changes we have already made, and the additional revisions we intend to make in the coming months, including the completion of several new experiments we propose in the revision plan below, our manuscript will be of interest to a broad genetics audience.

2. Description of the planned revisions

Planned Revisions based on comments from Reviewer #1

• __The authors found that expressing FSHR-1 in intestinal cells was sufficient to compensate for the fshr-1 mutation phenotype, suggesting that intestinal cell FSHR-1 can regulate neuromuscular junction (NMJ) function across tissues. However, the molecular mechanism remains unexplored. Since the downstream signaling pathways of FSHR-1 are clear, analyzing the gain-of-function (gf) mutations of gsa-1 and acy-1 in different tissues can help elucidate the signaling pathways transmitted across tissues. __ We completely agree that tissue-specific pathway analysis is important for understanding the molecular mechanism underlying the ability of FSHR-1 to control neuromuscular function from its location in distal tissues, like the intestine. Because of the complexity of these questions and the time required for us to generate strains to perform tissue-specific protein depletion or overexpression experiments, we intend these studies to be the focus of a future manuscript However, in lieu of performing a full suite of tissue-specific analyses of FSHR-1 downstream components, we will perform intestine-specific RNA interference experiments (as we did for fshr-1 in Figure 4B) of gsa-1, acy-1, and sphk-1 in wild type worms and in animals overexpressing fshr-1 in the intestine (which causes increased swimming behavior, Figure 3A) to determine if these downstream players are required for the effects of intestinal fshr-1 on the NMJ. __ __We appreciate the reviewer's suggestion to address these important questions regarding the site of action of the downstream players.

• The images of neurons should be presented in higher resolution and magnification to provide clearer visualization. __ We appreciate the reviewer's request for increased visualization of the neurons; however, because the current larger, lower resolution images show several release sites and were used for the quantitative analyses we present, we would like to keep the images as they are. __However, *we will provide higher resolution insets for the images in Figures 2A, 2C-F, and 4C, as requested. *

• It is unclear whether the glycoprotein subunit orthologs act in the intestine to regulate NMJ function with FSHR-1. This should be investigated and clarified in the manuscript. __ We fully agree that determining where and how the glycoproteins GPLA-1 and GPLB-1 interact with FSHR-1 - and if this is happening at the level of the intestine - is an important outstanding question. Based on prior work, it is known that these subunits are not expressed in intestinal cells, but they are found in several gut-associated neurons and tissues. Specifically, gpla-1 is expressed in neurons of the gastrointestinal tract, including M1, M5, I5 and NSM pharyngeal motor neurons, as well the AVL and DVB excitatory motor neurons that control defecation contractions in the hindgut. gplb-1 is also expressed in the DVB neuron, as well as in non-neuronal tissues (head mesodermal cells and the hindgut enteric muscles), and both glycoprotein genes show reporter expression in the RME motor neurons in the head (Kenis et al 2023). We will complete experiments testing whether the effects of intestinal FSHR-1 overexpression require the ligands, as suggested by Reviewer #2. __We intend that our future work will explore the glycoprotein-FSHR-1 interactions more deeply in a variety of contexts.

• __In Figure 4C, there are no error bars, and individual values should be shown in all statistical analyses to provide a complete representation of the data and its variability. __ We again thank the reviewer for catching this error in Figure 4C. We have replaced the graph with the complete one that includes error bars. We will replace the graphs in 1B, 1C, 3D, 4A, 4C, 5A, 5B, and 6E, as well as Supplemental Figure 5A, 5B, 6A, 6B, 7A, and 7C with bars overlaid with the individual data points. We are unable to do this for Figures 2A-F or Supplemental Figures 2A-C, 7B or 7D because these analyses were run using Custom-written Igor software (Burbea et al 2002) that does not provide individual values, only mean values and cumulative probability plots of the datasets. We recently showed consistency between the Igor analysis program and the newer Fiji plug-in we used for our more recent imaging data, supporting concordance of results despite not having the individual data points in Igor (Hulsey-Vincent et al 2023).

