Author response:

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

eLife assessment

The authors provide convincing experimental evidence of extended motivational signals encoded in the mouse anterior cingulate cortex (ACC) that are implemented by the orbitofrontal cortex (OFC)-to-ACC signaling during learning. The results are valuable to the field of motivation and cognition. The experimental methods used were state-of-the-art. The manuscript would further benefit from theory-driven analyses to inform a mechanistic understanding, particularly for the single-cell calcium imaging results. These results will be of interest to those interested in cortical function, learning, and/or motivation.

We thank the reviewers for their thoughtful reading of our paper and providing constructive feedback. We have made the relevant changes to the manuscript to improve the writing and figures. We provide responses below to each of the reviewer’s comments.

Reviewer #1 (Public Review):

(1) An important conclusion (Figure 4) is that when mice are trained to run through no reward (N) cues in order to reach reward (R) cues, the OFC neurons projecting to ACC each respond to different specific events in a manner that ensures that collectively they tile the extended behavioural sequence. What I was less sure of was whether the ACC neurons do the same or not. Figure 3 suggests that on average ACC neurons maintain activity across N cues in order to get to R cues but I was not sure whether this was because all individual neurons did this or whether some had activity patterns like the OFC neurons projecting to ACC.

We agree that it remains uncertain what individual ACC neurons do during the extended behavioral sequence. We now include a few sentences in the discussion about what we hypothesize, as we did not perform the cellular resolution imaging to determine this:

“While we did not perform single-cell imaging of ACC in our task, we hypothesize that individual ACC neurons could encode the distribution of actions/opportunities47 (i.e. stop, run, lick, suppress lick) taken during R or N cues. ACC neurons could compute the relative value of the action taken such that more ACC neurons become recruited once mice learn to run out of N cues. The sustained increase in bulk ACC activity across N cue trials (Figure 2) could come from a stable sequence of individual neurons that encode the timescale of the actions taken. In this way, OFC projections would encode current motivation across N cues before learning, which then triggers ACC to compute the valuebased actions. Motivational signals in OFC would thus represent state since past rewards/goals, while in ACC these signals represent actions taken to pursue rewards/goals in the future.”

(2) Figure 1 versus Figure 2: There does not seem to be a particular motivation for whether chemogenetic inactivation or optogenetic inhibition were used in different experiments. I think that this is not problematic but, if I am wrong and there were specific reasons for performing each experiment in a certain way, then further clarification as to why these decisions were made would be useful. If there is no particular reason, then simply explaining that this is the case might stop readers from seeking explanations.

Thank you for this comment and we agree that clarification on this is important. We performed chemogenetic inhibition of ACC in Figure 1 to take a broad survey of behavioral effects throughout a 40-min long behavioral session, and performed optogenetic inhibition in Figure 2 because we wanted to restrict our inhibition to the few seconds of cue presentation during a behavioral session and across days. Furthermore, we wanted to combat any potential off-target effects that would come from repeated administration of CNO over the several days of training (Manvich et al 2018). We have included a couple sentences on page 4 to clarify this:

“We proceeded to test whether these motivation related signals in ACC are required for learning. To restrict our inhibition to cue presentation portions of our task, and combat any potential off-target effects of CNO31 from repeated administration across several days of training, we used optogenetic inhibition.”

(3) P5, paragraph 2. The authors argue that OFC and anteriomedial (AM) thalamic inputs into ACC are especially important for mediating motivation through N cues in order to reach R cues. Is this based on a statistical comparison between the activity in OFC or AM inputs as opposed to the other inputs?

We determined that OFC and AM thalamic inputs to ACC are particularly important by comparing the pre-cue activity in a reward-no reward-reward trial sequence (RNR; Figure 3B). Specifically, we performed paired t-tests comparing pre-cue activity between N and R cues, and found a statistically significant increase for R cues but only for the OFC and AM inputs, not for the BLA or LC inputs.

(4) P3, paragraph 2. Some papers by Khalighinejad and colleagues (eg Neuron 2020, Current Biology, 2022) might be helpful here in as much as they assess ACC roles in determining action frequency, initiation, and speed and mediating the relationship between reward availability and action frequency and speed.

We thank the reviewer for bringing these relevant papers to our attention. We have included these papers in our citations in this paragraph.

(5) Paragraph 1 "This learning is of a more deliberate, informed nature than habitual learning, as they are sensitive to the current value of outcomes and can lead to a novel sequence of actions for a desired outcome1-3." Should "they" be "it"?

This is correct, we have edited this in the manuscript.

Reviewer #2 (Public Review):

Impact:

The findings will be valuable for further research on the impact of motivational states on behaviour and cognition. The authors provided a promising concept of how persistent motivational states could be maintained, as well as established a novel, reproducible task assay. While experimental methods used are currently state-of-the-art, theoretical analysis seems to be incomplete/not extensive. We thank the reviewer for these comments. In our paper, we performed single-cell calcium imaging of OFC projection neurons to ACC to build a mechanistic understanding for the bulk ramp-like response we identified in these neurons with photometry. We identified ensembles of neurons that tile sequences of trials that match the bulk response, in particular a subset of neurons that are active at the time a reward (R) cue is reached after 2 no-reward (N) cues. We included a paragraph in the discussion to address future theory-driven analyses to address how computation is achieved by OFC projection neurons:

“We linked the ramp-like increase in neural activity in OFC to motivation, but several questions still remain about how motivation is computed and why it would be represented as a ramp. Motivation could be computed as a combination of several variables such as time since last reward, value of reward, and effort to reach future rewards. Future theorydriven analyses could determine how motivation is computed, and whether individual variables of time, value, and effort, are encoded as clusters of similar tuned neurons, or mixed and collectively represented at the population level. In either case, it is likely that a combined map of task space and value-information carried by OFC are being used to inform downstream regions, such as ACC, for adjusting behavior. ”

Reviewer #2 (Recommendations for the Authors):

Overall, the layout of the figures seems a little bit chaotic and makes it hard to understand the boundaries between panels.

We agree that the figure layout could be improved upon to aid the reader in moving from panel to panel. We have edited two of the main figures with layouts that are most irregular (Figures 2 and 4) to help with this.

Figures/text should include the promoters used for protein expression so that readers understand which cell types would be affected.

We have made sure to edit the figures to include the promoter of the viruses we used, and edited the text to include both the AAV serotype and promoter.

Discuss why it is necessary for multiple prefrontal areas to be involved in maintaining motivational signals.

We thank the reviewer for this comment. We believe that prefrontal areas would be recruited as tasks to study motivational states become more complex and require animals to keep track of task structure and perform value-guided actions. We have included a couple sentences in the final paragraph of the discussion about this:

“Our work showed the recruitment of multiple frontal cortical areas in this process, which is to be expected as animals are required to build, maintain, and use representations of task structure and value to drive learned, motivated behaviors47. Future work can build upon the task we developed here to determine how the frontal cortex maintains motivational states across many more cue-outcome associations, and how these associations may dynamically change across time48”.

Additionally, we included a short discussion on how in motivational signals differ between OFC and ACC in our work. We suggest OFC encodes current motivation before and after learning, which then leads ACC to represent learned actions taken and thus have a longer timescale motivational response (see response to Reviewer 1).

Minor: Page 4, Line 1: "increase" instead of "increases".

This is correct, we have edited this in the manuscript.

Author response:

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

eLife assessment

This study provides important insights into the role of neurexins as regulators of synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body and the lateral superior olive, key components of the auditory brainstem circuit involved in computing sound source location from differences in the intensity of sounds arriving at the two ears. Through an elegant combination of genetic manipulation, fluorescence in-situ hybridization, ex vivo slice electrophysiology, pharmacology, and optogenetics, the authors provide convincing evidence to support their claims. While further work is needed to reveal the mechanistic basis by which neurexins influence glycinergic neurotransmission, this work will be of interest to both auditory and synaptic neuroscientists.

We appreciate the recognition of the significance of our study in shedding light on the role of neurexins in regulating synaptic strength and timing at the glycinergic synapse. Indeed, further investigations are warranted to delve deeper into the specific role of each different variant of neurexins in the future. We hope that our work will spark more interest and collaboration in unraveling the complexities of molecular codes of synaptic function.

Public Reviews:

Reviewer #1 (Public Review):

Jiang et al. demonstrated that ablating Neurexins results in alterations to glycinergic transmission and its calcium sensitivity, utilizing a robust experimental system. Specifically, the authors employed rAAV-Cre-EGFP injection around the MNTB in Nrxn1/2/3 triple conditional mice at P0, measuring Glycine receptor-dependent IPSCs from postsynaptic LSO neurons at P13-14. Notably, the authors presented a clear reduction of 60% and 30% in the amplitudes of opto- and electric stimulation-evoked IPSCs, respectively. Additionally, they observed changes in kinetics, alterations in PPR, and sensitivity to lower calcium and the calcium chelator, EGTA, indicating solid evidence for changes in presynaptic properties of glycinergic transmission.

Furthermore, the authors uncovered an unexpected increase in sIPSC frequency without altering amplitude. Despite the reduction in evoked IPSC, immunostaining revealed an increase in GlyT2 and VGAT in TKO mice, supporting the notion of an increase in synapse number. However, the reviewer expresses caution regarding the authors' conclusion that "glycinergic neurotransmission likely by promoting the synapse formation/maintenance, which is distinct from the phenotypes observed in glutamatergic and GABAergic neurons (Chen et al., 2017; Luo et al., 2021)", as outlined in lines 173-175. The reviewer suggests that this statement may be overstated, pointing out the authors' own discussion in lines 254-265, which acknowledges multiple possibilities, including the potential that the increase in synapses is a consequence rather than a causal effect of Nrxn deletion.

We appreciate the reviewer’s thoughtful evaluation of our study. We agree that our conclusion regarding the promotion of synapse formation/maintenance may have been overstated and recognize the need for a more nuanced interpretation of our findings. Accordingly, we have revised our interpretation by discussing carefully the various possibilities that may cause the observed increase in synapse number in line 256-266.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Jiang et al., explore the role of neurexins at glycinergic MNTB-LSO synapses. The authors utilize elegant and compelling ex vivo slice electrophysiology to assess how the genetic conditional deletion of Nrxns1-3 impacts inhibitory glycinergic synaptic transmission and found that TKO of neurexins reduced electrically and optically evoked IPSC amplitudes, slowed optically evoked IPSC kinetics and reduced presynaptic release probability. The authors use classic approaches including reduced [Ca2+] in ACSF and EGTA chelation to propose that changes in these evoked properties are likely driven by the loss of calcium channel coupling. Intriguingly, while evoked transmission was impaired, the authors reported that spontaneous IPSC frequency was increased, potentially due to an increased number of synapses in LSO. Overall, this manuscript provides important insight into the role of neurexins at the glycinergic MNTP-LSO synapse and further emphasizes the need for continued study of both the non-redundant and redundant roles of neurexins.

We thank the reviewer for the strong comments and support of our work.

Strengths:

This well-written manuscript seamlessly incorporates mouse genetics and elegant ex vivo electrophysiology to identify a role for neurexins in glycinergic transmission at MNTB-LSO synapses. Triple KO of all neurexins reduced the amplitude and timing of evoked glycinergic synaptic transmission. Further, spontaneous IPSC frequency was increased. The evoked synaptic phenotype is likely a result of reduced presynaptic calcium coupling while the spontaneous synaptic phenotype is likely due to increased synapse numbers. While neuroligin-4 has been identified at glycinergic synapses, this study, to the best of my knowledge, is the first to study Nrxn function at these synapses.<br />

We again appreciate the positive feedback on the strengths of our study. We agree that the observed reduction in evoked synaptic transmission and the increase in spontaneous IPSC frequency provide intriguing insights into the function of neurexins in regulating glycinergic synaptic activity.

Weaknesses:

The data are compelling and report an intriguing functional phenotype. The role of Neurexins redundantly controls calcium channel coupling has been previously reported. Mechanistic insight would significantly strengthen this study.

We wholeheartedly agree with the reviewer that understanding how neurexins control calcium channel coupling at the presynaptic active zone is crucial for elucidating their role in synaptic transmission. While our current study has provided compelling evidence for the functional phenotypes of pan-neurexin deletion, we recognize the importance of investigating the underlying molecular mechanisms in future research. Exploring these mechanisms would undoubtedly enhance our understanding of neurexin function at various synapses and contribute to advancing the field.

The claim that triple KO of Nrxns from MNTB increases the number of synapses in LSO is not strongly supported.

We agree. Echoing the suggestion made by reviewer 1 (as mentioned above), we acknowledge that the claim regarding the increase in synapse numbers in the LSO following the triple knockout of neurexins from the MNTB was overstated. Consequently, we have revised our conclusions more carefully to reflect this adjustment.

Despite the stated caveats of measuring electrically evoked currents and the more robust synaptic phenotypes observed using optically evoked transmission, the authors rely heavily on electrical stimulation for most measurements.

We acknowledge that optogenetic stimulation offers crucial advantages, and we have provided a balanced discussion of the caveats associated with both methods in our manuscript. Additionally, we have conducted new optogenetic experiments specifically for measuring the paired-pulse ratio in control and Nrxn123 TKO mice. These results have been included as a new supplementary figure (Figure S2).

For experiments involving EGTA and low Ca2+ manipulations, we opted for electrical stimulation due to concerns regarding potential side effects of optogenetics, including the phototoxicity and photobleaching during prolonged light exposure.

The differential expression of individual neurexins might indicate that specific neurexins may dominantly regulate synaptic transmission, however, this possibility is not discussed in detail.

We thank the reviewer for bringing up this important point. The differential expression of individual neurexins indeed suggests that specific neurexins may play dominant roles in regulating synaptic transmission. While our study primarily focused on the collective impact of ablating all neurexins, we acknowledge the significance of exploring the specific contributions of individual neurexin isoforms in the future. Understanding the distinct roles of each neurexin isoform could provide valuable insights into the precise mechanisms underlying synaptic function and plasticity. We have added discussion in our revised manuscript Line223-230.

Reviewer #3 (Public Review):

Summary:

The authors investigate the hypothesis that neurexins serve a crucial role as regulators of the synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body (MNTB) and the lateral superior olivary complex (LSO). It is worth mentioning that LSO neurons are an integration station of the auditory brainstem circuit displaying high reliability and temporal precision. These features are necessary for computing interaural cues to derive sound source location from comparing the intensities of sounds arriving at the two ears. In this context, the authors' findings build up according to the hypothesis first by displaying that neurexins were expressed in the MNTB at varying levels. They followed this up with the deletion of all neurexins in the MNTB through the employment of a triple knock-out (TKO). Using electrophysiological recordings in acute brainstem slices of these TKO mice, they gathered solid evidence for the role of neurexins in synaptic transmission at this glycinergic synapse primarily by ensuring tight coupling of Ca2+ channels and vesicular release sites. Additionally, the authors uncovered a connection between the deletion of neurexins and a higher number of glycinergic synapses in TKO mice, for which they provided evidence in the form of immunostainings and related it to electrophysiological data on spontaneous release. Consequently, this investigation expands our knowledge on the molecular regulation of synaptic transmission at glycinergic synapses, as well as on the auditory processing at the level of the brainstem.

Strengths:

The authors demonstrate substantial results in support of the hypothesis of a critical role of neurexins for regulating glycinergic transmission in the LSO using various techniques. They provide evidence for the expression of neurexins in the MNTB and consecutively successfully generate and characterize the neurexin TKO. For their study on LSO IPSCs the authors transduced MNTB neurons by co-injection of virus-carrying Cre and ChR2 and subsequently optogenetically evoke release of glycine. As a result, they observed a significant reduction in amplitude and significantly slower rise and decay times of the IPSCs of the TKO in comparison with control mice in which MNTB neurons were only transduced with ChR2. Furthermore, they observed an increased paired pulse ratio (PPR) of LSO IPSCs in the TKO mice, indicating lower release probability. Elaborating on the hypothesis that neurexins are essential for the coupling of synaptic vesicles to Ca2+ channels, the authors show lowered Ca2+ sensitivity in the TKO mice. Additionally, they reveal convincing evidence for the connection between the increased frequency of spontaneous IPSC and the higher number of glycinergic synapses of the LSO in the TKO mice, revealed by immunolabeling against the glycinergic presynaptic markers GlyT2 or VGAT.

We thank the reviewer for the thoughtful and thorough evaluation of the significance of investigating the role of neurexins in glycinergic transmission at the MNTB-LSO synapse, particularly in the context of auditory processing and sound localization. The positive feedback is greatly appreciated.

Weaknesses:

The major concern is novelty as this work on the effects of pan-neurexin deletion in a glycinergic synapse is quite consistent with the authors' prior work on glutamatergic synapses (Luo et al., 2020). The authors might want to further work out novel aspects and strengthen the comparative perspective. Conceptually, the authors might want to be more clear about interpreting the results on the altered dependence of release on voltage-gated Ca2+ influx (Ca2+ sensitivity, coupling).

Regarding the reviewer’s concern about the novelty of our work, we acknowledge that our previous work has explored the effects of pan-neurexin deletion on glutamatergic synapses (Luo et al., 2020). However, we would like to point out that a novelty of our present study indeed stems from the exploration of how different types of synapses converge to employ the same mechanism of synaptic function, particularly in the context of neurexin-mediated regulation. Our previous study focused on glutamatergic synapses, the current study delves into the realm of glycinergic synapses, which represent a distinct population with unique properties and functions. Despite the differences between these synapse types, our findings reveal a commonality in the underlying mechanisms of synaptic regulation mediated by neurexins. This convergence of mechanisms across different synapse types highlights the fundamental role of neurexins in synaptic function and plasticity. By elucidating how neurexins regulate synaptic transmission at both excitatory and inhibitory synapses, we provide valuable insights into the general principles governing synaptic function. In addition, this comparative perspective may shed light on the complex interplay between excitatory and inhibitory neurotransmission, which is crucial for maintaining the balance of neuronal activity and network dynamics.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

During the developmental period spanning P3-P12, the MNTB-LSO synapses undergo a transition from GABAergic to glycinergic transmission. It is well-established that Neurexin plays a role in modulating GABAergic transmission. In the authors' experimental system, AAV was injected at P0, likely impacting GABAergic transmission, including potentially influencing synapse number, before subsequently affecting glycinergic transmission. A thoughtful discussion of how the experimental interventions might have influenced this developmental process and glycinergic transmission would enhance the clarity and interpretation of their findings.

We thank the reviewer for raising the interesting topic of the transmitter switch during neurodevelopment. Strong evidence using gerbils and rats as animal models demonstrates that the MNTB-LSO synapses undergo a shift from GABAergic to glycinergic during the early development. However, in a more recent study by Friauf and colleagues (Fisher et al., 2019), patch-clamp recordings in acute mouse brainstem slices at P4-P11 combined with pharmacological blockade of GABAA receptors and/or glycine receptors clearly demonstrated no GABAergic synaptic component on LSO principal neurons, suggesting the transmitter subtype switch may be species different. We add a discussion in our revision to clarify this topic.

Reviewer #2 (Recommendations For The Authors):

The data are compelling and report an intriguing functional phenotype. Mechanistic insight into how this phenotype manifests would significantly strengthen this study. For example, which neuroligin is found at these MNTB-LSO synapses?

We agree that investigating the underlying molecular mechanisms, particularly the specific function of each variant of neurexins and their respective ligands on the postsynaptic neurons, is crucial. Exploring these mechanisms, which extend beyond the scope of our current study, would undoubtedly enhance our understanding of neurexin function at various synapses and foster advancements in the field.

Does the TKO alter the ability of MNTB inputs to induce AP firing in LSO neurons?

Activation of the MNTB inputs does not directly induce AP firing in LSO neurons, because the MNTB-LSO synapses are glycinergic and serve to inhibit neuronal activity.

We think the reviewer was to ask whether pan-neurexin deletion in the MNTB neurons alter their ability to impact the firing of LSO neurons. Indeed, the weakening of glycinergic transmission due to pan-neurexin ablation in MNTB neurons could potentially alter the excitation-inhibition (E/I) balance, thereby impacting the overall excitability of LSO neurons. We have conducted preliminary experiments to investigate this aspect and found that the E/I balance at LSO neurons was notably increased in TKO mice. We are currently preparing a manuscript to comprehensively address the role of neurexins at the auditory circuit and behavior levels.

Additional calcium measurements using GECIs would provide insight into whether nanodomain calcium or total calcium is altered at these synapses.

We appreciate the valuable suggestion provided by the reviewer. However, distinguishing between Ca2+ nanodomain and Ca2+ microdomain using Ca2+ imaging techniques requires advanced systems such as two-photon STED microscopy, which are beyond the scope of our current research.

It is unclear why fluorescence intensity is quantified instead of the number of synaptic clusters in LSO. In addition to changes in synapse numbers, fluorescent intensity can indicate a number of other possible morphological changes.