Planned Revisions based on comments from Reviewer #2

• __Fig 4B: An intestinal site of action seems likely for fshr-1 and is nicely supported by the intestine-specific RNAi experiment in Fig 4B. Does intestine-specific knockdown of fshr-1 also cause the aldicarb and SNB-1 defects seen in the mutant? Including this data especially for the synaptic markers would strengthen the gut to neuron inter-tissue signaling model that is proposed here (OPTIONAL). __ We appreciate the reviewer's suggestion to include additional intestine-specific knockdown data for the aldicarb, SNB-1::GFP, and other imaging data. We have the reagents to perform the intestine-specific knockdown of fshr-1 in the aldicarb assay and will complete these experiments as part of our revision plan. Although performing the same experiments in the imaging strains requires first crossing each imaging line to the intestine-specific RNAi line, which may may prove challenging, we are currently working to cross the intestinal RNAi line with nuIs152, the cholinergic SNB-1::GFP line and, assuming the cross goes well, will include results in our revised manuscript.

• Fig 5A: The authors show that G alpha s and adenylyl cyclase function downstream of fshr-1, but it is unclear whether these are direct fshr-1 effectors or whether they function less directly. Does expressing gsa-1(gf) or acy-1(gf) transgenes specifically in the intestine (or neurons) suppress the fshr-1 defects? (OPTIONAL) __ As stated in our response to Reviewer #1, we completely agree that tissue-specific pathway analysis is important for understanding the molecular mechanism underlying the ability of FSHR-1 to control neuromuscular function from its location in distal tissues, like the intestine. While the complexity of these questions and the time required for us to generate strains to perform tissue-specific protein depletion or overexpression experiments is likely more than is suitable for the revision time frame of this manuscript (and will be the focus of future work), in lieu of these experiments we will perform intestine-specific RNA interference experiments (as we did for fshr-1 in Figure 4B) of gsa-1, acy-1, and sphk-1 in wild type worms and in animals overexpressing fshr-1 in the intestine (which causes increased swimming behavior, Figure 3A) to determine if these downstream players are required for the effects of intestinal fshr-1 on the NMJ. __We appreciate the reviewer's suggestion to address these important questions regarding the site of action of the downstream players.

• __Fig 6A-D: The authors propose that fshr-1 is activated by its ligands for locomotion, but no evidence is presented to support this. This could be experimentally addressed with the reagents that are used in this study by determining whether the increased locomotion caused by overexpressing fshr-1 in the intestine (reported in Fig 3A), is dependent upon gpla-1 and/or gplb-1 activity. This experiment would help to distinguish whether gpla-1 and/or gplb-1 indeed are fshr-1 ligands or whether fshr-1 functions in a ligand-independent manner, and would justify the sentence on line 526 "...ligands...act upstream in this context..." __ We agree with the reviewer that the question of GP ligand activation of FSHR-1 in this context is an important and interesting question. We plan to cross the intestinal fshr-1 transgene into the gpla-1, gplb-1, and gpla-1gplb-1 mutants, as suggested and then will test their swimming behavior to see if the overexpression effect depends upon the ligands. We thank the reviewer for this experimental suggestion.

Planned Revisions based on comments from Reviewer #3

• __Within Figure 6, the authors state that an experiment was run 2-3X which seems inconsistent with other figure panels. It would be better if three times was consistently used. Adding in another run seems appropriate. To add another experimental run where needed within Figure 6 A-D seems realistic. The strains, reagents and skills are all in place, so the only significant investment is time. These experiments should be able to be completed in a few weeks/months. __ We appreciate the reviewer's desire for consistency in terms of the number of replicates. We will ensure all swimming experiments, which were the experiments in question in Figure 6, have been completed at least 3 times as part of our revision plan.

• The authors findings would be strengthened by doing further work to delineate in which tissues the downstream factors act, by doing tissue specific epistasis basically for gsa-1, acy-1 etc. This would entail a lot of work and would delay publication significantly. I do not see this as necessary unless the authors wish for a big impact journal publication. __ As stated in our response to Reviewers #1 and 2, we agree that tissue-specific pathway analysis is important for understanding the molecular mechanism underlying the ability of FSHR-1 to control neuromuscular function from its location in distal tissues, like the intestine. While the complexity of these questions and the time required for us to generate strains to perform tissue-specific protein depletion or overexpression experiments is likely more than is suitable for the revision time frame of this manuscript (and will be the focus of future work), in lieu of these experiments we will perform intestine-specific RNA interference experiments (as we did for fshr-1 in Figure 4B) of gsa-1, acy-1, and sphk-1 in wild type worms and in animals overexpressing fshr-1 in the intestine (which causes increased swimming behavior, Figure 3A) to determine if these downstream players are required for the effects of intestinal fshr-1 on the NMJ. __We appreciate the reviewer's suggestion to address these important questions regarding the site of action of the downstream players.