We appreciate the valuable suggestion from the reviewer. We have re-analyzed our imaging data to compare synaptic density. The results, as included in Fig.3f and 3h, confirm an increase in the number of glycinergic synapses after pan-neurexin deletion.

The most robust synaptic phenotypes were produced by measuring light-evoked oIPSCs and the authors acknowledge that electrically-evoked eIPSCs might be contaminated by uninfected fibers or by other sources of glycinergic inputs. I suggest that IPSC PPRs, EGTA, and low Ca2+ experiments be performed using optogenetics.

As discussed in our response to Public Reviews, we acknowledge that optogenetic stimulation offers crucial advantages, and we have provided a balanced discussion of the caveats associated with both methods in our manuscript. Additionally, following the reviewer’s suggestion, we have conducted new optogenetic experiments specifically for measuring the paired-pulse ratio in control and Nrxn123 TKO mice. We included this new dataset in supplementary Figure S2, which is consistent with our result obtained with electrically fiber stimulation.

For experiments involving EGTA and low Ca2+ manipulations, we opted for electrical stimulation due to major concerns regarding potential side effects of optogenetics, including the phototoxicity and photobleaching during prolonged light exposure.

It is sometimes confusing which type of evoked stimulation is being used (e.g. PPR, EGTA, and low Ca2+ experiments). To aid in the interpretations of these experiments, it would help to clarify.

We appreciate the reviewer's suggestion regarding the clarity of the evoked stimulation methods used in our experiments. We have revised the manuscript to provide clearer descriptions of the specific types of evoked stimulation employed in each experiment. Thank you for guiding towards this clarification.

The comparisons to Chen et al 2017 and the senior author's 2020 paper seem disjointed and do not contribute to the findings, which alone, are quite interesting. Given the prevailing notion that neurexins control different synaptic properties depending on the brain region and/or synapse studied, is it surprising that the findings observed here differ from previous studies of different synapses (glutamatergic and GABAergic)?

By comparing previous studies at different types of neurons/synapses, our findings reveal a commonality in the underlying mechanisms of synaptic regulation mediated by neurexins. This convergence of mechanisms across different synapse types highlights the fundamental role of neurexins in synaptic function and plasticity. In addition, this comparative perspective may shed light on the complex interplay between excitatory and inhibitory neurotransmission, which is crucial for maintaining the balance of neuronal activity and network dynamics.

Despite Nrxn3 being the most abundant Nrxn mRNA in MNTB neurons, the possible contributions of this highly expressed protein are not discussed.

We thank the reviewer for bringing up this important point. The differential expression of individual neurexins indeed suggests that specific neurexins may play dominant roles in regulating synaptic transmission. While our study primarily focused on the collective impact of ablating all neurexins, we acknowledge the significance of exploring the specific contributions of individual neurexin isoforms in the future. Understanding the distinct roles of each neurexin isoform could provide valuable insights into the precise mechanisms underlying synaptic function and plasticity. We have added discussion in our revised manuscript Line223-230.

Reviewer #3 (Recommendations For The Authors):

• There are several instances of spaces missing and typos, please carefully check the manuscript.

We greatly appreciate the reviewer's helpful feedback on the text that could be clarified or improved. We have meticulously edited the manuscript to address these concerns.

• While studying the properties of IPSC, apart from optogenetic stimulation, the authors performed experiments with electrical fiber stimulation. Their findings showed a slightly significant reduction of the IPSC amplitude and no effect on the IPSCs kinetics when comparing the TKO and control. One weakness is the discrepancy between the results from the optogenetic and fiber stimulation experiments, which the authors contribute to inefficient transfection in the fiber stimulation experiments. The authors state that they tried to optimize their protocols for virus injection protocols. However, they do not elaborate on how the transfection rates could be improved in the discussion section. Moreover, it would be good to further address the reasons for the difference in amplitude between the control IPSCs in the optogenetic and fiber stimulation experiments.

Echoing the suggestion by Reviewer 2 (see above), we acknowledge that optogenetic stimulation offers certain advantages, and we have provided a balanced discussion of the caveats associated with both methods in our manuscript. In addition, we have performed a new set of optogenetic experiment for the paired-pulse ratio measurement in control and Nrxn123 TKO mice and included as a new figure in supplementary figure S2.

For experiments involving EGTA and low Ca2+ manipulations, we opted for electrical stimulation due to major concerns regarding potential side effects of optogenetics, including the phototoxicity and photobleaching during prolonged light exposure.

We added the detail of virus injection strategy that optimized the transfection rates in the method section “To enhance virus infection efficiency, we decreased the dosage per injection while increasing the frequency of injections. Additionally, we ensured the pipette remained immobilized for 20-30 seconds to guarantee virus absorption at injection sites. As a result of this strategy, we estimated that the vast majority of MNTB neurons were inoculated by AAVs.” See line288-290.

• Abstract: "ablation of all neurexins in MNTB neurons reduced not only the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons."

Changed as suggested.

• Consider revising to "The synaptic dysfunctions primarily resulted from an altered dependence of release on voltage-gated Ca2+ influx."

We appreciate the reviewer's suggestion, which helps improve the clarity of our manuscript. We have revise the phrasing as follows: "The synaptic dysfunctions primarily resulted from an impaired calcium sensitivity of release and a loosened coupling between voltage-gated calcium channels and synaptic vesicles."

• Line 39 should be vertebrates.

Revised as suggested.

• Line 49 it would sound better to say "which further points to the diverse actions of neurexins in specific neurons."

Revised as suggested.

• Line 60 - this paragraph could include information about GABA signaling from the MNTB to the LSO, because on line 113 you mention LSO neurons receive inhibitory GABAergic/glycinergic inputs, but when you do not mention blocking of GABA currents to isolate the glycinergic ones.

We thank the reviewer for the thoughtful and detailed suggestion. We revised the text in line 60 to “In the mature mammalian auditory brainstem” and in line 113, we removed GABAergic to emphasize the nature of glycinergic synapse, particularly in the mouse brainstem where no GABAergic components are found (Fisher et al., 2019).

• Line 72/73 it should be adeno-associated virus; line 73: "combining this with the RNAScope technique" sounds better.

Changed as suggested.

• Line 91 using the RNAScope technique; lines 97, 119 as a control; line 108 the functional organization.<br />

Changed as suggested.

• Line 113 should be a pharmacological approach; line 122 optogenetically evoked.

Changed as suggested.

• Line 132, 160: the control.

Changed as suggested.

• Line 147 thus were infected; line 148 likely to be present but were obscured .

Changed as suggested.

• Line 154 which has been routinely used.

Changed as suggested.

• Line 155 It is not supposed to be Figure 2h but 2i; following that Figure 2i should be 2j; in my opinion, Figure 2i does not display a strong depression for the TKO mice.

Changed as suggested.

• Line 171 a better flow is achieved by saying: together these data show.

Changed as suggested.

• EC50 rather than IC50 of [Ca2+].

Changed as suggested.

• 180 it is better to say "we approached the matter by..."; line 183 while recording;

Changed as suggested.

• Line 203 were much stronger than the effect at control synapses; line 206 tightly clustering.

Changed as suggested.

• Line 212 sounds like they provide evidence for retina and spinal cord as well, should be made clear.

Changed as suggested.

• Line 289 previously.

Changed as suggested.

• Line 295 should be 30 min.

Changed as suggested.

• Line 336, 337 confocal microscope.

Changed as suggested.

• Please provide the number of data points also in figure captions or in the results section.

Added in the captions as suggested.

• Line 533, a better phrasing would be: the blocking effect of 0.2 mM Ca on IPSC amplitude.

Changed as suggested.

• Explain either in the methods or result section how was the EC50 of Ca2+ calculated.

Added in the methods as suggested.

Reviewer #3 (Public Review):

Summary:

This study proposes visual homogeneity as a novel visual property that enables observers perform to several seemingly disparate visual tasks, such as finding an odd item, deciding if two items are same, or judging if an object is symmetric. In Exp 1, the reaction times on several objects were measured in human subjects. In Exp 2, visual homogeneity of each object was calculated based on the reaction time data. The visual homogeneity scores predicted reaction times. This value was also correlated with the BOLD signals in a specific region anterior to LO. Similar methods were used to analyze reaction time and fMRI data in a symmetry detection task. It is concluded that visual homogeneity is an important feature that enables observers to solve these two tasks.

Strengths:

(1) The writing is very clear. The presentation of the study is informative.<br /> (2) This study includes several behavioral and fMRI experiments. I appreciate the scientific rigor of the authors.

Weaknesses:

(1) My main concern with this paper is the way visual homogeneity is computed. On page 10, lines 188-192, it says: "we then asked if there is any point in this multidimensional representation such that distances from this point to the target-present and target-absent response vectors can accurately predict the target-present and target-absent response times with a positive and negative correlation respectively (see Methods)". This is also true for the symmetry detection task. If I understand correctly, the reference point in this perceptual space was found by deliberating satisfying the negative and positive correlations in response times. And then on page 10, lines 200-205, it shows that the positive and negative correlations actually exist. This logic is confusing. The positive and negative correlations emerge only because this method is optimized to do so. It seems more reasonable to identify the reference point of this perceptual space independently, without using the reaction time data. Otherwise, the inference process sounds circular. A simple way is to just use the mean point of all objects in Exp 1, without any optimization towards reaction time data.

(2) Visual homogeneity (at least given the current from) is an unnecessary term. It is similar to distractor heterogeneity/distractor variability/distractor statics in literature. However, the authors attempt to claim it as a novel concept. The title is "visual homogeneity computations in the brain enable solving generic visual tasks". The last sentence of the abstract is "a NOVEL IMAGE PROPERTY, visual homogeneity, is encoded in a localized brain region, to solve generic visual tasks". In the significance, it is mentioned that "we show that these tasks can be solved using a simple property WE DEFINE as visual homogeneity". If the authors agree that visual homogeneity is not new, I suggest a complete rewrite of the title, abstract, significance, and introduction.

(3) Also, "solving generic tasks" is another overstatement. The oddball search tasks, same-different tasks, and symmetric tasks are only a small subset of many visual tasks. Can this "quantitative model" solve motion direction judgment tasks, visual working memory tasks? Perhaps so, but at least this manuscript provides no such evidence. On line 291, it says "we have proposed that visual homogeneity can be used to solve any task that requires discriminating between homogeneous and heterogeneous displays". I think this is a good statement. A title that says "XXXX enable solving discrimination tasks with multi-component displays" is more acceptable. The phrase "generic tasks" is certainly an exaggeration.

(4) If I understand it correctly, one of the key findings of this paper is "the response times for target-present searches were positively correlated with visual homogeneity. By contrast, the response times for target-absent searches were negatively correlated with visual homogeneity" (lines 204-207). I think the authors have already acknowledged that the positive correlation is not surprising at all because it reflects the classic target-distractor similarity effect. But the authors claim that the negative correlations in target-absent searches is the true novel finding.

(5) I would like to make it clear that this negative correlation is not new either. The seminal paper by Duncan and Humphreys (1989) has clearly stated that "difficulty increases with increased similarity of targets to nontargets and decreased similarity between nontargets" (the sentence in their abstract). Here, "similarity between nontargets" is the same as the visual homogeneity defined here. Similar effects have been shown in Duncan (1989) and Nagy, Neriani, and Young (2005). See also the inconsistent results in Nagy& Thomas, 2003, Vicent, Baddeley, Troscianko&Gilchrist, 2009.<br /> More recently, Wei Ji Ma has systematically investigated the effects of heterogeneous distractors in visual search. I think the introduction part of Wei Ji Ma's paper (2020) provides a nice summary of this line of research.

I am surprised that these references are not mentioned at all in this manuscript (except Duncan and Humphreys, 1989).

(6) If the key contribution is the quantitative model, the study should be organized in a different way. Although the findings of positive and negative correlations are not novel, it is still good to propose new models to explain classic phenomena. I would like to mention the three studies by Wei Ji Ma (see below). In these studies, Bayesian observer models were established to account for trial-by-trial behavioral responses. These computational models can also account for the set-size effect, behavior in both localization and detection tasks. I see much more scientific rigor in their studies. Going back to the quantitative model in this paper, I am wondering whether the model can provide any qualitative prediction beyond the positive and negative correlations? Can the model make qualitative predictions that differ from those of Wei Ji's model? If not, can the authors show that the model can quantitatively better account for the data than existing Bayesian models? We should evaluate a model either qualitatively or quantitatively.

(7) In my opinion, one of the advantages of this study is the fMRI dataset, which is valuable because previous studies did not collect fMRI data. The key contribution may be the novel brain region associated with display heterogeneity. If this is the case, I would suggest using a more parametric way to measure this region. For example, one can use Gabor stimuli and systematically manipulate the variations of multiple Gabor stimuli, the same logic also applies to motion direction. If this study uses static Gabor, random dot motion, object images that span from low-level to high-level visual stimuli, and consistently shows that the stimulus heterogeneity is encoded in one brain region, I would say this finding is valuable. But this sounds like another experiment. In other words, it is insufficient to claim a new brain region given the current form of the manuscript.

REFERENCES<br /> - Duncan, J., & Humphreys, G. W. (1989). Visual search and stimulus similarity. Psychological Review, 96(3), 433-458. doi: 10.1037/0033-295x.96.3.433<br /> - Duncan, J. (1989). Boundary conditions on parallel processing in human vision. Perception, 18(4), 457-469. doi: 10.1068/p180457<br /> - Nagy, A. L., Neriani, K. E., & Young, T. L. (2005). Effects of target and distractor heterogeneity on search for a color target. Vision Research, 45(14), 1885-1899. doi: 10.1016/j.visres.2005.01.007<br /> - Nagy, A. L., & Thomas, G. (2003). Distractor heterogeneity, attention, and color in visual search. Vision Research, 43(14), 1541-1552. doi: 10.1016/s0042-6989(03)00234-7<br /> - Vincent, B., Baddeley, R., Troscianko, T., & Gilchrist, I. (2009). Optimal feature integration in visual search. Journal of Vision, 9(5), 15-15. doi: 10.1167/9.5.15<br /> - Singh, A., Mihali, A., Chou, W. C., & Ma, W. J. (2023). A Computational Approach to Search in Visual Working Memory.<br /> - Mihali, A., & Ma, W. J. (2020). The psychophysics of visual search with heterogeneous distractors. BioRxiv, 2020-08.<br /> - Calder-Travis, J., & Ma, W. J. (2020). Explaining the effects of distractor statistics in visual search. Journal of Vision, 20(13), 11-11.

Reviewer 3. Arang Rhie

Comments to Author: 1. In the introduction, add recent marker based graph phasing algorithms in long-reads, such as hifiasm trio and verkko trio mode after the T2T-Y. They are different from trio-binning, which tries to phase the reads upfront. Graph based phasing is using markers to determine haplotype specific paths to traverse. a. T2T-Y chromosome should be referencing Rhie et al., Nature 2023. Verkko is a successor of the manual efforts taken in T2T-Y, which should be also noted in the introduction. b. Reference for sexPhase program is still missing. Also, some rephrasing of the sentence is needed, as the way it is currently written is easily misleading to be understood as sexPhase was part of the methods used in the assembly of the T2T-Y. 2. There are other approaches for phasing genomes taken in plants, for example the poly ploid potato phasing using many siblings of the child by Mari et al. bioRxiv 2022.3. "But only one male and one female could suffer from sampling error" - this part is unclear. Please clarify. 4. Reference for the mason_simulator, badread software is missing. 5. Provide the accession (HG02982) for the "African human Y" in the main text. 6. I appreciate that the authors compared assemblies to T2T-Y as I requested before. However, fundamentally, mapping to T2T-Y and comparing length of each sequence classes is comparing apples to oranges, particularly in the heterochromatic region and ampliconic region of the Y. It is known to have variable copy numbers and size differences between two individuals. Frequent inversions have been reported in the ampliconic regions across different Y haplogroup. The number, size, and distribution of the repeat arrays composing the heterochromatic region has been shown to vary among different Y haplogroups in Hallast et al., Nature 2023. This can be also seen in Fig. 3c; the overall depth of the flow sorting in the heterochromatic region is below 1 - indicating the Yqh is shorter than T2T-Y, as it is in Fig. 3b. To make the benchmark legit, the authors should compare SRY and the flow sorting method using samples from the same individual. HG02982 and HX1 are presumably having very different sequence compositions given the diverged population history (African vs. Asian). Comparing total length of the assembled region against a 3rd different Y haplogroup (HG002Y) makes things more complicated, especially on regions that are known to vary a lot. If the authors think flow sorting based method needs to be compared, it should be benchmarked on the same individual to make an apple-to-apple comparison. I do agree results from read sorting (i.e. portion of reads sequenced from non-Y chromosomes in SRY vs. flow-sorting) is an important finding. However, I'd still argue comparing assemblies from the two different Y haplogroups is a stretch. The authors could have performed the same assembly length comparison on the T2T-Y using results from their SRY sorted reads with Verkko of HG002 vs. Verkko assembly using trio-binned markers. 7. In the section where assemblies are compared, the authors point to Table 1, which contains results from HG01109. HG01109 has never been mentioned before. I thought the authors were comparing assemblies from SRY sorted reads of HX1? I am not sure why the authors suddenly added a 3rd PUR genome with no context. Was this a mistake? Add results from HX1 to Table 1. 8. Please add divider lines in Table 1 between All / Ampliconic / X-degenerate / X-transposed / PAR / Het / Others. It is hard to see which rows belong to which category. 9. The last result section where authors compare results from Verkko, it is unclear how the verkko assembly was run. The authors say "default option", and later "in trio mode" in the methods. Did the authors collect parental reads from HG002 (HG003 and HG004)? How was "trio mode" performed? Did the authors used trio binning to sort the reads, then run Verkko? Or used the homopolymer compressed parental kmers and used that in the Rukki step of Verkko (and this should be benchmarked)? Was the HG002 trio assembly taken from Rautiainen et al. paper? Please clarify and add the missing parts to the main text and methods. 10. Related to the above section, it is hard to see in Fig. 4a the "two approximately 1 Mb contigs aligning to the same region of the Y chromosome". An enlarged inset of the dotplot may be helpful. Also, add legends and scale to the X and Y axis of the dotplots. 11. Note there is a mis-assembly reported on T2T-Y palindrome P5 (https://github.com/marbl/CHM13-issues/blob/main/v2.0_issues.bed), which the entire P5 should be inverted. I don't see this in the dotplots of Fig. 4. 12. In the discussion, the authors are mentioning results from the 10 trios that have been removed from the previous results. Please add the 10 trio results to the main text if it was a mistake, or remove the irrelevant results from the Discussions and Supp. Tables. 13. The authors discuss the suboptimal performance of SRY in the PAR is contributed by the restricted data types. I thought it was contributed by the lower density of the markers? The PAR parental marker density was very similar to that of autosomes, with stretches of runs of homozygosity, presumably to maintain enough homology for recombination. What was the marker density in the PAR? Was it below their 7 kmer / 1kb? 14. The authors mentioned there are no ZW genomes available to test SRY. There is a Zebra finch trio (ZW, female, bTaeGut2) and a male sample (ZZ, male, bTaeGut1) available with HiFi of the child (bTaeGut2) and Illumina of all the genomes from the Vertebrate Genomes Project (Rhie et al., Nature, 2021). Perhaps the authors could apply SRY on this individual, and compare the W chromosome results to what has been released on https://www.genomeark.org/vgp-all/Taeniopygia_guttata.html.

Re-review: The authors have addressed most of my concerns. The revised manuscript reads much better than before. Regarding my last comment and response from the authors about the W chromosome, I was hoping to see comparable coverage of the W chromosome to the reference, as a proof of principle that SRY could be applied to non-human, highly diverged genomes. The assembly looks very fragmented though. Was it only the similarity to the Z chromosome that caused the fragmentation? Are there no other factors contributing to the discontinuity of the W chromosome? A few minor comments below to the revised version: 1. Please indicate which genome was compared in the legend of Supp. Table 5. 2.When using et al notations, please use the last name. Mari et al should be Serra Mari et al., Mikko et al should be Rautiainen et al. Also, Serra Mari et al is now published in Genome Biology: https://doi.org/10.1186/s13059-023-03160-z. Please update the reference. 3. There are a few grammar corrections to make.