• __Figures:____ Overall the authors have presented everything in a clear and thorough manner. Some modification of the Y-axes on several aldicarb resistance graphs & body bend bar graphs could improve the clarity. Trying to standardize the Y axis range and the tick mark locations would make it easier to read and compare between figures and panels. __ We appreciate the reviewer's attention to detail here and will work to further standardize the Y-axes on the graphs as requested.

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

Revisions made to the manuscript in response to comments by Reviewer #1

• __The authors should demonstrate the expression of FSHR-1 in various tissues, as this is essential for analyzing its function. __ We appreciate the reviewer's request for additional clarity regarding the sites of tissue-specific FSHR-1 expression and agree that this information was not sufficiently clear in the text. It is already known that FSHR-1 is expressed in various tissues (e.g., head neurons, glia, intestine) from prior studies (Cho et al, 2007; Kenis et al 2023; Hammarlund et al 2018); thus, we would like to defer to Reviewer #3's suggestion about the expression information and have added a description of FSHR-1 expression patterns to lines 129 -130 within the Introduction of the paper. (Reviewer #3: "In the discussion there is a section about the reported areas of endogenous fshr-1 expression. I would have appreciated knowing that information much earlier in the paper. Without being reminded of the reported normal expression pattern it is difficult to fully appreciate why how the neuronal and glial expression could be at work") This expression information is also mentioned in the Results section lines 420-421 when we first discuss the tissue-specific rescue experiments.

• __Figure 4A appears to be the same as Figure S5B. The authors should ensure that the figures are correctly labeled and distinct from each other. __ We thank the reviewer for noticing this oversight. We apologize for the inadvertent duplication. We have replaced the graphs in Figure 4A with the correct rescue experiment using the Pges-1, ibtEx35-expressing strain.

Revisions made to the manuscript in response to comments by Reviewer #2

• Fig 3: Using transgenic rescue experiments the authors observe rescue when expressing fshr-1 under promoters for the intestine as well as glia and neurons. Is it possible that the apparent rescue using glia and neuronal promoters may arise from leaky expression of these transgenes in the intestine? Leaky intestinal expression is a reported caveat for rescue experiments. This possibility should be discussed. We appreciate the reviewer's note regarding the potential caveat of leaky intestinal expression. We have added a mention of this possibility to the discussion (lines 612-616) where we outline other potential explanations for the ability of multiple transgenes to rescue the neuromuscular phenotype. This possibility is why we feel most confident in the intestine site of action given that we have intestine-specific RNA interference data showing fshr-1 necessity in this tissue. We also acknowledge the need for tissue-specific depletion studies to address requirements for fshr-1 in the other distal tissues. We hope to be able to address these other potential sites of action in our future work.

• __Fig 4B: Please clarify at what stage the intestine-specific knockdown of fshr-1 was conducted. It would be informative to treat animals with fshr-1 RNAi at various developmental stages to distinguish whether fshr-1 plays a developmental or post-developmental role in this process (OPTIONAL). __ We thank the reviewer for bringing to our attention the omission of details regarding the feeding RNA interference experiments. We have added an "RNA Interference" subsection with this information to the Materials and Methods section of the manuscript. Briefly, the intestine-specific knockdown was performed by feeding worms at the L4 stage HT115(DE) bacteria containing L4440 empty plasmid or one targeting fshr-1. Worms were grown for 4 days on NGM agar plates containing Ampicillin and IPTG, then offspring of the treated worms were assayed at the young adult stage. Thus, the knockdown animals we tested had been exposed to the RNAi for their lifetime. We are very interested in exploring the developmental timing of fshr-1 expression and function in future work; thus, we thank the reviewer for this suggestion. However, we feel that a detailed panel of developmental knockdown effects of fshr-1 is beyond the scope of the current study.

• Fig 4C: Is rescue significant? p values are not shown. In figure 4C, p-values are only shown for statistically significant differences, as noted in the figure legend. A Tukey's post-hoc test indicates that the Intestinal Rescue strain is not significantly different from either the wild type or the fshr-1 mutants, indicating partial rescue. While we cannot fully explain the discrepancy between the partial rescue of the SNB-1::GFP phenotype in light of the full behavioral rescue in the swimming, aldicarb, and crawling assays, we suspect it may be due to the fact that synaptic vesicle release has been sufficiently restored to recover neuromuscular signaling even though synaptic vesicle localization is not fully returned to wild type levels, given the variable and likely non-endogenous levels of fshr-1 re-expression from the tissue-specific transgenes. We have noted this discrepancy in the Discussion (lines 633-639) when considering the levamisole and SNB-1::GFP data in light of the aldicarb and swimming results. * "*For some tissue-specific fshr-1 expression experiments, we observed partial rescue of the swimming and crawling fshr-1 mutant phenotypes without a restoration of normal synaptic vesicle localization (e.g., cholinergic motor neurons, GABAergic motor neurons, glial cells, Supplemental Figures 6 and 7). We conclude that GFP::SNB-1 accumulation may not solely report on rates of synaptic vesicle release and/or that there are compensatory mechanisms for increasing muscle excitation (e.g. upregulation of postsynaptic ACh receptors or muscle excitatory machinery."

• __Fig 6E. There are two bars in this graph labeled gpla-1; gplb-1 that show significantly different amplitudes. Please clarify and define the different colors that each graph is outlined with. __ We thank the reviewer for catching this error. The third bar from the left should say "gpla-1;fshr-1". We have corrected this in the manuscript. We have also added descriptions of the colors to the figure legend indicating the following: dark blue = wild type, yellow = fshr-1; green = glycopeptide mutants; blue = glycopeptide;fshr-1 mutants. Similar clarification has been added to the legend for the bar graph in Figure 3D.

Revisions made to the manuscript in response to comments by Reviewer #3

Suggested Text Revisions: I have some suggestions to consider.

• In the abstract the term expression analysis is used to analyses of areas of FSHR-1 function using tissue specific rescue experiments. Expression analysis often means directly exploring mRNA, localization, or levels using transcriptomic approaches or reporter genes so some revision of language could improve accuracy in the abstract. We appreciate the reviewer's point and have removed the phrase "expression analysis" from the summary at the end of the Introduction section where it initially appeared.

• __In Figure 1, the authors do not comment on the overexpression phenotype or why this strain was included. __ We thank the reviewer for noticing this oversight. We have added a sentence describing the overexpression experiment and its implications in our description of Figure 1 in the Results section (lines 337-339).

• __In the discussion there is a section about the reported areas of endogenous fshr-1 expression. I would have appreciated knowing that information much earlier in the paper. Without being reminded of the reported normal expression pattern it is difficult to fully appreciate why how the neuronal and glial expression could be at work. __ We appreciate the reviewer's request for additional clarity regarding the sites of tissue-specific FSHR-1 expression and agree that this information was not sufficiently clear in the text prior to the discussion. We have added a description of FSHR-1 expression patterns to lines 129 -130 within the Introduction of the paper. It is also mentioned in Results section lines 420-421 when we first discuss the tissue-specific rescue experiments.

• __The section on tissue specific rescue could be written more strongly. The use of many "transition" phrases dilutes the importance of the findings in this paragraph. __ We are grateful for the reviewer's suggestions to improve the clarity of the text, specifically regarding the tissue-specific rescue section. We have tightened up the text in this section of the Discussion (lines 547-621) to remove some of the transitional phrases. We believe this has enhanced the readability of the manuscript and the impact of our findings.

• Figures: __ 3 panel D: it is not clear what the last 2 bars (Neuronal rescues) are being compared to, its it w.t.? Were the differences between fshr-1 and these rescues not significantly different? __ We appreciate the reviewer bringing this point of confusion to our attention with Figure 3D. We have clarified in the figure legend that the Neuronal rescue bars are compared to wild type and that there is no significant difference from the fshr-1 mutants for these two lines, further supporting our central focus on the intestine as the best-supported site of FSHR-1 action.