Reviewer 2. Nicolas Farrugia

Comments to Author: Summary of review This paper fills a very important gap in the literature investigating time-varying functional connectivity (or dynamic functional connectivity, dFC), by measuring analytical flexibility of seven different dFC methods. An impressive amount of work has been put up to generate a set of convincing results, that essentially show that the main object of interest of dFC, which is the temporal variability of connectivity, cannot be measured with a high consistency, as this variability is of the same order of magnitude or even higher than the changes observed across different methods on the same data. In this very controversial field, it is very remarkable to note that the authors have managed to put together a set of analysis to demonstrate this in a very clear and transparent way. The paper is very well written, the overall approach is based on a few assumptions that make it possible to compare methods (e.g. subsampling of temporal aspects of some methods, spatial subsampling), and the provided analysis is very complete. The most important results are condensed in a few figures in the main manuscript, which is enough to convey the main messages. The supplementary materials provide an exhaustive set of additional results, which are shortly discussed one by one. Most importantly, the authors have provided an open source implementation of 7 main dfc methods. This is very welcome for the community and for reproductibility, and is of course particularly suited for this kind of contribution. A few suggestions follow. Clarification questions and suggestions : 1- How was the uniform downsampling of 286 ROI to 96 done ? Uniform in which sense ? According to the RSN ? Were ROIs regrouped with spatial contiguity ? I understand this was done in order to reduce computational complexity and to harmonize across methods, but the manuscript would benefit from having an added sentence to explain what was done. 2- Table A in figure 1 shows the important hyperparameters (HP) for each method, but the motivations regarding the choice of HP for each method is only explained in the discussion (end of page 11, "we adopted the hyperparameter values recommended by the original paper or consensus among the community for each method"). It would be better to explain it in the methods, and then only discuss why this can be a limitation, in the discussion. 3- The github repository https://github.com/neurodatascience/dFC/tree/main does not reference the paper 4- The github repository https://github.com/neurodatascience/dFC/tree/main is not documented enough. There are two very large added values in this repo : open implementation of methods, and analytical flexibility tools. The demo notebook shows how to use the analytical flexibility tools, but the methods implementation is not documented. I expect that many people will want to perform analysis using the methods as well as comparison analysis, so the documentation of individual methods should not be minimized. 5 - For the reader, it would be better to include early in the manuscript (in the introduction) the presence of the code for reproductibility. Currently, the toolbox is only introduced in the final paragraph of the discussion. It comes as a very nice suprise when reading the manuscript in full, but I think the manuscript would gain a lot of value if this paragraph was included earlier, and if the development of the toolbox was included much earlier (ie. in the abstract). 6 - We have published two papers on dFC that the authors may want to include, although these papers have investigated cerebello-cerebral dFC using whole brain + cerebellum parcellations. The first paper used continuous HMM on healthy subjects, and found correlations with impulsivity scores, while the second papers used network measures on sliding window dFC matrices on a clinical cohort (patients with alcohol use disorder). I am not sure why the authors have not found our papers in their litterature, but maybe it would be good to include them. Authors need to update the final table in supplementary materials as well as the citations in the main paper. Abdallah, M., Farrugia, N., Chirokoff, V., & Chanraud, S. (2020). Static and dynamic aspects of cerebro-cerebellar functional connectivity are associated with self-reported measures of impulsivity: A resting-state fMRI study. Network Neuroscience, 4(3), 891-909. Abdallah, M., Zahr, N. M., Saranathan, M., Honnorat, N., Farrugia, N., Pfefferbaum, A., Sullivan, E. & Chanraud, S. (2021). Altered cerebro-cerebellar dynamic functional connectivity in alcohol use disorder: a resting-state fMRI study. The Cerebellum, 20, 823-835. Note that in Abdallah et al. (2020), while we did not compare HMM results with other dFC methods, we did investigate the influence of HMM hyperparameters, as well as perform internal cross validation on our sample + null models of dFC.

Minor comments 6 - "[..] what lies behind the of methods. Instead, they reveal three groups of methods, 720 variations in dynamic functional connectivity?. " -> an extra "." was added (end of page 10).

Reviewer 2. Darrin Lemmer, M.S.

Comments to Author: This paper describes a method for improving the accuracy and efficiency of extracting a pathogen of interest (M. tuberculosis in this instance, though the methods should work equally well for other pathogens) from a "clinical" metagenomic sample. The paper is well written and provides links to all source code and datasets used, which were well organized and easy to understand. The premise – that using a pangenome database improves classification -- seems pretty intuitive, but it is nice to see some benchmarking to prove it. For clarity I will arrange my comments by the three major steps of your methods: dataset generation, human read removal, and Mycobacterium read classification. 1. Dataset generation -- I appreciate that you used a real-world study (reference #8) to approximate the proportions of organisms in your sample, however I am disappointed that you generated exactly one dataset for benchmarking. Even if you use the exact same community composition, there is a level of randomness involved in generating sequencing reads, and therefore some variance. I would expect to see multiple generations and an averaging of the results in the tables, however with a sufficiently high read depth, the variance won't likely change your results much, so it would be nice, and more true to real sequencing data, to vary the number of reads generated (I didn't see where you specified to what read depth for each species you generated the reads for), as it is rare in the real world to always get this deep of coverage. Ideally it would also be nice to see datasets varying the proportions of MTBC in the sample to test the limits of detection, but that may be beyond the scope of this particular paper. 2. Human read removal -- The data provided do not really support the conclusion, as all methods benchmarked performed quite well and, particularly when using the long reads from the Nanopore simulated dataset, fairly indistinguishable with the exception of HRRT. The short Illumina reads show a little more separation between the methods, probably due to the shorter sequences being able to align to multiple sequences in the reference databases, however comparing kraken human to kraken HPRC still shows very little difference, thus not supporting the conclusion that the pangenome reference provides "superior" host removal. The run times and memory used do much more to separate the performance of the various methods, and particularly with the goal of being able to run the analysis on a personal computer where peak memory usage is important. The only methods that perform well within the memory constraints of a personal computer for both long reads and short leads are HRRT and the two kraken methods, with kraken being superior at recall, but again, kraken human and kraken HPRC are virtually indistinguishable, making it hard to justify the claim that the pangenome is superior. Also, it appears your run time and peak memory usage is again based on one single data point, these should be performed multiple times and averaged. Finally, as an aside, I did find it interesting and disturbing that HRRT had such a high false negative rate compared to the other methods, given that this is the primary method used by NCBI for publishing in the SRA database, implying there are quite a few human remaining in SRA. 3. Mycobacterium read classification -- Here we do have some pretty good support for using a pangenome reference database, particularly compared to the kraken standard databases, though as mentioned previously, a single datapoint isn't really adequate, and I'd like to see both multiple datasets and multiple runs of each method. Additionally, given the purpose here is to improve the amount of MTB extracted from a metagenomic sample, these data should be taken the one extra step to show the coverage breadth and depth of the MTB genome provided by the reads classified as MTB, as a high number of reads doesn't mean much if they are all stacked at the same region of the genome. Given that these are simulated reads, which tend to have pretty even genome coverage, this may not show much, however it is still an important piece to show the value of your recommended method. One final comment is that it should be fairly easy to take this beyond a theoretical exercise, by running some actual real world datasets through the methods you are recommending to see how well they perform in actuality. For instance, reference #8, which you used as a basis for the composition of your simulated metagenomic sample, published their actual sequenced sputum samples. It would be easy to show if you can improve the amount of Mycobacterium extracted from their samples over the methods they used, thus showing value to those lower income/high TB burden regions where whole metagenome sequencing may be the best option they have.

Re-review.

This is a significantly stronger paper than originally submitted. I especially appreciate that multiple runs have now been done with more than one dataset, including a "real" dataset, and the analysis showing the breadth and depth of coverage of the retained Mtb reads, proving that you can still generally get a complete genome of a metagenomic sample with these methods. However kraken's low sensitivity when using the standard database definitely impacts the results, making a stronger argument for using a pangenome database (Kraken-Standard can identify the presence of Mtb, but if you want to do anything more with it, like AMR detection, you would need to use a pangenome database). I really think that this should be emphasized more, and perhaps some or all of the data in tables S9-S12 be brought into the main paper. It is maybe worth noting, that the significant drop in breadth, I would imagine, is a result of dividing the total size of the aligned reads by the size of the genome, implying a shallow coverage, but the reality is still high coverage in the areas that are covered, but lots of complete gaps in coverage. I did also like the switch to the somewhat more standard sensitivity/specificity metrics, though I do lament the actual FN/FP counts being relegated to the supplemental tables, as I thought these numbers valuable (or at least interesting) when comparing the results of the various pipelines, particularly with human read removal, where the various pipelines perform quite similarly.

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Reply to the reviewers

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

*The study examined the mechanisms behind the nuclear transport of capsid proteins of various flaviviruses. The study used mass spectrometry to identify the interaction partners of JEV capsid protein and found Importin 7 as the top hit. After validating this interaction with IP-western blotting, using IPO7 knock-out cells they showed that the nuclear accumulation of capsid is dependent on IPO7. Moreover, they also observed nearly 10-folds reduction in titre of virus produced from knock out cells without reduction in virus replication or particle assembly.

The study needs improvements to bring it to publication standards. Some overaarching problems include, all capsid localization studies being done with GFP-tagged capsid, and not wild type capsid produced during authentic infection, lack of quantitation of most of the localization data and not showing capsid localization from infection experiments in knock out cells, and no in-depth analysis of the potential mechanisms behind the observed reduction in titre in knock out cells etc.

Thank you for your constructive comments. We have sincerely answered all of them, as shown below. We hope you are satisfied with our additional data and the revised manuscript.

The major comments are

Fig 1B: Please add quantitation and statistical analyses of the ratio of nuclear and cytoplasmic capsid protein of all different capsids used. Also include western blot to prove that there is no cleavage between Capsid and GFP and the green signal indeed comes from the fusion protein. Ideally you should use capsid alone instead of a fusion protein for at least selected few constructs to prove that the Capsid-GFP behaves identical to Capsid alone.

Following the reviewer’s comments, we have added quantification and statistical data in Figure 1D. We have added CBB data and western blot data in Figures 1B and S1. Because recombinant proteins of low molecular weights were artificially translocated into the nucleus through diffusion, less than 20 kDa proteins are typically used as GFP or GST fusion proteins for the IJ and PM experiments. Instead of IJ and PM experiments, we have added data on the translocation of the non-tagged core using IFA and its statistical data in Figure 1A. Although in vitro data on the translocation of capsid protein differ somewhat from IFA data, the data on nuclear translocation of core proteins are consistent across different experiments.

Fig 1C: It is unclear from the figure legends the WT JEV capsid means GFP-Capsid or Capsid alone. You should clearly state the GFP part if the construct includes GFP. Quantitation and statistics are missing and the information on how many independent experiments were performed is also not included in the figure legend.

Following the reviewer’s suggestion, we have described that the JEV proteins fused GFP as follows: “AcGFP-JEVCoreWT or AcGFP-JEVCoreGP/AA” (Line. 771). We added quantification and statistical analysis as shown in Figure 1E. IJ and PM experiments were performed three times independently and described in the legend of Figure 1 in the revised manuscript (Lines 773–774).

Fig 2B: Quantitation and statistics are missing. Ideally, the data need to be reproduced with Capsid alone instead of Capsid-GFP. A positive control is needed for the activity of Bimax to prove that the drug was working in the assay.

We have added quantitative and statistical data in the revised Figure 2B. As mentioned above, capsid alone is potentially translocated into the nucleus artificially using the IJ and PM assay. Bimax binds to importin alpha but not importin beta, specifically inhibiting the importin alpha/beta pathway. The RanGTP mutant binds to the importin beta family, including importin beta 1, and widely inhibits importin beta-dependent nuclear import. These inhibitors are well-characterized and recognized in the field. We cited the following reference: Tsujii et al., JBC, 2015.

Fig 2C: How do you reconcile the IP mass spectrometry data that Importin b1 is the second strongest hit with the lack of IP interaction you observed in fig 2C?

As shown in Figure 2C, importin b1 does not interact with the JEV core. Importin b1 is the most abundant member of the importin beta family. Thus, it might be a non-specific interaction between importin b1 and the JEV core. Therefore, we excluded importin b1 from further analyses. We added a sentence to explain why importin b1 was excluded on Line 145.

Fig 3C: How many independent confirmations of this experiment was performed?

All IJ and PM experiments were performed thrice independently. We described this in the legend of Figure 3 in the revised manuscript (Line; 794).

Fig 4A and B: Add quantitation for the western blot. 4A-D Include data on the number of biological repetitions. 4C-D: Add quantitation and statistical analyses of the ratio of nuclear and cytoplasmic capsid protein.

We have added quantification data, as shown in Figures 4A and 4B. All experimental results shown in Figures 4A, 4B, 4C, and 4D were performed thrice independently, as described in the legend of Figure 4 of the revised manuscript (Lines; 810-812).

Fig 5B. This data should be shown in the context of infection with untagged Capsid at least for 1-2 viruses. This is a serious drawback of the present study as there is no clear evidence presented that the native capsid protein in an infection context depend on importin 7 for nuclear accumulation and behave similar to the GFP-Capsid constructs being used.

Following the reviewer’s concerns, we used an un-tagged JEV and DENV core to examine core translocation in WT or IPO7KO Huh7 cells. As shown in Figures 5C and 5D and their quantitative data, nuclear translocation of JEV and DENV core protein was inhibited in IPO7KO Huh7 cells. We tested the translocation of core protein upon infection with DENV as shown in Figure 5F. Although we could not examine ZIKV infection because we could not find appropriate antibodies against the ZIKV core, these data are consistent in that nuclear translocation of flavivirus core protein largely depends on IPO7.

Fig 5 A-D: Two repetitions are insufficient; a minimum of three biological repeats and statistical analysis need to be included. 5E-F: You cannot do statistics on two repeats, need minimum of three repeats to perform statistical analysis. 5G-H: I presume three repetitions based on the data points shown, this should be clearly stated in the figure legend.

We repeated three independent experiments, shown in Figures 5A and 5C-5F, and indicated them on Lines 823. We have added statistical data in Figures 5B-5F. We have corrected the statement of biological repeats in Figures 6A and 6B (Lines; 843-844).

Fig 5E-G: Taking the data of 5E and 5G together it seems Importin 7 functions as the level of particle release and not particle assembly or maturation. Have you checked for the specific infectivity of the particles released from knock out cells to determine the reason behind the reduction in virus titre? You could look at the prM maturation by furin cleavage to check it this is altered in the IPO7 knock out cells.

We determined the ratio of infectious titer per 103 copies of viral RNA in Figure 6F. The proportion of infectious viruses targeting extracellular JEV RNA was decreased in IPO7KO cells. Simultaneously, no difference was observed in the proportion of infectious viruses targeting intracellular JEV RNA between WT and IPO7KO cells. Although we could not find appropriate antibodies against the JEV core, we checked prM expression using the DENV virus. The expression of prM was slightly increased in JEV-infected IPO7-KO Huh7 cells (Figure S3D). This result suggests that the efficiency of prM cleavage by furin was partially involved in the impairment of infectious virus release in IPO7KO Huh7 cells.

Fig 5H: Have you checked if the observation regarding intracellular RNA levels in 5F is applicable to these viruses as well.

We checked the intracellular RNA levels of DENV and ZIKV-infected cells. In contrast to JEV, intracellular ZIKV or DENV RNA showed no difference in IPO7-KO Huh7 cells (Figure 6H). We discuss it in Discussion section (Lines; 269-271)

Fig 6: The figure legend "Data are representative of two (A, B) independent experiments and are presented as the mean {plus minus} SD of three independent experiments (C)" is confusing. The sentence should be reworded to state the repetitions separately for independent experiments. Fig 6C should show original titres and not percentages.

We have corrected Figure legends according to the reviewer’s comments. We have showed the original titers in Figures 6C and 6E.

Fig 7B: This experiment should be performed in IPO7 knock out cells to confirm that the observed reduction of core mutant is mainly contributed from its lack of interaction with IPO7 and not from any other confounding factors.

Following the reviewer’s suggestion, we performed SRIP experiments for GP/AA mutation using IPO7KO Huh7 cells. As shown in Figure 7C, the SRIPs harboring WT core were impaired in IPO7KO Huh7 cells; no difference was observed in the SRIPs harboring GP/AA mutations in WT and IPO7KO cells. These results suggest that IPO7-dependent nuclear translocation of core protein is important for the viral release.

Reviewer #1 (Significance (Required)): While the authors could convincingly demonstrate the interaction between capsid and IPO7, how that interaction results in the observed reduction in viral titre is largely unexplored. As all the localization data used a GFP-tagged capsid outside an infection context, this reviewer is not confident that all the reported observations will hold in an infection setting. This need to be urgently addressed to rise the confidence about the observation. The current data is insufficient to confidently attribute the change in titre to the interaction between capsid and IPO7 and the capsid localization to the nucleus. Knocking out IPO7 could have pleotropic effects independent of capsid nuclear accumulation that could lead to the observed titre reduction. This need to be addressed further before linking both these phenotypes. Certain key experiments needed to address these questions are currently missing. While the interaction of Capsid with IPO7 is certainly intriguing, the implications of this interaction on virus biology needed further investigation before clear conclusions can be drawn regarding this observation.

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

Summary: In this study Itoh and colleagues investigate the mechanism, role and impact of the nuclear localization of the flavivirus core protein. The import of the core protein has long been observed and investigated and herein the authors use some novel approaches to identify potential cellular binding partners that facilitate nuclear import. Via proteomics and biochemical approaches they determine that importin-7 plays a crucial role in the import of the core protein that appears to be conserved across Flavivirus members. In general the findings and conclusions are sound but there are some significant omissions and caveats that warrant further investigation.

Major comments: - one of the major caveats of the study is that the flavivirus NS5 protein also translocates to the nucleus in an Importin-alpha/beta dependent manner. Therefore how can the authors discount any impact of preventing NS5 import, in addition to core, on virus and SRIP replication and production. Some discussion, if not additional experiments are required here ie. NS5 localization in the KO cells during virus infection

We examined the localization of NS5 using IPO7KO Huh7 cells. As shown in Figure S2D and S2E, we confirmed that IPO7 was not involved in the nuclear localization of NS5.

• the localization is predominantly nucleolus rather that nucleoplasm when compared to the SV40 NLS. What are the sequence differences between the flavivirus proteins that potentially could account for this? A protein known to localize solely to the cytoplasm should also be used eg. NS1 or NS3.

The JEV core does not contain a consensus nucleolar localization signal. Nuclear localization of NS5 depended on importin-α similar to the SV40 NLS, while flavivirus core proteins were independent of importin-α. Gly42 and Pro43 are critical amino acids for the nuclear localization of the core protein, as shown in Figures 1C and 1D. The Gly42 to Pro43 of core proteins were well-conserved in the core proteins of the Flaviviridae family.

• controls for Figure 2? Ie. a protein known to be inhibited by Bimax but not the RanGTP mutant and vice versa.

Bimax binds to importin alpha but not importin beta and specifically inhibits the importin alpha/beta pathway. The RanGTP mutant binds to the importin beta family, including importin beta 1, and widely inhibits importin beta-dependent nuclear import. These inhibitors are well-characterized and recognized in the field. Therefore, we have cited the following references: Tsujii et al., JBC, 2015.

• Fig 5. Difference with WNV and DENV in nucleoplasm localization but also WNV still appeared to have Core in the nucleus in the KO cells

We agree with the reviewer’s comment about differences in nuclear localization among the viruses using the IJ assay. We have added new data to examine the localization of the DENV core after DENV infection. Nucleolar localization of the DENV core following DENV infection was observed, as shown in Figure 5F. Therefore, differences in nucleoplasm or nucleolar localization among different viruses shown in Figure 1C and Figure 5B might be artifacts of recombinant proteins. One possibility is that the localization of core proteins using IJ assay was detected by anti-GFP antibodies. Although purified GFP-core proteins, as shown in Figure 1B and S1, were observed as a single band of fusion proteins, core proteins of WNV and DENV might be cleaved during IJ experiments, and GFP alone might be detected at nucleoplasm, as shown in Figure 5B. Because our study focused on the nuclear translocation of flavivirus core proteins, the detailed localization of each core protein in the nucleus will be studied in the future.

• Fig 5C still has substantial JEV and DENV core but not WNV and ZIKV. Why is the DENV and WNV localization pattern different to Fig 5B?

We appreciate the reviewer’s suggestion; we re-checked all our data presented in Figure 5B and other data shown in Figure 5B. We quantified the ratio of nuclear localization as shown in the right of Figure 5B. Our quantification data showed that the nuclear transport of all core proteins used in this study was dependent on IPO7. In contrast, Figure 5A shows that nuclear translocation of WNV core protein is partially dependent on IPO7. This discrepancy might be explained that nuclear translocation of WNV core protein might be regulated by several nuclear carriers. We described this in discussion section (Line; 250-254).

• Fig 5F, does the KO also restrict NS5 from entering the nucleus and could this then results in increase polymerase activity confined to the cytoplasm resulting in more viral RNA?