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

Comment from Reviewer #1

• __The article concludes that the fshr-1 mutation affects the release of acetylcholine vesicles. However, using fluorescent proteins to label key proteins released by vesicles may introduce artifacts. Therefore, electron microscopy should be used to analyze vesicle accumulation for more reliable results. __ We thank the reviewers for this suggestion and acknowledge the potential value of EM to definitively show vesicle accumulation in fshr-1 mutants. However, these experiments are technically demanding, involve specialized high-pressure freezing, and would require us to establish new collaborations to complete; thus, we would not be able to be complete such experiments in a timeframe reasonable for revision. While the fluorescence microscopy experiments admittedly offer less resolution, this approach has been used with great success in numerous other studies to identify alterations in synaptic vesicle localization in motor neurons that correlate with electron microscopy, electrophysiology, and aldicarb data that more directly measure numbers of synaptic vesicles and synaptic function (Jorgensen et al 1995; Jin et al 1999; Nonet et al 1999). Thus, we believe that the pHluorin experiments, coupled with the SNB-1::GFP imaging, are sufficient to demonstrate defects in vesicle release, regardless of the specific effects on vesicle clustering. We have been mindful not to overstate our conclusions (lines 371-372: "Together, these data demonstrate that FSHR-1 signaling promotes the localization and/or release of cholinergic synaptic vesicles.") We hope the reviewer will agree that our analysis provides meaningful information about SV organization in the absence of EM level experiments.

• __The authors analyzed the release of vesicles from GABA and acetylcholine (Ach) neurons separately to demonstrate that the fshr-1 mutation specifically affects Ach neuron vesicle release. However, while GFP::SNB-1 and GFP::SYD-1 accumulated in GABA neurons, mCherry::UNC-10 did not change significantly in GABA neurons. To fully understand vesicle release, the authors should also use synaptopHluroin (SpH) to analyze GABA neuron vesicle release. __ We agree that our data indicate that, in addition to effects on cholinergic synaptic vesicle release, there may be effects on release of vesicles from GABAergic neurons, and we acknowledge this in the manuscript. However, while we are interested in potentially exploring the effects of fshr-1 in GABAergic neurons, we believe this question requires extensive additional work that is beyond the scope of this manuscript, which is focused on fshr-1 effects on cholinergic signaling. Moreover, given that fshr-1-deficient animas are aldicarb resistant (Figure 1A), it is unlikely that GABA release is decreased. If GABA release was decreased, we would expect hypersensitivity to aldicarb. Thus, while it is still possible there are different effects on GABA vesicles, our data suggest the most physiological relevant effect is on cholinergic signaling. We do acknowledge in the Discussion that it will be of interest to determine the relevance of effects in the GABA neurons (lines 649-651).

Author response:

Reviewer #1 (Public Review):

Summary:

[...] This study is a fundamental step towards our better understanding of the mechanisms underlying light effects on cognition and consequently optimising lighting standards.

Strengths:

While it is still impossible to distinguish individual hypothalamic nuclei, even with the high-resolution fMRI, the authors split the hypothalamus into five areas encompassing five groups of hypothalamic nuclei. This allowed them to reveal that different parts of the hypothalamus respond differently to an increase in illuminance. They found that higher illuminance increased the activity of the posterior part of the hypothalamus encompassing the MB and parts of the LH and TMN, while decreasing the activity of the anterior parts encompassing the SCN and another part of TMN. These findings are somewhat in line with studies in animals. It was shown that parts of the hypothalamus such as SCN, LH, and PVN receive direct retinal input in particular from ipRGCs. Also, acute chemogenetic activation of ipRGCs was shown to induce activation of LH and also increased arousal in mice.

Weaknesses:

While the light characteristics are well documented and EDI calculated for all of the photoreceptors, it is not very clear why these irradiances and spectra were chosen. It would be helpful if the authors explained the logic behind the four chosen light conditions tested. Also, the lights chosen have cone-opic EDI values in a high correlation with the melanopic EDI, therefore we can't distinguish if the effects seen here are driven by melanopsin and/or other photoreceptors. In order to provide a more mechanistic insight into the light-driven effects on cognition ideally one would use a silent substitution approach to distinguish between different photoreceptors. This may be something to consider when designing the follow-up studies.