Following the reviewer’s suggestion, we examined NS5 localization during viral infection and plasmid transfection, as shown in Figure S2D and S2E. Previous data regarding the nuclear localization of NS5 depended on importin-α. Our data are consistent with previous reports that IPO7 was not involved in the nuclear localization of NS5. In contract to JEV, we also confirm that intracellular ZIKV or DENV RNA showed no difference in WT and IPO7-KO Huh7 cells (Figure 6H). As described in the discussion, other factors, such as antiviral factors, might be involved in IPO7-mediated nuclear transports in JEV infected cells (Line; 269-271).

• Why was WNV infection not performed in Fig 5H? What where the viral tires compared to for the relative % values?

Because our institution does not have a BSL3 facility, we could not use WNV. Following the reviewer’s comment, we showed viral titers in Figure 6G.

• Fig 6B, still a significant amount of core present in the nucleolus. Also WT cells have (almost?) no cytoplasmic staining for core where this could be clearly observed in the WT cells in Fig 5D. Why the difference?

Plasmid transfection of AcGFP-Core WT showed that almost all core proteins were located in the nucleus. We assumed that AcGFP might influence nuclear exports of core proteins or the efficiency of nuclear transports as shown in other data of in vitro experiments. However, our finding that IPO7 was involved in the nuclear transport of core proteins is consistent.

• In Fig 7B, D and E, when were the SRIPs collected and what was the time period after subsequent infection?

Following the reviewer’s comments, we have added more details on SRIP experiments in Materials & Methods (Line; 521-523).

• In Fig 7C was the luciferase measured from the initial transfection and how did it correlate with RNA production? A 15-fold increase in replicon RNA actually seems quite low over a 48h period

Because large amounts of in vitro-transcribed replicon RNA were injected into cells in this experiment, we observed that significant amounts of luciferase values were detected after 4 h. However, the 15-fold enhancement in luciferase value was consistent with previous reports (PMID: 30413742, PMID: 17024179). We have added references in the revised manuscript.

• quantitation is required throughout all of the experimental IFA data provided

Following reviewer comments, we have quantified all IFA data and showed their results.

Reviewer #2 (Significance (Required)):

The nuclear translocation of flavivirus protein has long been studied and it has been observed that the core, NS5 (RNA polymerase) and potentially the NS3 (helicase/protease) proteins all translocate the nucleus. Importin alpha and beta have been shown to facilitate this process. The authors aim to extend this to identify importin-7 as a major cellular factor enabling nuclear translocation. Overall the experiments have been performed well but there is a lack of quantitation for many of the results an suitable controls are required.

I am a researcher in the field of flavivirus replication

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

In the presented study the authors identified and mechanistically investigated how Flaviviruses including Japanese encephalitis virus (JEV), Dengue virus (DENV), and Zika virus (ZIKV) commonly use importin-7 (IPO7), an importin-β family protein, as a cellular carrier protein to facilitate nuclear core protein translocation. The authors evaluated how the production of infectious viruses is regulated by IPO7 using cellular infection models including IPO7-deficient knockout cells. In the submitted manuscript, the authors provide evidence that IPO7 facilitates viral core protein import into the nucleus of infected cells, which is essential for effective Flavivirus replication. Taken together, the study is interesting to a broader readership with interest in molecular virology, and its findings are informative for potential future targeting of IPO7 to affect flavivirus replication using small molecule drugs. The manuscript is well-written and easy to follow, the methods are appropriate, the structure is logical, and statistical analysis is adequate.

Major comments:

• It is unclear why the authors specifically used Ala substitution at Gly42 anb Pro43 to obtain the abolishment of nuclear core protein localization. It would be helpful to put this into more context and explain the approach.

Mutations of Gly42 and Pro43 to Ala were previously reported and characterized by the same research group (PMID: 15731239). Following the reviewer’s comment, we have added more details of GP mutations in the text (Lines 66–70).

• In Figure 4, the authors claim that the binding between IPO7 and RPS7 is disrupted upon the addition of RanGTPQ69L. This is not clearly evident from the pulldown experiment and should be proven experimentally with additional experiments (e.g. by using an imaging approach) to underline the statement that the binding mode of IPO7 to the JEV core protein is similar to that of RPS7. Loading controls for pulldown blots should be added.

As described in response to the comment by reviewer#2 regarding Figure 2, the RanGTPQ69L mutant inhibits the interaction between the importin beta family, including IPO7 and its substrates, by directly binding to importin beta proteins. For the benefit of readers without knowledge of the typical Ran-dependent nuclear transport mechanism, we have described its effects with several cited references (Dickmanns et al., 1996; Tachibana et al., 2000). We referred to a study that showed that IPO7 transports RPL proteins, including RPS7 (Jäkel and Görlich, 1998). The data in Figures 4A and 4B demonstrate that adding RanGTPQ69L remarkably reduces the binding of IPO7 to the Core proteins and that the effect is more robust than that for RPS7. We believe that these results are experimentally valid, indicating that nuclear transport of Core proteins by IPO7 is achieved through a typical Ran-dependent pathway.

• Most methods used are presented logically but require some more details so that they can be reproduced. In particular, the difference between Figure 4 E and 4H is confusing. What is the difference? Is 4E showing intracellular viral titers and 4H infectious viral titers in the supernatant of cells? Clarification needed. Put relevance of these experiments in context of the hypothesis.

We apologize for the confusion regarding the data in Figures 5E and 5H (we assume). These data were derived from the same experiments, except for the time-course data presented in Figure 5E. We have removed Figure 5E to simplify our results.

• Identical phenotypes induced by IPO7 knockout in a number of HuH7 clones are shown in Figures 6A to 6C. This data does not add to the overall understanding and should be moved to supplementary figures. Why are 293T cells used in experiments shown in Figure 6D and 6E? What is the relevance of kidney cells to Flavirius infections?

Following the reviewer’s comments, we have moved Figure 6 to supplementary figures. We used 293T cells because of efficient JEV propagation and gene-deficient efficiency. We wanted to demonstrate that our data are not Huh7-dependent through experiments in 293T cells.

• Prior studies are referenced appropriately, however, in a recent study it was demonstrated that IPO7 is stabilized upon Epstein-Barr Virus infection and that IPO7 presence is required for the survival of host cells (Yang YC, Front Microbiol. 2021 Feb 16;12:643327. doi: 10.3389/fmicb.2021.643327).

We deeply appreciate the publications in these fields. Following the reviewer’s comment, we have cited these references.

This important study about the physiological relevance of IPO7 during viral infections has not been cited by Itoh and colleagues in the presented study. However, the results of the uncited study are very relevant to the provided manuscript, since Itoh and colleagues are using IPO7 knockout cells to investigate its function in Flavivirus core protein nuclear import. Hence, the authors should perform cell survival and cellular fitness experiments to demonstrate that observed phenomena of reduced viral replication and virus export in IPO7 knockout cells are independent of compromised cellular fitness due to IPO7 deficiency.

We evaluated cellular fitness between WT and IPO7KO Huh7 cells using PI (Propidium Iodide) staining through flow cytometry. As shown in Figure S2F, no differences were observed in cell viability between WT and IPO7KO Huh7 cells. It suggests that viral titers reduced in IPO7KO Huh7 cells are not involved in cellular fitness.

Minor comments:

• Describing Figure 3B, the authors state that they focused on IPO7 among the core binding proteins belonging to the importin-b family, because IPO7 "was identified the most peptides" in the mass spectrometry approach. This requires a more detailed explanation. Also, an explanation of why HEK293T cells were used for this approach and not HuH7 cells, as used predominately in most parts of the study, would provide more clarity to the reader.

We focused on IPO7 because it had the highest number of detected peptides, and we found that the second most detected peptide, IPOB1, did not bind to JEV core proteins as shown in Figure 2C. Therefore, we included the lack of interaction between IPO7 and IPOB1 as part of the rationale.

• In Figures 4E and 4F, colour coding is missing.

We have indicated color coding in this data. Thank you for your comments.

Reviewer #3 (Significance (Required)):

The provided manuscript 'Importin-7-dependent nuclear localization of the Flavivirus core protein is required for infectious virus production' by Itoh and colleagues investigates a topic with important scientific relevance. The presented study builds on previous findings by the authors where they have demonstrated that Flavivirus core protein nuclear localization is actually conserved among Flaviviridae and represents a potential target for broad-range antiviral small molecule drugs (Tokunaga et al., Virology, 2020 Feb;541:41-51). However, our understanding of Flavivirus core protein nuclear localization during viral replication and how the processes could potentially be targeted using novel therapeutic drugs remains elusive. Here, the provided manuscript addresses a mechanistic investigation of how the Flavivirus core protein is actually translocated from the cytoplasm to the nucleus of infected cells. The study is informative particularly for virologists with expertise in Flavivirus replication.

However, from my point of view as a virologist investigating host-pathogen interactions with a strong interest in clinical translational, the manuscript requires a more careful evaluation and interpretation of some results of key experiments. In addition, some of the results need to be more precisely described for clearer understanding by a broader readership.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Summary: In the manuscript entitled "Importin-7-dependent nuclear localization of the Flavivirus core protein is required for infectious virus production", by combining proteomics, CRISPR/Cas9 gene KO, CLSM and standard virology techniques, Yumi Itoh report novel data concerning the involvement of IPO7 in the nuclear and nucleolar localization of Flaviviridae core nuclear and nucleolar localization and viral particle release. Surprisingly, IMPa/b1 inhibition via Bimax2 does not affect core nuclear transport, whereas both RanQ69L and WGA did so. The authors try to identify the cellular transporters involved in core nuclear import, and to this end performed a MS spec analysis of JEV core interactors, which yielded IPO7 as the most likely candidate. After confirming the result by Co-IP, the authors go on showing most core proteins require IPO7 for nuclear delivery using Huh7 and HEK7 IPO7-KO cells, with the exception of WNV core which was able to partially enter the nucleus. In such cells, upon infection, extracellular (but not intracellular) viral titers were strongly reduced, a phenotype which was observed with a JEV core mutant bearing the Gly42 and Pro43 to Ala substitutions in a previous study.

Major comments: - The major conclusions of the study are:

1.IPO7 is the main driver of core nuclear transport 2.Core nuclear localization is somehow important for viral particle release Both conclusions are well-supported by experimental evidence.

Methods are clear and precise, the study appears to have been produced with high quality standards, and so is the presentation of the results. A few controls however should be added to increase the reliability of the results presented here (see below)

Since the authors attempt to link the phenotype observed on virus release upon IPO7 KO to defects on core nuclear import by making a parallelism with core GP/AA mutant, it would be important to know the behavior of such virus in Huh7 wt and Huh IPO7 KO cells. In other words, is GP/AA JEV released efficiently in Huh7 IPO7 KO cells?

We have added new data examining the propagation of the GP/AA JEV mutant in IPO7KO Huh7 cells (Figure 6F). Our new data showed that there were no differences in the propagation of the GP/AA mutant in WT and IPO7-KO Huh7 cells.

A similar approach can be applied to data shown in Figure 7 (effect on release on a capsid nuclear deficient mutant). This would help understand if IPO7 KO, viral release defects and core nuclear import are somehow linked.

We produced SRIPs harboring GP/AA core using WT and IPO7KO Huh7 cells and demonstrated that the number of infectious viruses produced by WT and IPO7KO Huh7 cells was the same (Figure 7C).

Minor comments:

INTRODUCTION • “Flaviviruses...are mosquito-borne human pathogens" What about tick borne encephalitis virus?

We have corrected it (Line; 43-44).

• " replication.... occur in the endoplasmic reticulum (ER)" This sentence is a bit inaccurate. Flaviviridae RNA replication occurs in so-called viral replication factories, double membrane vesicles which are partly derived from the ER. see "PMID: 26958917".

We have corrected this sentence according to the reviewer’s comment (Line; 60-62).

• "it is known that some flavivirus core proteins are translocated from the cytoplasm into the nucleus" o I think the first evidence of core in the nucleus dates back to 1989, and here it might be appropriate to cite the original reference: "PMID: 2471810". o It might be worth mentioning that NS5 has also been reported in the nucleus (See "PMID: 28106839")

We have corrected the sentence according to the reviewer’s comment (Line; 63-65).

• "In the cytoplasm, NLS-containing proteins are recognized by importin-α " o This is true only for classical NLSs, not every NLS binds IMPa, as the authors confirm in this study! Indeed, we have also PY-NLS, IPO7 specific NLSs, IPOb1 NLSs, etc. I therefore suggest rephrasing.

Thank you for pointing out the exact description of NLS. We agree with the reviewer’s comment that “NLS” includes all types of signal sequences, such as PY-NLS. To clearly distinguish between the CLASSICAL nuclear transport pathway by importin α/β1 and the various nuclear transport pathways by the importin β family, such as transportin, we refer to NLS as classical NLS (cNLS) in the document. We have modified the following sentence by adding “such as transportin” and “without importin-α.”

RESULTS

• Fig. 1. o it is not clear what is new here, with respect to what has been already published. The authors should clearly differentiate novel findings from confirmatory results

Thank you for your suggestion. We would like to introduce our new assay using recombinant virus core proteins, as shown in Figures 1C and 1D. The data shown in Figure 1 are crucial for understanding our data in Figure 2, and we believe this figure is required for broad-ranging readers.

Fig. 2 and 4 o Proteins whose nuclear transport is dependent on IMPa/IMPb1 (such as SV40 NLS) are lacking here

Bimax binds to importin alpha but not to importin beta and specifically inhibits the importin alpha/beta pathway. The RanGTP mutant binds to the importin beta family, including importin beta 1, and widely inhibits importin beta-dependent nuclear import. These inhibitors are well-characterized and recognized in the field. Therefore, we have cited the following references: Tsujii et al., JBC, 2015.

• Fig.5 o It would be important to know the effect on total virus infectivity (intracellular + extracellular) and total viral RNA. It would also be important the effect on RNA replication by using a subgenomic viral replicon (with deletion of the env gene for example). The question here is if IPO7 depletion affects to any extent viral genome replication, and this is impossible to assess in a fully assembling system. We determined the ratio of infectious titer per 103 copies of viral RNA in Figure 5D. The proportion of infectious viruses targeting extracellular JEV RNA was decreased in IPO7KO cells, and there was no difference in the proportion of infectious viruses targeting intracellular JEV RNA between WT and IPO7KO cells. We examined the effects of IPO7 on viral RNA replication of subgenomic replicon. We showed that the deficiency of IPO7 enhanced viral RNA replication as shown in Figure 7E. As described in the Discussion section, IPO7 may transport other factors possessing antiviral activity against flaviviruses. These data will be investigated in the future.

o Panels A-F legend is missing, consider adding it?

We have added more details to Figure 5A-5F following the reviewer’s suggestion.

• Fig.7 o I did not completely understand how NLuc is the readout here To quantify RNA replication, we quantified Nluc values using a plate reader. We have added more details on the reporter assay in Materials and Methods (Line; 521-523).

o Also, I do not understand if the effect of GP/AA substitution of panel B has already been reported or if it is a novel finding

Previous reports regarding the effect of GP/AA substitution of JEV showed the impairment of infectious virus release. However, the SRIP assay was performed to examine the viral release step. Our detailed data showed that the lack of IPO7-mediated nuclear transport of core proteins impaired infectious viral release, and our new results using SRIPs harboring GP/AA core showed that the lack of nuclear transport of core proteins also impaired the release of infectious viruses. Our data strongly suggest that the lack of nuclear transport of core proteins influences the viral release.

• All CLSM figures lack quantification (Fn/c; Fno/n)

We have added quantitative data for IFA experiments in our revised manuscript.

DISCUSSION

• "The nuclear entry of viral genomic DNA has been demonstrated to involve IPO7" o It would be nice to know which viruses the authors are freeing to here

We have added the virus name and corresponding references.

• "While RNA viruses, including flaviviruses, are considered to replicate in the cytoplasm of mammalian cells, increasing evidence suggests nucleolar localization of the viruses " o I suspect Rawlinson did not propose the viruses localize to the nucleolus, as this sentence seems to imply. Rather, a trafficking of viral proteins to nucleoli, to manipulate cell function, is more realistic. I suggest considering rephrasing. We have corrected this sentence.

Reviewer #4 (Significance (Required)):

SECTION B - Significance ========================

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. As alluded to above, this work presents several advances of current knowledge in the field of viral proteins nuclear trafficking, and in Flavivirus biology. The finding of most core proteins depending on IPO-7 is novel and intriguing, and opens the question of what makes WNV core special. Indeed, this protein nuclear targeting is only partially inhibited in IPO7 deficient cells. The fact that the authors extend their findings to several Flaviviruses adds significance. The role of nuclear core for virus release is also intriguing, but appears poorly characterized. In this respect a mechanistic explanation of the phenomenon would be highly desirable to increase the significance of the work presented here.

In this context I would have a few suggestions:

A) The authors performed MS spec on JEV core, this most likely resulted in a long list of "hits". However, they only report IMPb superfamily members. This is perfectly fine, since they focus at identifying partners responsible for nuclear import. However, it might be helpful for understanding the role of nuclear core. By comparing MS of wt core and GP/AA core, and or wt core in wt and IPO7KO cells, authors could identify core biding partners in the nucleus (in the nucleolus?) which are important for virus release. This could be subsequently addressed by knocking down these factors and study the effect on virus life cycle.

We appreciate the reviewer’s valuable comments. We did not perform MS analysis on GP/AA core protein and core protein using WT or IPO7KO Hun7 cells. To report IPO7-mediated core translocation simply, we would like to cite our manuscript focusing on IPO7. To clarify the importance of nuclear transport of core protein on the viral life cycle, we will perform wide-ranging proteomics.

1. B) Further, the authors should try to address the role of core in the nucleus (and nucleolus). Does it interact with cellular/nucleolar proteins? Does it deliver viral RNA to sites of assembly? Does it interfere with rRNA synthesis? All these findings would be easily obtainable using the GP/AA virus and/or Huh7 KO cells, and tremendously increase the impact of the study, which at the moment is limited at points 1 and 2 in the first section of the current report.

Thank you for your valuable comments. We agree that we should clarify the roles of the nucleus or nucleolar localization of the core protein. We tested the effects of rRNA synthesis on JEV core expression. Our data showed that core protein expression slightly impaired the maturation of rRNA synthesis, as shown here. However, the core expression did not influence protein translation. We focused on the phase separation capacity of core protein localized in the nucleolar or nucleus. From our accumulating data, we hypothesized that the acquisition of phase separation capacity of core protein might be involved in an efficient virus release step. We hope that these data will be reported in the near future.

Overall, this work should be interesting for both cell biologists interested in trafficking of viral proteins, and virologists interested in virus-host interactions. The antiviral approach at the moment is a bit less convincing, but the manuscript might be interesting for scientists trying to develop new antiviral strategies. (In this context it might be worth reading and possible discussing the very recent paper from the Bartenschlager group "PMID: 37702492." Also, I think that it would be worth discussing the recent discovery that a closely related virus belonging to the Hepacivirus genus within the Flaviviridae family, mediated re-localization of Nups to viral replication factories, where they are believed to control access to DMVs interior, thereby regulating virus replication and assembly. Could the core IPO7-interaction have any role in core delivery to DMVs? See "PMID: 26150811".

Thank you for your valuable comments. We have added several sentences in the Discussion section (Line; 297-305). We will investigate the role of nuclear transports in viral life cycles in the future.

Since I am a molecular virologist studying viral nucleocytoplasmic trafficking, virus-host interactions, and antiviral drug-discovery I think I have sufficient expertise for an informative and helpful revision of this work.

I chose this section because it’s extremely relatable to me as a STEM student who’s spent countless nights up staying up completing assignments or studying. Most community college STEM students are like me and want the perfect 4.0 to transfer to a nice University, so we all get our assignments to look “beautiful and wonderful” to get a perfect grade on it. This article is important to me because I’m more aware of neurodivergent students and the challenges they face, with my new knowledge I’ll be able to be more supportive towards my fellow future STEM classmates. I also think this article is important for STEM students because some of them could be neurodivergent students and this article could help them manage challenges they may have like work balancing. Now I’m not personally a neurodivergent student but I’ve noticed tons of the same traits are relatable to me which is interesting, overall I’m glad I chose this article because it’s really informative and helps you gather information about other people's struggles.

i have chosen this quote because not only can i personally relate to it, but i know many of my friends could relate to it too. when talking to a friend that i share a class with, we often wonder why everyone else understands the concepts much easier even though we are doing everything we can to try and understand. its very frustrating and often times makes me think i'm missing something, like everyone automatically knows what to do and im behind somehow, which all in all can lower confidence in classwork. so i could definitely relate to this paragraph. i think this article connects the idea of designing for equity and inclusion by giving a voice to the fact that neurodivergent students have felt this way for a long time and gave examples of ways they have had to adapt to the "ideal students" world.

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.

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

Manuscript number: RC-2024-02438

Corresponding author(s): Ryusuke, Niwa

1. General Statements [optional]

This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

Below are quotes from the Reviewers' overall evaluations:

As might be expected based on the authors' skills and expertise, the study is well executed, nicely documented with perfect microscopy images, and well presented. It has been easy to follow. However, suitability for publication depends on where the authors aim to place their paper. Although I like the paper very much, it might seem incomplete for high-end journals.

This is a very nice paper and solid piece of work.

Its major strength is the focus on poorly studied the male reproductive organ and identification of Ldh as a novel target of JH activity in the seminal vesicles.

While the developmental roles of insect Juvenile Hormone (JH) are very well studied, its adult functions are largely unknown. Target genes of JH signaling are poorly described. This study adds significant insight into both of these aspects. The study underscores the usefulness of the JHRE-GFP reporter that identifies JH function, and not just JH presence since the reporter is only expressed after JH binding to Met and Gce, a prerequisite for JHRE reporter activation.

The authors have identified the epithelial cells of the ____Drosophila____ seminal vesicle as a JH target tissue. The authors nicely extended this finding by mining already existing expression data to identify a specific JH induced gene in these cells.

This small study reports new but limited results (one tissue of one stage, one hormone) that could be useful for specialists. The work is solid and includes controls and interpretable data.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

1) The study suggests an important role for JH signaling in the SV, likely affecting reproductive capacity of males. The authors depleted the JH receptors through RNAi, achieving a loss in the expression of the WT JHRE-GFP reporter as well as of the authentic target Ldh. Surprisingly, no phenotypic consequences of the double KD of Met and gce are presented. Does that mean that there were none? The authors only discuss a potential impact of Ldh loss for metabolism. Unless I am missing something, the study reports molecular phenotypes that clearly document JH signaling in the SV but no physiological impact of loss of this JH signaling, suggesting that there may be no obvious biological role for JH in this context. I think this is unlikely. Have the authors check fertility of the males, sperm viability and quality, mating competitiveness of the RNAi males? Loss of JH epoxidation (only methyl farnesoate present) made mosquito males less fit and less reproductively competitive relative to epox+ controls (Nouzova et al., 2021, PNAS) -- btw, I think the authors should discuss this paper.

Our response: We will conduct the following experiments to answer these criticisms.

1) We will examine the male fertility by counting the number of offspring from wild-type mothers crossed with males of the seminal vesicle-specific ____Met _& _gce____ double RNAi and with males of control RNAi.

2) We will also examine the mating competitiveness of the RNAi males. In more detail, we will cross ____w1118_ (white eye) wild-type background females with (i) a mixed population of males of _w1118_ wild-type background males and_ w+_ (red eye) control RNAi males, and (ii) a mixed population of males of _w1118_ wild-type background males and_ w+ Met _& _gce____ double RNAi males. We can distinguish between the progenies from RNAi males and those from wild-type males by eye colors.

By conducting plans 1) and 2), we will also indirectly evaluate sperm viability and quality.

In addition, we will also discuss the paper of Nouzova et al. PNAS 2021 in the Discussion section.

2) The authors seem to have made no effort to distinguish between Met and Gce functions. It is always the results from the double knockdown of both paralogs that are presented. Does this mean that single-KD had no effect, thereby indicating entirely redundant functions of both proteins in the studied context? Even if so, it would be of interest to document this redundancy by showing the single-gene KD data. However, I would be surprised if both proteins were equally important in the SV. The authors checked mRNA/protein expression levels. Was any of the two paralogs prevalent in the SV?

Our response: To address this criticism, we will conduct a single transgenic RNAi experiment to knock down either Met or gce separately and assess JHRE-GFP signals in the seminal vesicles.

__ Regarding the expression of Met and gce in the seminal vesicles, a previous study (Baumann et al. Scientific Reports 7: 2132, DOI:10.1038/s41598-017-02264-41) has already reported that GFP signals are observed in the seminal vesicles of _Met-T2A-GAL4>UAS_-GFP and gce-T2A-GAL4>UAS-GFP animals. These results strongly indicate that both Met and gce are expressed in the seminal vesicles. We will describe and discuss this point in our revised manuscript. In addition, we plan to check and analyze gene expression of Met, gce, and Ldh in the seminal vesicles using a publicly-available single-cell RNA-seq database, such as _DRscDB (https://www.flyrnai.org/tools/singlecell/web/).

3) The authors argue for direct regulation of Ldh by Met/Gce (again by which one?). Oddly, the statement in the Results (l.187-188; "suggests ... direct target") is stronger than in the Discussion (l.214, "leaving open the possibility"). The putative JHREs upstream and within the Ldh gene are identified but not tested in a functional study. At least a simple luciferase reporter assay and mutagenesis of the JHREs should be attempted.

Our response: To address this criticism, we plan to conduct a luciferase-based promoter/enhancer analysis in Drosophila S2 cultured cells. A similar system was used for a JH-responsiveness of the JHRE promoter in a previous study (Jindra et al. PLoS Genetics 11: e1005394, DOI: __10.1371/journal.pgen.1005394). We will generate plasmid constructs carrying the luciferase coding regions. In these plasmids, the luciferase coding regions will be fused with the upstream region and the first intron region of Ldh possessing the intact E-boxes or the mutated E-boxes. Then, we will determine whether the luciferase activity is enhanced by the presence of a JH analog (methoprene) when E-boxes are intact. __

__ For this revision, a new collaborator, Ryosuke Hayashi (a graduate student in the Niwa lab), will participate in this analysis. Thus, he becomes a co-author in the revised manuscript.__

l.232-233. It is not surprising that the JHRR-lacZ reporter shows a different expression pattern relative to JHRE-GFP, as these are really different constructs. The problem is that JH-dependent activation of the JHRR-lacZ transgene has not been tested as thoroughly as that of JHRE-GFP. Is it inducible by added JH or methoprene?

Have the authors examined whether JHRE-lacZ expression increases with Methoprene?

Our response: We have yet to do this analysis. To address this important point from Reviewers #1 and #2, we will examine whether JHRR-lacZ expression is upregulated in the seminal vesicles of virgin males fed methoprene-supplemented food. The lacZ signals will be visualized by immunostaining with an anti-LacZ antibody.

Document testis staining of JHRE-GFP. I think the authors missed a chance by not providing a clear/nice picture of the testis staining. Stainings of testes squashed on a slide is easy and would nicely document in which cells the reporter is activated. Similarly, extracting sperm from the seminal vesicle and examining whether the sperm express JHRE-GFP would be informative.

Our response: As the reviewer suggested, we will assess JHRE-GFP signal in sperm in squashed testis samples.

Did the authors try to analyze the 66 genes identified in seminal vesicle whether they had JHRE elements? This could yield additional significant information about other JH responsive genes in the seminal vesicle.

Our response: We have yet to do this analysis. We will follow the reviewer's suggestion and examine whether the 66 genes identified in the seminal vesicle have JHRE elements.

3a. Doublestaining would further confirm that pd8-Gal4 (crossed to UAS-dsRed) and JHRE-GFP overlap.

3b. Similarly, Doublestaining would further confirm that pd8-Gal4 (crossed to UAS-dsREd) and JHRE-GFP overlap.

Our response: To address this question, we will generate males of Pde8-GAL4; UAS-red fluorescent protein (RedStinger, RFP, or DsRed); JHRE-GFP and observe the overlap between the red fluorescent signals and green fluorescent (JHRE-GFP) signals in the seminal vesicle epithelial cells.

Minor comments:

Fig.1a could be in a supplement.

__Our response: At this point, we are unsure whether to follow this reviewer's suggestion. This is because there are no supplemental figures in the current manuscript, so we hesitate to create a supplemental figure just for this one figure. On the other hand, three reviewers now ask us to perform various additional experiments, thus some of the new data may be shown as supplemental figures. In this case, Fig. 1a can be moved to a supplemental figure, but we would like to wait on this decision. __

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

l.25,91,117, and throughout, "JH analog" or "JHA". The authors only use methoprene, so it would be better to specifically talk about methoprene, which is a proven agonist ligand of the JHR proteins (reference 10 and/or Jindra and Bittova, 2020 [Arch Insect Biochem Physiol] for a review). This would land more credibility to using methoprene than just referring to a "JHA".

Our response: According to the reviewer's suggestion, we have replaced "JHA" with "methoprene" as many as possible. In Figures, we used "MTP" instead of "methoprene" due to space limitations.

l.42,44, "paralogs". I believe in this case the authors refer to orthologs of Met in other species. Paralogs result from gene duplications within species, such as Met and gce in cyclorrhaphous flies or Met 1 and 2 in the Lepidoptera. I recommend a recent review on all bHLH-PAS proteins featuring reconstruction of the phylogenetic position of Met/Gce (Tumova et al., 2024 in J Mol Biol).

Our response: As suggested, we have replaced "paralogs" and "paralogous" with "orthologs" and "orthologous," respectively on P3. We have also cited Tumova et al. J. Mol. Biol. 2023 as a new Ref 12.

l.54, "Met and Gce act redundantly to regulate JH-responsive gene expression". Ref 10 should be cited here as it provides functional cell-based and genetic rescue evidence for each paralog.

Our response: We have cited Ref 10 as suggested.

l.66, It would be better to start "In this study" or "Here" to distinguish from the last cited paper.

Our response:____ We created a new paragraph with the sentence "In this study..." at the beginning. We hope we understand the reviewer's suggestion correctly.

l.175, levels were

Our response: We have fixed this error in the transferred manuscript.

l.209, might be evolutionarily among.... conserved ??

Our response: We have fixed this error in the transferred manuscript.

l.226, study has

Our response: We have fixed this error in the transferred manuscript.

l.227-229. The authors are missing a paper by Shin et al., 2012 (PNAS) that shows physical interaction of Met with Cycle and their regulation of circadian gene activity and another paper by Bajgar et al., 2013 (PNAS) which describes photoperid-dependent seasonal regulation of circadian genes by Met, Clk and Cyc.

On the other hand, the cited reference [51] does NOT demonstrate Met:Clk heterodimer since coIP is by no means adequate to address complex stoichiometry. In fact, it is suspicious that Met would heterodimerize and either Cyc or Clk, as they present class II and class I bHLH-PAS proteins.

Our response: In response to both comments from Reviewer #1, ____we have cited these references and rewritten the discussion on P10-11 as below: "An interesting previous study has reported that the seminal vesicle expresses multiple clock genes such as period, Clock (Clk), and timeless, all of which are necessary for generating proper circadian rhythm [52]. In the case of the mosquito Aedes aegypti female, it is reported that JH controls gene expression via a heterodimer of Met and circadian rhythm factor Cycle (CYC) [53]. It was also suggested that Met binds directly to CLK in D. melanogaster [54]. In addition, in the linden bug, Pyrrhocoris apterus, JH alters gene expression via Met, CLK, and CYC in the gut [55]. Considering these previous reports and our results, circadian rhythm factors and JH may cooperate to regulate gene expression in the seminal vesicles."

l.245. It is not "whether", but for sure the existing reporters only reflect limited JHR activity, being based on Kr-h1 JHREs. These reporters likely uncover only a small subset of JH activity in vivo.

Our response: We have rewritten the sentence as follows: "..., more comprehensive JH reporter strains will be needed in D. melanogaster as well as other insects in future studies."

reference 10/11 is duplicated.

Our response: We have fixed this error in the transferred manuscript.

Have the authors done a careful comparison of JHRE-GFP expression and the Met/gce reporter expression described by Baumann et al (Scientific Reports | 7: 2132 | DOI:10.1038/s41598-017-02264-4)? Would be nice to add a few more sentences in the discussion.

Our response: As suggested, we have added some sentences to explain this point on Page 11 as below: "P____revious studies reported that ____Met-T2A-GAL4_ and _gce-T2A-GAL4_ labeled male accessory glands, ejaculatory duct, and testes as well as seminal vesicles. On the other hand, in our results, JHRE-GFP only labels cells in seminal vesicles and testes [21]. Considering that Met and Gce are expressed in almost all cell types of male reproductive tracts [21], more comprehensive JH reporter strains will be needed in _D. melanogaster____ as well as other insects in future studies."

• In the discussion:*

6.1 Would have liked to see a more in depth discussion of the role of the seminal vesicle. How could that be supported by JH / metabolic processes? Does it have secretory functions that might be induced by JH? Important functions relative to sperm storage? How could that relate to the finding that JH response is enhanced by mating?

Our response: Unfortunately, the function of the seminal vesicles is largely unknown. However, ____in response to the reviewer's suggestion, we have added some sentences to discuss this point and cited some references describing the seminal vesicles in insects other than the fruit fly, as follows on P9-10: "Furthermore, in some insects other than D. melanogaster, morphological and ultrastructural studies revealed that secretory vesicles were observed in the epithelial cells of the seminal vesicles [37,38,40,44]. JH is known to stimulate secretory activity in the male accessory glands of many insects [45]. Based on the JH response in the seminal vesicles, it is possible that JH signaling affects the secretory activity of the seminal vesicles in D. melanogaster."

The arrow in figure is not defined

Our response: We believe that the reviewer pointed out the arrow in Figure 1e. We have added a sentence to define the arrow in the Figure legend as "The arrow indicates the cell with a GFP signal."

Figure 2b graph labels are flipped

Our response: We have fixed the error.

Line 624: Change "Allow heads" to "Arrowheads"

Our response: We have fixed this error in the transferred manuscript.

Major Comments:

The work uses standard methods and strains. Although the specific findings are new and believable, the authors interpret them beyond what is appropriate. For example, based on increased amounts of a single RNA, they propose that JH regulates metabolism in seminal vesicles and because circadian rhythm genes were known to be expressed in this tissue they propose that JH and circadian systems work together there.

Our response: In response to the reviewer's criticisms, we have discussed our arguments more appropriately in the Discussion. For example, we have mentioned circadian rhythm more carefully on Pages 10-11 as follows: "An interesting previous study has reported that the seminal vesicle expresses multiple clock genes such as period, Clock (Clk), and timeless, all of which are necessary for generating proper circadian rhythm [52]. In case of mosquito Aedes aegypti female, it is reported that JH controls gene expression via a heterodimer of Met and circadian rhythm factor Cycle (CYC) [53]. It was also suggested that Met binds directly to CLK in D. melanogaster [54]. In addition, in the linden bug, Pyrrhocoris apterus, JH alter gene expression via Met, CLK and CYC in the gut [55]. Considering these previous reports and our results, it is possible that circadian rhythm factors and JH cooperatively regulate gene expression in the seminal vesicles."

__ Regarding Ldh, we have added a sentence on Page 10 as "Also, the biological significance of the induction of Ldh expression by JH signaling is not clear."__

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

l.244, tract

Our response: We have carefully checked out the usage of "tract" and "tracts" not only on Page 11 but also throughout the manuscript. We have decided to use "tracts," but not "tract," throughout the manuscript.

6.2 What do epithelial cells of spermatheca do?

Our response: We agree with the reviewer that this is a very interesting question. However, please note that this paper focuses on males, and females are beyond our current scope. We plan to examine JHRE-GFP signals in the spermatheca in a different project. We do appreciate the reviewer's kind understanding.

6.3 How do the authors envision that JH enters the epithelial cells?

__Our response:____ We don't have any hypotheses on this point. Transporters may exist to achieve intracellular permeability of JH, but we do not think this point has been discussed in current insect physiology. Furthermore, since this issue is related to all JH-responsive cells, not just seminal vesicle epithelial cells, we do not feel the need to discuss it in this paper. __

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.

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

We thank the reviewers for their time and effort to improve and clarify our manuscript. We now have addressed the reviewers’ suggestions in full on a point-by-point basis. Revisions in the manuscript file are highlighted in yellow.

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

Supernumerary centrosomes are observed in the majority of human tumors. In cells they induce abnormal mitosis leading to chromosome missegregation and aneuploidy. In animal models it is demonstrated that extra centrosomes are sufficient to drive tumor formation. Previous work studying the impact of centrosome amplification on tumor formation in vivo used Plk4 overexpression to drive the formation of supernumerary centrosomes. In this manuscript Moussa and co-workers from the Krämer group developed a mouse model in which centrosome amplification is triggered by the overexpression of the structural centrosomal protein STIL rather than the kinase Plk4 in order to a) assess the potential for centrosome amplification induced by STIL overexpression to drive tumor formation and b) to rule out any potential non-centrosomal related effects of the kinase Plk4 on tumor formation.* The authors show that STIL ovexrexpression in cells (MEFs) drives centrosome amplification and aberrant mitosis (Fig. 1), leading to chromosome missegregation and aneuploidy (Fig. 2). They also show that STIL overexpression is linked to reduced cellular proliferation and apoptosis (Fig 3). The authors then present in vivo experiments performed in mice. They observed that STIL expression causes embryonic lethality, microcephaly and a reduced lifespan (Fig 4). Despite increased STIL mRNA levels they do not detect elevated STIL protein levels in adult tissues except for the spleen. They do not detect significant increase of centrosome amplification or aneuploidy in animal tissues (Fig 4) and they conclude of a STIL translational shut down in most adult tissues. The authors then assess the impact of STIL overexpression on tumor formation. They observed a reduced spontaneous tumor formation despite elevated STIL mRNA levels in both healthy and tumor (lymphomas) tissues of mice overexpressing STIL. They don't detect increased centrosome amplification and aneuploidy in lymphomas from STIL overexpressing mice compared to lymphomas naturally occurring in control animals (Fig 5). Finally, they found that STIL overexpression suppresses chemical skin carcinogenesis using a combination of tamoxifen induction of STIL in the skin with DMBA/TPA carcinogenic treatment (Fig 7). They link this effect to an increased number of centriole and a reduction in cycling cells number in the skin of STIL overexpressing mice (Fig 6).

The manuscript is written in a clear manner. The experimental approaches are properly designed and the experimental methods are described in sufficient details. Most of the experimental data present a good number of replicates. The figures are generally well assembled despite some errors in a few panels/legends (see major and minor points). Most of the conclusions are supported by the experimental data. However, a few specific points or interpretations are not convincingly supported by the experimental data (see major points) and will need to be revised and/or reformulated.

Major points:

1. Figures 1D and F show that MEFs hemizygous (CMV-STIL+/-) and homozygous (CMV-STIL+/+) for STIL present similar level of centrosome amplification and aberrant mitosis. Although, despite these similarities the homozygous MEFs display about two time more micronuclei and chromosomes aberrations (Fig. 2). The authors explain this discrepancy by the fact that MEFs homozygous for STIL have reduced proliferation and an increased propension to stay in interphase compared to hemizygous MEFs (Fig. 3). I don't understand why an interphase arrest would lead to a higher chromosomal instability resulting in higher micronuclei formation and abnormal karyotypes since those phenotypes are the consequences of abnormal mitosis occurring in cycling cells. I would rather argue that Homozygous MEFs are more prone to cell cycle arrest because of mitotic errors, but those mitotic errors cannot be explained by the centrosome status or the mitotic figures quantified in homozygous MEFs. Therefore, the authors explanation written as: "Graded inhibition of proliferation and accumulation of cells in interphase explains why CMV-STIL+/- and CMV-STIL+/+ MEFs contain increasing frequencies of micronuclei and aberrant karyotypes (Fig. 2) despite similar levels of supernumerary centrosomes" is not right for me. The authors should reformulate this section of the manuscript so their conclusion fit their data. The differences between hemi and homozygotes MEFs regarding chromosome stability could come from mitotic errors they did not spot using fixed immunofluorescence images of mitotic MEFs. Thus, as an optional additional experiment, analyzing live mitosis of MEFs could potentially help reconciliate results from mitotic figures and from karyotypes.*

We basically agree with the reviewer and have therefore reanalyzed our data on centriole numbers in a time-dependent manner. As already shown in Figure 3L of the initial manuscript version, the number of both CMV-STIL+/- and CMV-STIL+/+ MEFs with supernumerary centrioles increases with passaging from passage 3 (p3) to p6. Also, in this experiment amplified centrioles were more frequent in CMV-STIL+/+ compared to CMV-STIL+/- MEFs in both passages (p3 and p6) analyzed. We have therefore now pooled the data and substituted the former Figure panel 1D by these combined results. As the results of Figure 1F and especially those for the CMV-STIL+/+ MEFs had to rely on very low mitotic figure counts, because these cells only very rarely divide (as shown in Figure 3A; mitosis frequency of CMV-STIL+/+ MEFs 0.12%), we have now deleted Figure panel 1F from the manuscript. For the same reason - an extremely low proliferation and division rate of especially CMV-STIL+/+ MEFs - live cell imaging to detect different types of mitotic errors, is unfortunately not feasible.