We thank the reviewer for acknowledging the quality and interest of our work and agree with the weaknesses they pointed out.

Blue-enriched light illuminances were set according to the technical characteristics of the light source and to keep the overall photon flux similar to prior 3T MRI studies of our team (between ~1012 and 1014 ph/cm²/s) (Vandewalle et al. 2010 PNAS, Vandewalle et al. 2011 Biol. Psy.). The orange light was introduced as a control visual stimulation for potential secondary whole-brain analyses. It’s photopic illuminance should ideally have been set similar to the low illuminance blue-enriched light condition, but it was not the case. For the present region of interest analyses, we discarded colour differences between the light conditions and only considered illuminance as indexed by mel EDI lux. This constitutes indeed a limitation of our study as it does not allow attributing the findings to a particular photoreceptor class.

The revised version of the manuscript will include a better explanation as to the choice of illuminances and spectra. The discussion will make clear that these choices limit the interpretation about the photoreceptors involved. The discussion will also point out that silent substitution could be used in the future to resolve such question.

Reviewer #2 (Public Review):

[...] By shedding light on these complex interactions, this research endeavors to contribute to the foundational knowledge necessary for developing innovative therapeutic strategies aimed at enhancing cognitive function through environmental modulation.

Strengths:

(1) Considerable Sample Size and Detailed Analysis: The study leverages a robust sample size and conducts a thorough analysis of hypothalamic dynamics, which enhances the reliability and depth of the findings.

(2) Use of High-Resolution Imaging: Utilizing 7 Tesla fMRI to analyze brain activity during cognitive tasks offers high-resolution insights into the differential effects of illuminance on hypothalamic activity, showcasing the methodological rigor of the study.

(3) Novel Insights into Illuminance Effects: The manuscript reveals new understandings of how different regions of the hypothalamus respond to varying illuminance levels, contributing valuable knowledge to the field.

(4) Exploration of Potential Therapeutic Applications: Discussing the potential therapeutic applications of light modulation based on the findings suggests practical implications and future research directions.

Weaknesses:

(1) Foundation for Claims about Orexin and Histamine Systems: The manuscript needs to provide a clearer theoretical or empirical foundation for claims regarding the impact of light on the orexin and histamine systems in the abstract.

(2) Inclusion of Cortical Correlates: While focused on the hypothalamus, the manuscript may benefit from discussing the role of cortical activation in cognitive performance, suggesting an opportunity to expand the scope of the manuscript.

(3) Details of Light Exposure Control: More detailed information about how light exposure was controlled and standardized is needed to ensure the replicability and validity of the experimental conditions.

(4) Rationale Behind Different Exposure Protocols: To clarify methodological choices, the manuscript should include more in-depth reasoning behind using different protocols of light exposure for executive and emotional tasks.

We thank the reviewer for recognising the interest and strength of our study. We agree that corrections and clarifications to the text were needed. We will address the weaknesses they pointed out as follows:

(1) As detailed in the discussion, we do believe orexin and histamine are excellent candidates for mediating the results we report. As also pointing out, however, we are in no position to know which neurons, nuclei, neurotransmitter and neuromodulator underlie the results. We will therefore remove the last sentence of the abstract as we agree our final statement in the abstract was too strong. We will carefully reconsider the discussion to avoid such overstatements.

(2) We are unsure at this stage how to address the comment of the reviewer without considerably lengthening the manuscript with statements which can only be putative. Hypothalamus nuclei are connected to multiple cortical (and subcortical) structures. The relevance of these projections will vary with the cognitive task considered. In addition, we have not yet considered the cortex in our analyses such that truly integrating cortical structures appears premature. We will nevertheless refer to the general statement that subcortical structures (and particularly those receiving direct retinal projections) are likely to receive light illuminance signal first before passing on the light modulation to the cortical regions involved in the ongoing cognitive process.