Figure 5 panel F does not support the claim of the main text and does not match the legend of the figure: In the text the authors wrote: "Ki67 immunostaining revealed that, ..., proliferation rates were elevated independent from lymphoma genotypes". If the authors claim and increased cell proliferation in lymphoma compared to lymph nodes, which is expected, they should show the data for the lymph node in the graph. In addition, in the legend the authors mentioned a "Percentage of Ki67-positive cells in healthy spleens and lymphomas from mice with the indicated genotypes." Since there are three genotypes and two tissue types but the figure presents a graph with only three bars did the Spleen and lymphoma data were combined? Or did some data were not inserted in the graph? Thus, since the data does not support the claim for an increased cell proliferation in lymphoma, the authors explanation for the increased protein level observed in these lymphomas (Fig. 5 panel E) is not supported. Therefore, the authors need to present the correct data in the figure or to change their conclusion. They will also need to correct the figure legend and to add a panel with images illustrating the Ki67 labelling in the different tissues in the figure.

We apologize for this mistake and have corrected the legend to Figure panel 5F, which now reads: “Percentage of Ki67-positive cells in two B6-STIL, two CMV-STIL+/- and one CMV-STIL+/+ lymphoma. For comparison, frequencies of Ki67-positive cells in healthy lymph nodes from B6-STIL mice are displayed. Data are means ± SEM from at least two independent immunostainings per lymphoma or healthy lymph node. P-values were calculated using the one-way ANOVA with post-hoc Tukey test for multiple comparison. For space reasons, only statistically significant differences are displayed”.

   We agree with the reviewer that for comparison Ki67 immunostainings of healthy lymph node tissue was missing in the graph and have therefore added this information to the figure panel, which shows increased proliferation of lymphoma compared to normal lymph node cells. Also, a panel with images illustrating Ki67 labelling in healthy lymph node and lymphomas from different genotypes has been added to the figure (panel 5G).

• *

__Minor points:____* * __1. In the introduction, page 4 paragraph 3, the authors wrote: "To assess the impact of centrosome amplification on CIN, senescence, lifespan and tumor formation in vivo without interfering with extracentrosomal traits,..." they need to clarify what they meant by extracentrosomal traits.

As requested by the reviewer we have modified the respective sentence, which now reads: “To assess the impact of centrosome amplification on CIN, senescence, lifespan and tumor formation in vivo with an orthologous approach without interfering with PLK4, we generated transgenic mouse models overexpressing the structural centrosome protein STIL, …”.

• *

In the 1st paragraph of the results, page 4, the authors wrote: "leads to ubiquitous transgene expression at levels similar to the CAG promoter used in most..." but there is no link to a figure presenting the mRNA levels in those mice (potentially Fig. 4F and Fig. S6). Also, in the references cited for comparison, to my knowledge, there was no measurement of Plk4 mRNA levels in tissues in the work from Marthiens and colleagues, in this work the authors assess the expression of the Plk4 transgene by investigating the presence of the protein.

To show STIL transgene expression levels in our system, we have now linked Figure panels 1A (STIL mRNA expression in MEFs), 1B (STIL protein expression in MEFs) and Supplemental Fig. S2 (Supplemental Fig. S6 of the previous manuscript version showing STIL mRNA levels in healthy mouse tissues) to this statement as suggested. In the references now cited for comparison (Kulukian et al. 2015; Vitre et al. 2015; Sercin et al. 2016) PLK4 transgene mRNA (Kulukian et al. 2015; Sercin et al. 2016) and protein levels (Vitre et al. 2015) are shown.

• *

Page 5 second line the authors wrote: "Despite the graded increase in Plk4 expression, CMV-STIL+/- and, CMV-STIL+/+ MEFs exhibited a similar increase in supernumerary centrioles". The authors must meant increase in STIL expression or do they have data not shown about an increase of Plk4 expression? Then they explain this absence of difference in supernumerary centriole by the ability of "excess Plk4" to access the centrosome, again they probably meant STIL. Regarding this point and related to Major Point 1 it might be worth for the authors to quantify actual extra centrosomes in mitosis rather than cells with more than 4 centrioles in interphase (as in Fig. 1C, D). They might find differences in the number of centrosomes in hemizygous versus homozygous MEFs.

We indeed meant STIL instead of PLK4 and have corrected the mistake. As described in our response to the reviewer’s major point 1 we have now reanalyzed our data on centriole numbers in a time-dependent manner. As already shown in Figure 3L of the initial manuscript version, the frequency of both CMV-STIL+/- and CMV-STIL+/+ MEFs with supernumerary centrioles increases with passaging from passage 3 (p3) to p6. Also, in this experiment amplified centrioles were more frequent in CMV-STIL+/+ compared to CMV-STIL+/- MEFs in both passages (p3 and p6) analyzed. We have therefore now pooled and substituted the former Figure panel 1D by these combined results.

Page 5, in the first paragraph the authors mention "the rate of respective mitotic aberrations..." without defining the mitotic aberrations. For instance, in panel 1E a metaphase with 4 centrosomes is shown for CMV-STIL+/- while an anaphase with an unknown number of clustered centrosomes is presented for CMV-STIL+/+. Classifying the different types of aberrant mitotic figures (i.e: multipolar anaphases versus bipolar with clustered centrosomes) might help the authors identify differences between hemi and homozygous MEFS that may explain the differences in the proportions of chromosomes aberrations they present in Fig. 2.

As described in our response to the reviewer’s major point 1 the number of mitotic figures that could be analyzed was extremely low, especially for CMV-STIL+/+ MEFs, which do only rarely divide (mitosis frequency of CMV-STIL+/+ MEFs 0.12%). Therefore, although certainly of value, classification of different types of mitotic aberrations is unfortunately not feasible.

• *

In Fig 4A the number of mice analyzed should be mentioned.

After mating of B6-STIL transgenic animals with CMV-CRE mice and further breeding of successive generations, we obtained a total of 198 pups over four generations, 162 of which were born alive: 116 B6-STIL wildtype animals, 27 CMV-STIL+/- and 19 CMV-STIL-/- mice. We have now added these numbers to the figure legend.

• *

In Fig. 5E, the band corresponding to STIL protein is difficult to visualize in the B6-STIL control, it is therefore difficult to compare its level to the level of STIL protein in the CMV-STIL hemizygotes and homozygotes. If possible, it would improve the manuscript to present a blot with clearer results.

We have tried to improve the quality by repeating the Western blot. Due to the small size of healthy mouse lymph nodes, resulting in low protein yields, only lysates from lymphomas were left, and these were of poor quality with a high lipid content. We therefore tried to delipidate the lymphoma lysates and hope that the result of the new blot is now somewhat clearer. Due to the low lymphoma frequency in CMV-STIL hemizygotes and homozygotes (only 2 in each case) we were unfortunately not able to prepare fresh lysates.

Related to Figure 6B the authors wrote a "5 to 10 fold-increased expression..." in the text while panel 6B show a maximum of 8 fold increase.

The respective statement has been rephrased according to the reviewer´s suggestion.

__Reviewer #1 (Significance (Required)): ______ *Centrosome amplification is a demonstrated cause of genomic instability and tumor development as shown in multiple previous work performed in mice. In this work, Moussa and co-workers developed a mouse model that does not depends on Plk4 to trigger centrosome amplification but which depends on the overexpression of the centrosome structural protein STIL. This effort is welcome as previous works could not formally rule out potential role of Plk4, not related to its centrosome duplication function, on tumor formation. The authors show that their system is functional in MEFs where STIL overexpression drives centrosome amplification and aneuploidy. Unfortunately, in vivo, despite elevated level of STIL mRNA they do not detect centrosome amplification in tissues and consequently, they do not observe an increase rate of aneuploidy and tumor formation. This result is not surprising as previous studies using strong promoters (comparable to the one used to drive STIL expression in this study) to induce Plk4 overexpression led to similar results, i.e. an absence of centrosome amplification in adult tissues and no effects on tumor formation. Therefore, the results and the concepts proposed in this work are not novel but they reinforce previous studies showing the deleterious effect of high level of centrosome amplification on cells. This work also confirms that strong mechanisms, here the authors propose a translational shut-down, are preventing the apparition or the persistence of high level of centrosome amplification in animal tissues. By complementing existing results with the use of an alternate experimental approach this study will be of interest for the scientific community working on the basic biological mechanisms driving aneuploidy and tumor development.*

__Reviewer #2 (Evidence, reproducibility and clarity (Required)):______ *In this manuscript, Moussa et al. describe the effects of over-expressing the centriole duplication factor STIL in whole mice and with expression restricted to the skin. They find that over expression of STIL, similar to that of PLK4, induces centriole overduplication, abnormal mitoses, and genetic instability leading to cell arrest. Additionally, over-expressing STIL results in microcephaly, perinatal lethality and a shortened lifespan. In addition, they do not find that expression of the p53 R127H mutant alleviates the cell growth defect. Moreover, overexpression of STIL does not lead to increased general tumour formation and suppresses tumour formation in an induced skin tumour model.

Although this is an interesting manuscript, the authors need address a number of issues before this manuscript can be recommend the manuscript for publication. Importantly, the manuscript lacks statistical analyses to support some of their conclusions, some figures should be quantified, and controls are missing in some cases. *

__Major Issues____* * __1. Many of the figure panels lack appropriate statistical analyses to support the conclusions (see details below). This needs to be rectified.

In view of the limited number of mice (due to an increased frequency of pups that died around birth) and the resulting impossibility of performing several (>3) independent experiments in many cases, we have decided to limit the statistics in the main text to a descriptive analysis without mentioning inferences (p-values). Nevertheless, we have now included the missing statistical analyses in the figure panels and/or legends. However, the reported p-values (*p≤0.05, **p≤0.01, ***p≤0.001; ns, not significant) should be interpreted as descriptive rather than confirmatory values.

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The authors suggest that the interpretation of PLK4 over-expression studies are hampered by the possibility of centriole/centrosome independent PLK4 roles and that STIL overexpression circumvents some of these issues. Although orthologous approaches to problems are always desired, STIL itself has also been implicated in other cellular processes, such as the Sonic hedgehog pathway (Carr AL, 2014) and in cell motility (Liu Y, 2020). In addition, the data presented in the manuscript are suggestive of a STIL function in the mouse that is independent of centriole number. The authors demonstrate that the amount of centriole over-duplication in MEFs containing a single copy of the STIL over-expression locus is equivalent to that of MEFs carrying two copies. However, in most other assays, the homozygous lines display more severe phenotypes, suggesting that STIL might have a function outside centriole duplication. The authors need to discuss this further in a revised manuscript.

As described in our response to major point 1 and minor point 3 of reviewer 1 we have now reanalyzed our data on centriole numbers in a time-dependent manner. As already shown in Figure 3L of the initial manuscript version, the number of both CMV-STIL+/- and CMV-STIL+/+ MEFs with supernumerary centrioles increases with passaging from passage 3 (p3) to p6. Also, in this experiment amplified centrioles were more frequent in CMV-STIL+/+ compared to CMV-STIL+/- MEFs in both passages (p3 and p6) analyzed. We have therefore now pooled the data and substituted the former Figure panel 1D by these combined results, which show that, similar to other models, also regarding STIL overexpression the homozygous line displays a more severe phenotype, which does therefore per se not argue for a STIL function outside the centrosome. However, as a few recent studies indeed suggest additional roles of STIL, we have amended the respective passages in the revised version of the manuscript accordingly.

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Why did the authors use the p53 R127H mutant instead of a p53 knockout or null allele system? The R127H mutant has a gain-of-function phenotype and cells expressing this mutant display different phenotypes than a p53 null. The primary conclusion in one of the references cited by the authors (Caulin C, 2007) is that p53R127H is a gain-of-function mutant and behaves distinct from loss-of-function p53 mutations, such as deletions using floxed alleles. Throughout the manuscript, the authors use terms that suggest the R127H allele is equivalent to a loss of function mutant. Given that supernumerary centriole growth arrest is universally suppressed by inactivation of p53 it is somewhat surprising that this pathway is not active in response to STIL over-expression. The authors should confirm this key conclusion by depleting p53 in MEFs using RNAi, or by using mice where complete inactivation of p53 can be achieved.

We agree with the reviewer that the p53-R172H mutant version of p53 is not equivalent to a p53 knockout. We have therefore and as suggested by reviewer 3 as well (see also our response to point 3 of reviewer 3) corrected the wording and have substituted “absence of p53” by “interference with p53 function” where appropriate. In addition, we now have added data to the manuscript, which show that neither p53 expression nor p53-S18 phosphorylation becomes induced during prolonged cultivation and passaging of CMV-STIL transgenic MEFs (see Figure 3B of the revised manuscript). Importantly, this finding is in line with a recent report showing that PLK4-induced extra centrosomes may not rely on p53 for tumor suppression and cell death induction (Braun et al.: Extra centrosomes delay DNA damage-driven tumorigenesis. Sci. Adv. 10: eadk0564, 2024). Similarly, it has been recently shown that centrosome amplification increases apoptosis independently of p53 in PLK4-overexpressing cells treated with DNA-damaging agents (Edwards et al.: Centrosome amplification primes for apoptosis and favors the response to chemotherapy in ovarian cancer beyond multipolar divisions. bioRxiv 2023.07.28.550973, 2023). Therefore, these findings and references have now been added to results and discussion sections of the revised manuscript.

   A plethora of p53-related findings in mouse models, including the majority of results on PLK4-induced tumor formation in mice, is based on p53 knockouts, a situation that is only rarely found in human cancers. In contrast, the p53-R172H missense mutation in mice corresponds to the p53-R175H mutation in human tumors, which has the highest occurrence in diverse human cancer types among all p53 hotspot mutations, and results in a transcriptionally inactive protein that accumulates in cells, similar to the majority of naturally occurring versions of mutant p53 (Yao et al.: Protein-level mutant p53 reporters identify druggable rare precancerous clones in noncancerous tissues. Nat Cancer 4: 1176-1192, 2023; Chiang et al.: The function of mutant p53-R175H in cancer. Cancers 13: 4088, 2021). We therefore believe that it more faithfully recapitulates the situation in p53-mutant tumors than a p53 knockout.

   Although basically an important and valid experiment, depleting p53 in STIL-transgenic MEFs using RNAi is not easily done as (i) transfection of MEFs per se is difficult and (ii) STIL-overexpressing MEFs do only slowly proliferate and are prone to senescence and apoptosis (see Figure 3), all phenotypes which are even further exacerbated after transfection. Generation of STIL-transgenic mice with complete inactivation of p53 on the other hand is an extremely time-consuming endeavor that would lead to a significant delay of publication of our results. Given that currently similar data are published by other groups (Braun et al.: Extra centrosomes delay DNA damage-driven tumorigenesis. Sci. Adv. 10: eadk0564, 2024; Edwards et al.: Centrosome amplification primes for apoptosis and favors the response to chemotherapy in ovarian cancer beyond multipolar divisions. *bioRxiv* 2023.07.28.550973, 2023), we do not think that this would be appropriate.

__Minor Issues and details____* * __Figure 1 1. Panel E. It is unclear what the authors are calling an 'aberrant mitosis'. Typically an aberrant mitosis refers to chromosomal abnormalities such as multipolar spindles, anaphase bridges or micronuclei (which they quantify in Figure 2). The aberrant mitotic figures presented in Figure 1E show a clustered metaphase with 4 centrosomes (2 per pole; 2 centrioles per centrosome) for CMV-STIL+/- MEFs and a clustered telophase with 2 centrosomes (1 per pole; 5 centrioles per centrosome) for CMV-STIL+/+ MEFs. This is now specified in detail in the legend to Figure 1E.

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Panel E. Please include images representing a normal mitosis from control cells derived from B6-STIL mice.

As suggested, we have now included a representative image of a normal mitosis from B6-STIL control mice.

Figure 2____ 1. Panels B, E and F. Statistical significance is not indicated between B6-STIL and CMV-STIL+/- or CMV-STIL+/- and CMV-STIL+/+. The authors indicated a 'graded' phenotype which is qualitatively apparent, but should be backed by statistical analysis.

We have now included a statistical analysis. However, and as already described in our answer to major issue 1 of this reviewer, the reported p-values should be interpreted as descriptive rather than confirmatory values due to the limited number of independent experiments.

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Can the authors indicate how they scored a tetraploid cell? Some of the cells are 100% tetraploid while others contain other aberrations.

According to the International System for Human Cytogenomic Nomenclature (ISCN) version from 2020, polyploidy is defined by the modal numbers of chromosomes in the karyotype. A number of 81-103 chromosomes is called near-tetraploid, at which a hypotetraploidy (81-91 chromosomes) is distinguished from a hypertetraploidy (93-103 chromosomes) (An International System for Human Cytogenomic Nomenclature, Karger (2020), Eds.: McGowan-Jordan, Hastings, Moore). For mouse karyotypes respective numbers were recalculated on the basis of a diploid chromosome content of 40 instead of 46 chromosomes. To be strictly in accordance with this nomenclature, we have exchanged the term "tetraploid" by "near-tetraploid".

__ Is the height of the rows in Panel D significant? What are the solid black rows?______ We thank the reviewer for this comment/observation. We have now increased the resolution of this part of the figure. Unfortunately, the resolution had deteriorated so much when the pdf file was created that individual lines were no longer recognizable. The height of the lines should be identical, as single lines correspond to the karyotypes of each metaphase cell analyzed, while chromosomes are plotted as columns. The solid black lines separate independently established MEF lines with the indicated STIL genotypes from each other. At least 20 metaphase cells per MEF line were analyzed. We have now explained these points in the figure legend.

Figure 3____ 1. Panels C, F, G, and K require statistical analyses.

We have now included the appropriate statistical analyses in the figure panels and/or legends. However, the reported p-values should be interpreted as descriptive rather than confirmatory values due to the limited number of independent experiments.

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Panel D should be quantified.

We have now included a quantification of the protein bands in panels B, E (former panel D), and K of the revised manuscript and explained the quantification procedure in detail in the methods section.

Panel E. mRNA expression is quantified in RPKM here, while GeTMM is used in Figures 3I and Supplementary Figures S2 and S6. Is there a reason this panel uses a different method? RPKM can be used for intra-sample comparisons, but is not ideal for comparison among different samples.

We now uniformly quantify mRNA expression in GeTMM in all figures of the revised manuscript version as requested.

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Panel G. Can the authors show the original FACS profiles in Supplementary material?

As requested, we have now included representative examples of original FACS profiles from the cell cycle analyses into Supplemental Figure S5.

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Panel H. Requires molecular weight markers

Molecular weight markers for the DNA ladder (L) with the corresponding bp size have now been included into the Figure panel (formerly 3H, 3I in the revised version of the manuscript).

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__ Panel J. Missing B6-STIL control. Quantify Western blots.______ We have now included an immunoblot showing STIL protein expression levels in passage p1-p5 of B6-STIL control MEFs as well as a quantification of the protein bands into the Figure panel (formerly 3J, 3K in the revised version of the manuscript). The quantification procedure has been explained in detail in the methods section of the revised manuscript version.

Figure 4____ 1. The authors mention 'Simultaneously, we found an increased frequency of pups that died around birth.' Can the data for this be included?

After mating B6-STIL transgenic animals with CMV-CRE mice and further breeding of successive generations, we obtained a total of 198 pups over four generations, of which 162 were born alive: 116 B6-STIL wildtype animals, 27 CMV-STIL+/- and 19 CMV-STIL+/+ mice. We have now added these numbers to the figure legend. Stillbirths increased over the generations: while in the first generation after mating B6-STIL animals with CMV-CRE mice all pups (B6-STIL wildtype animals and STIL heterozygotes) were born alive, in the fourth generation (from mating CMV-STIL transgenic mice with each other) 54% of the pups were stillborn. We have now included this observation into the main text to further emphasize the impact of STIL overexpression on perinatal lethality.

Panels B and D. Please include the data for CMV-STIL+/-.

We now have included a representative H&E-stained histological section of a CMV-STIL+/- mouse brain into Figure panel 4D as suggested by the reviewer. For space reasons we have not added an extra image of a CMV-STIL+/- total brain into Figure panel 4B, as this does not add novel information.

Panels C, F and K require statistics.

As requested, we have now included the appropriate statistical analysis in the figure panels and/or legends. However, the reported p-values should be interpreted as descriptive rather than confirmatory values due to the limited number of independent experiments.

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Panel F. Include statistical analysis.

We have now included the appropriate statistical analysis in the figure panels and/or legends. However, the reported p-values should be interpreted as descriptive rather than confirmatory values due to the limited number of independent experiments.

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Panel G/H. The levels of STIL in the CMV-STIL+/+ spleen are higher than the other samples, yet there is no concomitant increase in centriole overduplication. Can the authors comment on this?

Interestingly, we indeed found a higher STIL protein expression level in spleen tissue from CMV-STIL+/+ as compared to B6-STIL control and CMV-STIL+/- mice. Nevertheless, the amount of splenocytes with supernumerary centrioles was only marginally increased in these animals. A similar finding has recently been described for B lymphocytes with upregulated PLK4 expression after PLK4 transgene induction by exposure to doxycycline in vivo (Braun et al.: Extra centrosomes delay DNA damage-driven tumorigenesis. Sci. Adv. 10: eadk0564, 2024). Here, the lack of B cells with supernumerary centrioles despite increased PLK4 levels was explained by increased apoptosis and thereby selection against and rapid loss of PLK4-overexpressing cells. In line, we show that CMV-STIL+/+ MEFs have increased rates of senescence and apoptosis (Fig. 4).

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__ Panel J. The font within the plots is difficult to read. ______ We thank the reviewer for this comment/observation. We have now increased the resolution of this figure panel, and the font is now outside of the plots.

Figure 5____** s should be interpreted as descriptive rather than confirmatory values due to the limited number of independent experiments. No further statistical analysis can be done for panel D as in some cases (lymph node from B6-STIL mouse, lymphoma from CMV-STIL+/+ mouse) only one measurement exists.

Panel F. The legend indicates that these data are from spleens and lymphomas. Is this correct? Would the results from non-lymphoma cells in the spleen mask the results from lymphoma cells?

We apologize for this mistake and have corrected the legend to Figure panel 5F, which now reads: “Percentage of Ki67-positive cells in two B6-STIL, two CMV-STIL+/- and one CMV-STIL+/+ lymphoma. For comparison, frequencies of Ki67-positive cells in healthy lymph nodes from B6-STIL mice are displayed. Data are means ± SEM from at least two independent immunostainings per lymphoma or healthy lymph node. P-values were calculated using the one-way ANOVA with post-hoc Tukey test for multiple comparison. For space reasons, only statistically significant differences are displayed”.

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Panel F. The authors indicate that 'In line, assessment of lymphomas from B6-STIL control, CMV-STIL+/- and CMV-STIL+/+ mice by Ki67 immunostaining revealed that, corresponding to STIL protein levels, proliferation rates were elevated independent from lymphoma genotypes'. However, Ki67 levels, the marker for proliferation actually decreased in these samples indicating less proliferative cells. This needs to be clarified since the data shown appears to show the opposite of what is stated in the mansucript....

As noticed by the reviewer further below, differences in the percentages of Ki67-positive, proliferating cells between lymphomas from B6-STIL, CMV-STIL+/- and CMV-STIL+/+ mice were statistically not significant. However, we have now for comparison added the results of Ki67 immunostaining of healthy lymph node tissue to Figure panel 5F, which show increased proliferation of lymphoma compared to normal lymph node cells. Also, a panel with images illustrating Ki67 labelling in healthy lymph node and lymphomas from different genotypes has been added to the figure (panel 5G). These data reveal that, independent from the genotype, proliferation rates of lymphoma cells are increased as compared to healthy lymph nodes, thereby further corroborating our assumption that STIL protein levels in lymphomas are increased as a consequence of their increased proliferation and independent from STIL transgene expression.

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Corresponding to point 3 above, the authors suggest that 'STIL protein expression is a consequence of increased lymphoma cell proliferation.' This hypothesis cannot explain STIL protein levels if proliferation has actually decreased.

Please see our response to point 3 above.

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Corresponding to point 3 and 4 above, the actual data is marked as non-significant indicating there is actually no proliferative difference among the samples.

This is correct. See also our comments to point 3 and 4 above.

__ Panel 5I. The authors state that 'On the other hand, overall levels of chromosomal copy number aberrations were higher in lymphomas (mean gains + losses: 225.2 Å} 173.7 Mb) as compared to healthy tissues (mean gains + losses: 87.3 Å} 127.5 Mb; p=0.06), irrespective of their STIL transgene status (Fig. 4J; Fig. 5I), although the difference did not quite reach statistical significance.' The authors need to soften this statement since statistically, the samples are not different. For example, 'On the other hand, overall levels of chromosomal copy number aberrations appeared to trend higher in lymphomas as compared to healthy tissues irrespective of their STIL transgene status, although the difference did not quite reach statistical significance.'______ The statement was rephrased according to the reviewer´s suggestion.

Figure 6____ 1. Panels A, B, and C require statistical analysis.

We have now included the appropriate statistical analyses into panels A, B, and C in the figure panels and/or legends. However, the reported p-values should be interpreted as descriptive rather than confirmatory values due to the limited number of independent experiments.

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The figure legend references to panels C and D appear to be swapped.

We thank the reviewer for this comment/observation. We have corrected this mistake.

Panel F. Indicate that the samples are not significantly different.

We have now included the appropriate statistical analysis including the indication that the samples are not statistically significantly different.

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__ Corresponding to point 3, the authors indicate that 'the proportion of Ki67-positive cycling cells was lower in tamoxifen-treated... ... although the difference did not quite reach statistical significance.' The authors need to soften this statement to reflect that the samples are not statistically different (i.e. 'appeared lower' or similar).______ The statement was rephrased according to the reviewer´s suggestion.

__Figure 6 and 7 _ Do you have data for B6-STIL animals treated with and without tamoxifen? The experiments as shown demonstrate the differences between control and tamoxifen-treated animals of the same genotype, but it is unclear if any of these effects are due to the underlying genotypes or from tamoxifen itself. ___ The experiments presented in Figures 6 and 7 have not been performed in B6-STIL control mice with and without tamoxifen treatment.

Supplemental Figure 1____ 1. Please include molecular weight marker for this and all panels showing PCR products.

Molecular weight markers for the DNA ladder (L) with the corresponding bp size have now been included into all Figure panels showing PCR products as requested.

The B6-STIL and CMV-STIL+/- lines should contain a larger MW band corresponding to the STIL-F and STIL-R PCR product. Please show if possible.

We thank the reviewer for the important remark. We agree that there should be a large PCR product band at around 3000 bp containing the bacterial neomycin phosphotransferase gene (TK-neo-pA) and the STOP cassette in the B6-STIL control mice/MEFs, and two PCR product bands (large: 3000 bp, small: 410 bp) in the heterozygous CMV-STIL+/-mice/MEFs. When we began with genotyping, we did indeed observe both bands depending on the STIL background (see figure below). However, the band intensity of the larger PCR product was relatively weak (arrowheads) compared to the smaller PCR product, and its visibility was dependent on genomic DNA input and PCR efficiency. During the PCR optimization process, the PCR conditions were changed in such a way that the yield of the small band were increased despite small input amounts of genomic DNA, but at the expense of the large PCR product band (arrows). At the end of the optimization process the larger PCR product had almost disappeared, making the discrimination between heterozygous CMV-STIL+/- and homozygous CMV-STIL-/- DNA difficult. Therefore, we decided to additionally check for STOP cassette excision in a second PCR approach in parallel. In the genotyping results shown in Supplemental Figure S1B, which have been produced after PCR optimization, no larger STIL PCR product band was visible anymore.

__Supplemental Figure 6 _ 1. The 'Spleen' sample is missing the B6-STIL control data. 'Liver' is missing CMV-STIL+/+. Please include or indicate why they are missing. The plot order of the samples differs for 'Liver' (red, black) compared to the others (black, red, blue). Indicate statistical significances. ___ We apologize for this mistake, have corrected the Figure (formerly Supplemental Figure S6, S2 in the revised version of the manuscript), and have included the missing spleen and liver samples.

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General issues ____ 1. The materials and methods indicate that HPRT and PIPB were used as reference genes, but only HPRT is referred to in the qPCR figure legend.

We thank the reviewer for this comment/observation. As generally recommended (Vandesomele et al., Genome Biol 3(7): research0034.1-research0034.11, 2002; Kozer and Rapacz, J Appl Genet 54(4): 391-406, 2013) we used both reference genes for accurate normalization of qPCR in all experiments. We have now corrected this mistake in the figure legend.

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Figure panels 1F and 3C display 95% confidence intervals while others use SEM. Is there a reason for this?

In the two referenced figures (former Figure 1F has been deleted from the manuscript, see also our comment to point 1 of reviewer #1 for reasons; Figure 3C of the former manuscript is now Figure 3D in the revised manuscript version) the endpoint variable was defined by whether individual cells in a single experiment showed a certain property or not (binary variables). By definition, these kinds of variables show a nonsymmetric error structure, which cannot be expressed properly by a single value such as the standard error (SEM), but can be covered correctly by a confidence interval. For the same reason, Fisher’s exact tests were employed to obtain p-values in these situations. In the other figures, the relevant endpoint variables were roughly normally distributed, either directly, or due to them being an average of many values. In this case, a symmetric SEM was thus considered sufficient, and t-tests were used for p-values. To make this clear in the figures, we used different display options to distinguish between error bars showing SEM or 95% CI.

__Reviewer #2 (Significance (Required)): ______ *In this manuscript, Moussa et al. describe the effects of over-expressing the centriole duplication factor STIL in whole mice and with expression restricted to the skin. They find that over expression of STIL, similar to that of PLK4, induces centriole overduplication, abnormal mitoses, and genetic instability leading to cell arrest. Additionally, over-expressing STIL results in microcephaly, perinatal lethality and a shortened lifespan. In addition, they do not find that expression of the p53 R127H mutant alleviates the cell growth defect. Moreover, overexpression of STIL does not lead to increased general tumour formation and suppresses tumour formation in an induced skin tumour model. Although this is an interesting manuscript, the authors need address a number of issues before this manuscript can be recommend the manuscript for publication. Importantly, the manuscript lacks statistical analyses to support some of their conclusions, some figures should be quantified, and controls are missing in some cases. *

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): ______ Previously it has been proposed that supernumerary centrioles play important deleterious effects in vivo including increased tumorigenesis. However, the work was inconclusive because the way of inducing centriole amplification via the PLK4 kinase could have induced other effects besides supernumerary centrioles. To resolve this question, the authors generated a mouse model of centrosome amplification, in which the structural centriole protein STIL is overexpressed. Using this mouse model in vivo along with mutant mouse embryonic feeder (MEF) lines in vivo, the authors test out the role of centrosome amplification in vivo in animal development, lifespan, and tumorigenesis. They report both embryonic lethality, defects in brain development, and shortened life span in these mice. They also find that skin tumorigenesis is reduced in the mutant mice, and demonstrates that the STIL overexpression effects are not perturbed in a dominant negative p53 model. The authors demonstrate that STIL overexpression causes centrosome amplification accompanied by aneuploidy, which however is highly deleterious for cell fitness even in the absence of p53. Clearly, tissue corrective mechanisms lead to the elimination of cells with extra centrosomes and/or aneuploidy by impaired proliferation, senescence, and apoptosis. This finding is interesting and significant and seems worthy of dissemination to the broader readership.

This study is thorough and well executed and there is a significant body of work that leads to solid conclusions. The data is convincing, and the figure are well presented. It was refreshing to read this paper, as it was not so cluttered with data that the message gets murky, yet the data was clearly very substantial. The text is clear and easy to follow.

There really are only minor aspects of this paper that need correction, in my opinion. The text should be thoroughly checked for typos, few extra redundant words here and there, and a couple of confusing sentences.______ As suggested by the reviewer we have rechecked the manuscript for typos, redundancies, and confusing sentences and corrected where necessary and appropriate. __* *

For example, the last sentence in abstract is confusing 'These results suggest that supernumerary centrosomes... [result in]... tumor formation' because it should read 'reduced tumor formation' or 'impairs tumorigenesis' or otherwise be written more clearly because it seems to convey the opposite message the way it is right now. ______ We thank the reviewer for this comment and have corrected the sentence, which now reads: “These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation”. The p53 dominant negative mutant is not exactly a KO so it is not fair to say "in the absence of p53"; the verbiage should be corrected and checked throughout the paper - perhaps 'interfering with p53 normal function' is more appropriate.__ As suggested by the reviewer we have corrected the wording and have substituted “absence of p53” by “interference with p53 function” where appropriate. The sentence "Senescence- and apoptosis-driven depletion of the stem cell pool may explain reduced life span and tumor formation in STIL transgenic mice." from discussion is highly speculative and should be edited to clearly convey its speculative nature or removed entirely. ______ We agree with the reviewer and have deleted the sentence from the discussion section of the manuscript.

__Reviewer #3 (Significance (Required)): ______ Clearly, tissue corrective mechanisms lead to the elimination of cells with extra centrosomes and/or aneuploidy by impaired proliferation, senescence, and apoptosis. This finding is interesting and significant and seems worthy of dissemination to the scientific community. It adds to previous work on another centriole related protein PLK4 kinase that led to very different conclusions.

I chose this section because it gives another perspective on certain design choices. As we all are aware, design is relative and many people have a lot of their own personal preferences for a 'good' or a 'bad' design. In relation to designing for equity and inclusion, I chose this text since I find it interesting how different cultures may have different perspectives, opinions, and solutions to their designs. For example, as a freelance graphic designer, I myself often rely on my own experience and exposure of knowledge to digital media. If my client were to ask me to make a design for their packaging according to whatever I'd like it to be, I would make the packaging based on my knowledge, my opinion, and my belief on what I think would be a good design. However, if my client were to have a more specific request on what they would want the packaging to be, I would cater the design to how they want it to be. Sometimes, their preferences can come across as questionable or unflattering in my opinion, but at the end of the day I remind myself that again these people are requesting these designs according to their own experience and knowledge on their culture's exposure which would influence their preferences in design. Hence why, this text is very impactful to me as it encapsulate a designer's entire relationship with a client in one sentence. Having soft skills such as: open-mindedness, flexibility, and compromise to the clients' needs are also important factors in becoming a good designer, not only the technical skills matter. As we do this we are indirectly including people from different backgrounds, experiences, and abilities to be involved in the design process in order to create a more effective and relevant end product.

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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.

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*- 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.

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

RESPONSE TO REVIEWS_RC-2024-02383

We thank all the reviewers for their comments and suggestions. Our point-by-point response is shown below, in bold.

—----------------------------------------------------------------------------------------------------------------------------

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

Summary: the work presented by the authors detail how pharmacological inhibition of the rate limiting one carbon metabolic enzyme DHFR by the drug methotrexate increases the lifespan of yeast and worms. Furthermore, placing aged mice on dietary folate and choline restriction potentially enhanced metabolic plasticity but did not significantly increase lifespan with sex specific differences observed.

The findings in this manuscript are very interesting and important to our understanding of the conserved mechanisms that regulate longevity through one carbon metabolism. This is especially significant in light of the current folate intake and supplementation in the adult human population. The manuscript, however, requires major revisions. Please see comments below for details.

Major comments:

1. The overall tone in this manuscript is colloquial and conversational in nature. A third person academic style and tone, while avoiding the use of subjective descriptive terms would improve the quality of this text. Using terms such as "appeared less diverse", "results are remarkable ...strikingly more pronounced", "possibly positive outcomes" , "appear younger...for unknown reasons", "little Uracil", "tended to be higher", "roughly proportional", "slightly higher", "as a rough readout", and many other examples from the text should not be used in a scientific manuscript. The language should be academic, scientific, precise, and non-ambiguous. A thorough revision of the manuscript with substantial changes to the language and tone is necessary prior to publication. RESPONSE: Thank you for your feedback on the manuscript's tone. We revised most of the expressions mentioned by the reviewer. We note, however, that these phrases were used along with numbers and statistics. Hence, there was no lack of specifics, and readers could quickly evaluate the conclusions. We strive for a balance between scientific rigor and readability to maintain accessibility for a diverse audience.

In the results section, we find multiple instances where the results are interpreted and extensively discussed. This should be reserved for the discussion section. The results section should be used to simply report the findings in a detailed manner.

RESPONSE: We appreciate the suggestion on the integration of interpretation within the Results section. Upon review, we have clarified the presentation of our findings, ensuring a more distinct separation from interpretive commentary. Brief explanations remain to aid the reader's comprehension in light of the complex data, aiming to keep the flow and coherence of the manuscript and prevent overextension of the Discussion section (already ~1,300 words long). We welcome specific suggestions for further refinement.

The materials and methods section is severely lacking in details in some areas. For example, no details were provided regarding how the worm lifespans were conducted and previous work of collaborators were referenced instead. Important details such as worm numbers, biological and technical replicates, solid agar vs liquid culture, temperature, use of FUdR, antibiotics, transfer frequency, methods of scoring, etc... are lacking. Other details such as the preparation of the plates (Was MTX incorporated into the agar, seeded with the bacterial lawn, or liquid culture was used), storage conditions, age of the plates when lifespan started, how was the UV killing of the lawn verified etc...

many other methods subsections lack crucial details. Please carefully review the methodology and include sufficient pertinent details.

RESPONSE: The number of worms assayed in each case were shown in each figure, as described in the legend. We now also added all the information requested by the reviewer in the methods section. The text now reads:

“Briefly, the assays were done on solid agar nematode growth media (NGM) plates prepared fresh before each experiment. The bacterial lawn was exposed twice to a UV dose of 120mJ/cm2 using a UVC-515 Ultraviolet Multilinker (Ultra-Lum, Inc.). Streaking these UV-exposed bacteria to fresh LB agar plates (1% w/v tryptone, 0.5% w/v yeast extract, 1% w/v sodium chloride) produced no visible colonies. Methotrexate, or the ATIC inhibitor, was first dissolved in dimethyl sulfoxide (DMSO) and then added to the media used to prepare the plates after autoclaving (the media were kept in a 50°C water bath until the plates were poured). Mock-treated control plates contained only DMSO. At the start of each experiment, a sufficient number of eggs were collected from plates without any drugs and then placed on plates containing the indicated doses of each compound tested. After hatching and progression to the adult stage, animals were transferred to new plates (marked as the start of the lifespan assay) containing the drug tested and fluorodeoxyuridine (FUDR; dissolved in water), added at 50μM to block hatching of new animals. The plates were scored at least every other day until all the worms died. If an animal responded to gentle touch, it was scored as alive, otherwise a death was recorded, and the animal was removed from the plate. Worms were transferred to fresh plates as needed (e.g., if there was evidence of microbial contamination, dryness/cracks on the agar surface, consumption of the bacterial lawn, or hatching of new animals that escaped the FUDR block). The reported lifespans were compiled from several independent experiments done over several months (9-10 months for the methotrexate experiments and 4-5 months for the ATIC inhibitor), each scored by multiple individuals (4-5 persons per experiment). No experiments were excluded from the analysis.”

In the worms, interventions that impact germline proliferation can extend lifespan. Methotrexate is known to impact germline proliferation and can lead to toxic developmental effects and germline arrest. Was fecundity impacted by methotrexate using the dosages found to extend lifespan?

RESPONSE: We did not score fecundity in our experiments.

The authors stated that UV killed bacteria was used in the worm experiments but did not provide the reasoning for it. Virk had concluded that reduced bacterial pathogenicity is responsible for the lifespan extension and not the worm's OCM. How does your work agree with or refute these previous findings?

RESPONSE: The dose of methotrexate used by Virk et al was very high, so it is difficult to directly compare it to our experiment. Nonetheless, we do not think there is any contradiction. We added the following in the text to clarify this point:

“At higher doses (10-100μΜ), methotrexate did not extend lifespan (not shown), in agreement with (Virk et al., 2016), who treated adult animals with a very high dose of methotrexate (220μM). We also note that the bacteria used to feed the worms in our experiments were killed by ultraviolet radiation to exclude any impacts from bacterial folate metabolism, which is known to affect worm lifespan (Virk et al., 2016, 2012).”

The authors state that AICAR (100 uM administration to the worms (no experimental details were given) increases their lifespan and concluded that this is proof that manipulation of 1C metabolism promotes longevity. There are 2 concerns here; first, AMPK activation leads to inhibition of TOR and that has been shown to promote longevity in multiple models. While we agree that a significant crosstalk between TOR and OCM exists, this experiment does not necessarily contribute to the argument that the authors are making. Second, it has been established by multiple groups that inhibition (RNAi and pharmacological) of DHFR1, TYMS1, SAMS1 and possibly other OCM enzymes leads to lifespan extension in worms. These findings provide stronger evidence that OCM regulates organismal longevity.

RESPONSE: We acknowledged prior research on lifespan extension and do not claim our use of the ATIC inhibitor as the first evidence of 1C metabolism's impact on longevity. Rather, our findings complement existing studies from us and several other groups (including the examples mentioned by the reviewer, which we had cited) by introducing novel evidence of lifespan increase through this specific inhibitor in C. elegans. Please also note that we added a detailed description of the experiment in the Methods, as suggested in a previous comment.

In the mouse study, the authors do not provide a rationale on why a folate and choline deficient diet was adopted as opposed to only a folate deficient diet. Additionally, we assume that the diets did not contain antibiotics (succinyl sulfathiazole) to reduce microbiome folate production since it was not mentioned. Were wire bottom cages used to eliminate coprophagy? Were there any significant differences between male and female serum folate levels that could have contributed to the endpoints. Was only a subset of samples assayed for total folate? (fig 2b shows a possible n of 6 per group?). If no antibiotics and no wire bottom cages were used, mice can maintain adequate folate levels from coprophagy without developing signs of anemia. Please discuss these details as it helps clarify the conditions used.