(3) Illuminance and spectra could not be directly measured within the MRI scanner due to the ferromagnetic nature of measurement systems. The MR coil and the associated optic fibre stand, together with the entire lighting system were therefore placed outside of the MR room to reproduce the experimental conditions of the in a completely dark room. A sensor was placed 2 cm away from the mirror of the coil (mounted at eye level), i.e. where the eye of the first author of the paper would be positioned, to measure illuminance and spectra. The procedure was repeated 4 times for illuminance and twice for spectra and measurements were averaged. This procedure does not take into account inter-individual variation in head size and orbit shape such that the reported illuminance levels may have varied slightly across subjects. The relative differences between illuminance are very unlikely to vary substantially across participants such that statistics consisting of tests for the impact of relative differences in illuminance were not affected. We will report these methodological details in the supplementary material file associated to the paper.

(4) The comment is similar to the issue raised by reviewer 1 (and reviewer 3) so we refer to the response provided to reviewer 1 to address the final comment of reviewer 2.

Reviewer #3 (Public Review):

[...] The authors find evidence in support of a posterior-to-anterior gradient of increased blood flow in the hypothalamus during task performance that they later relate to performance on two different tasks. The results provide an enticing link between light levels, hypothalamic activity, and cognitive/affective function, however, clarification of some methodological choices will help to improve confidence in the findings.

Strengths:

The authors' focus on the hypothalamus and its relationship to light intensity is an important and understudied question in neuroscience.

Weaknesses:

I found it challenging to relate the authors' hypotheses, which I found to be quite compelling, to the apparatus used to test the hypotheses - namely, the use of orange light vs. different light intensities; and the specific choice of the executive and emotional tasks, which differed in key features (e.g., block-related vs. event-related designs) that were orthogonal to the psychological constructs being challenged in each task.

Given the small size of the hypothalamus and the irregular size of the hypothalamic parcels, I wondered whether a more data-driven examination of the hypothalamic time series would have provided a more parsimonious test of their hypothesis.

We thank the reviewer for acknowledging the originality and interest of our study. We agree that some methodological choices needed more explanations. We will address the weaknesses they pointed out as follows:

The first comment questions the choices of the light conditions and of the tasks. Regarding light conditions, since reviewer 1 (and reviewer 2) raised a similar issue, we refer to the response provided to reviewer 1. We agree that many different tasks could have been used to test our hypotheses. Prior work of our team showed that the n-back task and emotional task we used were successful probes to demonstrate that light illuminance modulates cognitive activity, including within subcortical structures (though resolution did not allow precise isolation of nuclei or subparts). When taking the step of ultra-high field imaging we therefore opted for these tasks as our goal was to show that illuminance affects subcortical brain activity across cognitive domains in general and we were not interested in tasks that would test specific aspects of these domains. The fact that one task is event-related while the other consists of a block design adds, in our view, to the robustness of our finding that a similar anterior-posterior gradient of activity modulation by illuminance is present in hypothalamus. We will update the discussion to highlight this aspect.

As mentioned in the text, the protocol also included an auditory attentional task that could have further broadened the potential generalisability of our findings, but it was not part of the analyses as it could only include 2 illuminance levels due to time constrains.

We agree that a data driven approach could have constituted an alternative means to tests our hypothesis. We opted for an approach that we mastered best while still allowing to conclusively test for regional differences in activity across the hypothalamus. Examination of time series of the very same data we used will mainly confirm the results of our analyses – an anterior-posterior gradient in the impact of illuminance - and may yield slight differences in the limits of the subparts of the hypothalamus undergoing decreased or increased activity with increasing illuminance. While the suggested approach may have been envisaged if we had been facing negative results (i.e. no differences between subparts, potentially because subparts would not correspond functional differences in response to illuminance change), it would now constitute a circular confirmation of our main findings (i.e. using the same data). While we truly appreciate the suggestion, we do not consider that it would constitute a more parsimonious test of our hypothesis now that we successfully applied GLM/parcellation and GLMM approaches.

The cooling cuff is attached to the injured sciatic nerve and then routed along the spine to a headcap. This headcap is placed on the rat's head and allows for further monitoring and control of the device. Figures 5C-D illustrate how the device interfaces with the rat's sciatic nerve and the pathway of the connections to the headcap. The injured nervous tissue in the rat causes a pain response when touched, which is measured each time contact is made. By applying cooling with the device, this pain response should decrease and eventually be eliminated as the temperature decreases.

Reviewer #3 (Public Review):

Summary:

In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:

The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42 . In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.