RESPONSE: Excellent points, and we have now added this information (see Material and Methods):

“We note that when designing experiments to assess the consequences of folate limitation, it is common to control both folate and choline intake to ensure that the observed effects are due to the restriction of folate (Beaudin et al., 2011) because the presence of choline can mask the effects of folate deficiency. Choline can be oxidized to betaine, which provides methyl groups for converting homocysteine to methionine, independent of the folate cycle. Choline can also be incorporated into phosphatidylcholine, a major methyl ‘sink’ in the cell, through the Kennedy pathway. Lastly, we did not use any antibiotics to interfere with the microbiome nor wire bottom cages to eliminate coprophagy. Wire bottom cages were used only in the metabolic chamber experiments.”

Were there any significant differences between male and female serum folate levels that could have contributed to the endpoints. Was only a subset of samples assayed for total folate? (fig 2b shows a possible n of 6 per group?).

RESPONSE: ____Regarding folate levels, no significant sex differences were observed. We assayed all the animals we had at 120 weeks of age, the euthanasia endpoint, as shown in Figure 2B. There were fewer females than males in both diets.

There are instances in the results section where statements were made implying that there are differences observed "slightly higher", "negative association" when it is not statistically significant. There can be either statistically significant differences/correlation or not. please be precise in your wording.

RESPONSE: We have revised the Results section to ensure that qualitative descriptions such as "slightly higher" are only used when supported by appropriate statistical evidence. We have listed____ all the relevant numbers in each case after performing thorough and robust statistical analyses. We note, however, that mentioning qualitative descriptors is not always unwarranted, as long as they are factual.

Graying was observed less significantly in the F/C- group according to the authors. However, no quantitative assessment was made, and it is merely observational.

RESPONSE: It is not clear how to quantify graying non-invasively. Hence, we simply took photographs.

Inference to inhibition of mTOR was made, but mTOR protein and phosphorylation levels were not performed. The authors did perform western blotting on ribosomal S6 protein, however no assessment of the downstream mTOR targets P70S6k1 and 4EBP are shown.

RESPONSE: This is a good suggestion.____ We added a new experiment, looking at 4EBP1 phosphorylation (see new Figure S2). The results mirror those looking at S6 phosphorylation.

Can the change in RER in F/C- mice compared to controls be explained by the increased adiposity in these animals?

RESPONSE: We do not know. The relationship between adiposity and respiratory exchange rate can be quite complex. The increased adiposity of male mice limited for folate may lead to higher RER, reflecting perhaps a greater reliance on carbohydrate metabolism. But this is very speculative, especially since these mice are not obese. It is unclear how the improved metabolic plasticity could be associated with adiposity for the females.

How was the microbiome normalized between groups prior to the beginning of the experiment? (fecal slurry gavage, bedding exchange, cohabitation, none of the above?). There is no mention of this crucial step in the materials and methods section. Furthermore, additional details regarding the microbiome analysis are required (analysis pipeline, read depth, denoising, software, data processing, PCA analysis, etc...). it is not sufficient to state that Zymo performed the analysis.

RESPONSE: We now revised the text and added a detailed description of the methods, as follows:

“There was no microbiome normalization between groups prior to the beginning of the experiment. Mouse fecal pellets were gathered by positioning the mice on a paper towel beneath an overturned glass beaker. A minimum of three fecal pellets from each animal were transferred into cryovials using sterile forceps. The samples were preserved at -80°C and shipped to Zymo Research, where they were processed and analyzed with the ZymoBIOMICS® Shotgun Metagenomic Sequencing Service (Zymo Research, Irvine, CA).For DNA extraction, the ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used according to the manufacturer’s instructions. Genomic DNA samples were profiled with shotgun metagenomic sequencing. Sequencing libraries were prepared with Illumina® DNA Library Prep Kit (Illumina, San Diego, CA) with up to 500 ng DNA input following the manufacturer’s protocol using unique dual-index 10 bp barcodes with Nextera® adapters (Illumina, San Diego, CA). All libraries were pooled in equal abundance. The final pool was quantified using qPCR and TapeStation® (Agilent Technologies, Santa Clara, CA). The final library was sequenced on the NovaSeq® (Illumina, San Diego, CA) platform. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.

Raw sequence reads were trimmed to remove low quality fractions and adapters with Trimmomatic-0.33 (Bolger et al., 2014): quality trimming by sliding window with 6 bp window size and a quality cutoff of 20, and reads with size lower than 70 bp were removed. Antimicrobial resistance and virulence factor gene identification was performed with the DIAMOND sequence aligner (Buchfink et al., 2015). Microbial composition was profiled with Centrifuge (Kim et al., 2016) using bacterial, viral, fungal, mouse, and human genome datasets. Strain-level abundance information was extracted from the Centrifuge outputs and further analyzed to perform alpha- and beta-diversity analyses and biomarker discovery with LEfSe (Segata et al., 2011) with default settings (p > 0.05 and LDA effect size > 2).”

What is an "easily distinguishable gut microbiome" and "appeared less diverse"?

RESPONSE: To clarify these points, __w__e now edited as follows:

“The different sex and diet groups had an easily distinguishable gut microbiome, occupying different areas of principal component analysis graphs (Figure 5A), based on Bray-Curtis β-diversity dissimilarity indices (Knight et al., 2018). The intestinal microbiome of male mice on the F/C- diet was not statistically less diverse (p=0.222, based on the Wilcoxon rank sum test; Figure 5 - Supplement 1).”

a two-dimensional plot using two principal components would be more suitable for image 5A and allow for better visualization of the clustering of the groups.

RESPONSE: We tried displaying the data on a multipanel (3 panels per group, 12 total) two-dimensional figure, but the result is more confusing. Since the sample number is small (n=6 animals per group), the 3D graphs are visually adequate and more pleasing. They are also the standard way of representing this kind of data.

Since the authors suggest that the microbiome could be a source of 1C metabolites (including natural folate), it is important to clarify if coprophagy is involved.

RESPONSE: We agree and have added the information as requested.

How are inflammatory cytokines and marker levels linked to reduced anabolism and immune function in non-challenged animals?

RESPONSE: ____We do not make any claims for such links if that is what the reviewer implied. If the intent was more towards speculation, we suspect one could imagine various situations. For instance, nutrients may be more heavily used during inflammation to support immune cell responses instead of central anabolic processes in other tissues, limiting the building blocks available for tissue growth and repair. Since we do not see major changes in inflammatory cytokines, we prefer not to speculate about possible links.

When discussing the epigenetic analysis, the authors state "no changes in the DNA methylation from liver samples.." and "groups appear younger than expected". Please clarify these statements. Additional details are needed regarding the analysis performed and the choice of methylated loci and methods. Please reference the epigenetic clock or model that was used and if was developed for the same strain and sub-strain of mice. Is it using a modified "Hovarth" mouse DNA age epigenetic clock? If so, provide the necessary details and a possible explanation for the discrepancy other than "unknown reasons"

__RESPONSE: ____The assay is based on the "Hovarth" mouse DNA age epigenetic clock, for the strain we used (C57BL/6). We have now added a detailed description, which we received from the company, as follows (see Materials and Methods): __

"Liver samples (~15mg) collected at euthanasia were placed in 0.75mL of 1X DNA/RNA Shield™ solution (Zymo Research, Irvine, CA), shipped to Zymo Research, and processed with DNAge® Service according to their established protocols. Briefly, after DNA extraction, the EZ DNA Methylation-Lightning Kit (Zymo Research, Irvine, CA) following the standard protocol was used for bisulfite conversion. Samples were enriched specifically for the sequencing of >1000 age-associated gene loci using Simplified Whole-panel Amplification Reaction Method (SWARM®), where specific CpGs are sequenced at minimum 1000X coverage. Sequencing was run on an Illumina NovaSeq instrument. Sequences were identified by Illumina base calling software then aligned to the reference genome using Bismark. Methylation levels for each cytosine were calculated by dividing the number of reads reporting a "c" by the number of reads reporting a "C" or "T". The percentage of methylation for these specific sequences were used to assess DNA age according to Zymo Research's proprietary DNAge® predictor which had been established using elastic net regression to determine the DNAge®."

As for a possible explanation for the discrepancy, since all our "groups appear younger than expected," unfortunately, other than "unknown reasons," we have none to offer. Nonetheless, the critical point for this study is that we saw no diet effects, regardless of where the company's assay draws the baseline.

Regarding Uracil misincorporation, the liver contains significant stores of folate as it is the main hub for several critical OCM reactions (Phospholipid methylation is a major one). Earlier studies used antibiotics with or without coprophagy prevention measures to induce a state of folate depletion to induce uracil incorporation in various tissues of rodent models. There is some controversy whether dietary folic acid restriction/methyl donor restriction alone will lead to uracil misincorporation when there is no apparent depletion or anemia. Please discuss your specific experimental procedures and how it agrees or disagrees with the published literature.

__RESPONSE: We have now added the experimental details, as suggested in a previous comment. Since we do not see uracil misincorporation, we prefer not to comment on the published literature for possible links between misincorporation and anemia. __

The section discussing RPS6 needs to be rewritten and it is difficult to understand.

RESPONSE: We revised the text, which now reads:

“____Immunoblot analysis of liver tissue samples gathered at the time of euthanasia revealed variability in the detected values across individual mice. When examining the male mice, we observed that, on average, those fed the F/C- diet had approximately half the amount of phosphorylated RPS6 (P-RPS6) compared to those on the F/C+ diet. However, due to high variability in the measured values, the overall differences in P-RPS6 levels between the two dietary groups did not reach statistical significance (Figure 7 - Supplement 1; p>0.05, based on the Wilcoxon rank sum test).”

Furthermore, as stated previously, considering phosphorylation of mTOR and its downstream targets 4EBP and S6K1 will give a clear indication of proliferative signaling.

RESPONSE:____ As we mentioned above, we have now added the suggested 4EBP experiment (see new Figure S2).

Additionally, these pathways are impacted by feeding status, diurnal cycles, and sex. Were these factors controlled prior to sacrifice? Were the animals sacrificed at the same time? In a fed or unfed state?

RESPONSE: The animals were sacrificed at the same time, with no feeding limitations.

The western blots provided in supplementary files show uneven protein loading across lanes (ponceau stain). No loading control is shown such as B-actin. A separate blot is used for total and phosphorylated proteins as opposed to gently stripping the membrane of the phosphorylated bolt and re-incubating with the antibody for total. While normalizing phosphorylated to total protein levels will eliminate some of the variability in the author's method. The uneven loading may introduce errors in the calculated ratios.

RESPONSE: The uneven loading across mouse samples is inconsequential. We report the ratio of phospho-RPS6 to the total amount of RPS6 ____within____ each mouse sample. These ratios were then compared among the different animals and diet groups. We also note that stripping could introduce other artifacts if it is not uniform across all the blot areas.

While the authors referenced older studies utilizing low dose methotrexate on rodents and provided a composite lifespan based on these findings, why was dietary folate and choline restriction used instead of a low dose methotrexate in mice in the current study? Please provide a rationale for this approach.

__RESPONSE: First, in the context of current folate fortification policies, we reasoned that testing dietary folate limitation late in life would be more informative. Second, three of us (M.P., B.K.K., and M.K.) proposed to the Interventions Testing Program at the National Institutes of Health to test whether low-dose methotrexate extends lifespan in mice. The proposal was accepted, and the study is ongoing (the ITP decided to test methotrexate at 0.2ppm, starting at 14 months of age; _https://www.nia.nih.gov/research/dab/interventions-testing-program-itp/supported-interventions_). __

Minor comments:

1. While the authors make compelling arguments that lower folate intake later in life may promote healthy aging, an important consideration in the human population that a considerable percentage of older individuals may be consuming an excessive amount of folate due the combination of fortification and voluntary supplementation. An alternate hypothesis that could apply to humans and lab models is that the existing levels of exposure to folate/folic acid may be accelerating the aging process and promoting disease in later life. __RESPONSE: Perhaps, but as we describe in the text (2nd paragraph in the introduction): __

“...analyses ‘did not identify specific risks from existing mandatory folic acid fortification’ in the general population (Field and Stover, 2018). This conclusion neither refutes nor contradicts the idea that a moderate decrease in folic acid intake among older adults may improve healthspan. Merely because high folic acid intake does not harm the health of older adults does not negate the possibility that a lower folic acid intake might enhance health.”

The common C57BL/6j is being referred to as the "long lived strain". Is this relative to mice in wild conditions? There are many transgenic C57bl/6 strains that live considerably longer. Please clarify if this is meant to describe the aged mice used in the experimental process.

RESPONSE: ____This was from a comprehensive comparison of many different inbred strains. We apologize for omitting the citation, which we have now added____ (Yuan et al, 2009).

While the authors state early in the manuscript that longevity was not a measured outcome in the mouse study, the manuscript contains statements discussing animal survival in the results and survival curves (figure 2). This gives the impression that the study was planned as a survival analysis initially and since no difference was observed between the experimental groups during the earlier stages, the secondary endpoints of health span analysis were adopted. Either approach does not detract from the significance of the study's findings. Further clarity on the approach would be beneficial to the readers.

RESPONSE: The study was designed, and the Animal Use Protocol was institutionally approved for healthspan, not lifespan. The number of animals we used did not have sufficient power to detect lifespan differences. Note that, at least for males, very few animals had died by 120 weeks, our approved euthanasia endpoint. However, it was important to report that folate limitation did not adversely affect overall survival during the analysis time frame.

For yeast culture conditions, what are the folate sources and content? Is there added folic acid similar to cell culture conditions where supraphysiological concentrations are used in standard mediums (RPMI and DMEM).

RESPONSE: The yeast media we used ____were undefined (YPD, see Materials and Methods). The source of folate in this media is “yeast extract,” which is generally considered to contain very high amounts of folate (it was used decades ago to treat anemia and folate deficiency in pregnant women). Note also that, unlike animals, yeast can synthesize folate.

In the metabolism section, the authors make statements such as "the differences were minimal" , "probably were due..", "minimal effects", "apparent increase", "tended to be", "little uracil" etc.. please refrain from using subjective language and use precise scientific terms.

RESPONSE: Please see our earlier response to this comment.

Figure 2-c, there is a typo, Weeks not months

RESPONSE: Corrected. Thank you!

** Referees cross-commenting**

while we generally agree with the other reviewer's concerns, we find that reviewer 3 rejection of the authors conclusion without considering the evidence presented in the context of what is currently known in the field potentially limiting. Multiple groups have shown that manipulation of OCM enzymes (DHFR, TYMS, SAMS) can extend lifespan in worms. the recent report Antebi's group (Annibal et al. Nature Com, 2021) provides strong evidence that OCM is central to longevity regulation in worms and mice and that folate intake can interact with and modulate organismal longevity. while this manuscript findings are not conclusive, I think it is premature to dismiss it completely. perhaps the alternative is to discuss the limitations of this approach and interpret the results (or the lack of significant differences) in order to help guide future research into this important subject. generalizing rodent results to human is always going to be a limiting factor in this type of work. Mice have significantly higher circulating folate. additionally, DHFR activity (the rate limiting enzyme in folate OCM) in rodents can be up to 100 times higher than its human equivalent. another consideration is that mice, similar to other rodents, engage in coprophagy, thereby recycling and supplementing bacterially produced folate in the absence of antibiotics in the diet. Therefore, mice placed of dietary folate restriction in the absence of antibiotics do not develop signs of anemia or deficiency. Therefore, it could be argued that there is no loss of nutrients in mice in this scenario and that supplementation at the arbitrarily recommended level of synthetic folic acid (2mg/kg day) or higher could impact health and aging. Similarly , in humans excess folate intake has been controversially associated with a number of deleterious health effects. It is important not to dismiss these reports and encourage further research into this subject that impacts a significant percentage of the human population due to the widespread use of supplements.

RESPONSE: We thank the reviewers for their evaluation of the work we presented. We have also added the following in the discussion, expanding the limitations of the study:

“Since mice engage in coprophagy, microbiome contributions to folate metabolism are bound to be substantial in this species. There are also significant differences in folate status between mice and people. For example, people have lower levels (~10-15 ng/mL) of serum folate than mice (Bailey et al., 2015), and the activity of DHFR, an enzyme essential for maintaining tetrahydrofolate pools -the folate form used in 1C reactions, maybe only 2% of that in rodents (Bailey and Ayling, 2009). Hence, mice are likely more refractory to a low folate dietary intake.”

Reviewer #1 (Significance (Required)):

Significance:

A major strength of this study is that the authors show that manipulation of OCM either through pharmacological inhibition or dietary restriction can impact organismal longevity in a conserved manner across species from yeast to worms and mammals. These findings provide compelling evidence that folate intake and metabolism in humans should be rigorously researched as potential regulator of aging. These findings complement and agree with a recent report by Antebi's group (Annibal et al. Nature Com, 2021) highlighting that long-lived worm and mice strains exhibit similar metabolic regulation of one carbon metabolism. In the same report low levels of folate supplementation partially or completely abrogated the lifespan extension in some models. This study provides additional evidence that restricting OCM through drugs or dietary restriction can significantly impact healthspan and lifespan. Additionally, it raises the question whether excessive folate intake in aged adults may have potentially deleterious effects on health and longevity. The limitations of this study can be seen in the overall lack of significant impact of the dietary intervention on the health metrics that were measured in mice. The study does not provide strong evidence that restricting folate and choline intake will produce favorable effects on health. Similarly, no significant impact on mice lifespan was observed based on the partial lifespan analysis. Further clarity is needed regarding the experimental procedures and methods used. The study, nonetheless, is an important step towards investigating the role of folate and OCM in regulating mammalian healthspan and lifespan. Future studies can expand on these findings and investigate whether OCM interventions that are started in early life can produce significant and measurable effects on longevity and health in mammals. The findings here provide a conceptual and incremental advance in our understanding of these complex interactions.

These findings are important to the research communities especially in the areas of longevity, metabolism, and nutrition.

RESPONSE: We appreciate the recognition of our work's significance in furthering understanding of longevity, metabolism, and nutrition. We would also like to stress that this study is not an incremental advance. We believe our study's focus on dietary folate limitation ____in aged mice____ represents a novel and more radical contribution, considering the lack of prior research in this specific context, underscoring the distinctiveness and importance of our findings.

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

Summary: In this manuscript they investigate whether disruption of the folate cycle can slow ageing/improve health in yeast, worms and mice. There are a few experiments in yeast and C. elegans but the rest is a meta analysis of some old data on folate-deprived mice and their own study of mice on a diet with and without folic acid and choline. The find that various interventions of the folate cycle extend lifespan in yeast and worms, that the old study suggest mice live longer without folic acid supplementation and that there is no change to healthspan with mice without folic acid and choline in the diet late in life and that these mice show some positive benefits. Analysis of the microbiome and the transcriptomics suggest small changes to the microbiota and changes in gene expression. Overall the authors conclude that biosynthetic processes have been inhibited without negative effects on healthspan.

Major comments

1. The two worm lifespan experiments in Fig 1 show very different controls despite the methods stating that the conditions were the same. Controls can vary from one experiment to another but the difference is striking. It would be good to have supplementary data about the number of repeats and other data about these experiments. RESPONSE: We also noted the difference. However, we believe our conclusions are valid and robust because we used only experiment-matched controls for each comparison. We now describe in detail how the experiments were done (see revised Materials and Methods). Lastly, the two compounds were tested years apart from different individuals, and the different lifespans of the controls could arise from differences in the media batches, temperature control, etc.

The diet lack folic acid and choline yet the conclusions are only about folate. The choline aspect of the diet needs to be acknowledged as a potential factor.

RESPONSE: As we mentioned above, we have now added this information (see Material and Methods):

“We note that when designing experiments to assess the consequences of folate limitation, it is common to control both folate and choline intake to ensure that the observed effects are due to the restriction of folate (Beaudin et al., 2011) because the presence of choline can mask the effects of folate deficiency. Choline can be oxidized to betaine, which provides methyl groups for converting homocysteine to methionine, independent of the folate cycle. Choline can also be incorporated into phosphatidylcholine, a major methyl ‘sink’ in the cell, through the Kennedy pathway. Lastly, we did not use any antibiotics to interfere with the microbiome nor wire bottom cages to eliminate coprophagy. Wire bottom cages were used only in the metabolic chamber experiments.”

The authors argue that the effects on the mice are not mediated effects on the diet by the microbiome because there is not a statistical effect on diversity. However they do show a clear difference at the metagenomic level that fits with a metabolic difference. It also ignores work in C. elegans showing that inhibition of bacterial folate synthesis increases lifespan, not by decreasing folate supply but because lowered bacterial folate prevents an age-accelerating activity in the bacteria (Virk et al 2016). It has also been shown that a breakdown product of folic acid can be taken up by bacteria and influence ageing (Maynard et al 2018). I do not think the evidence is strong enough to discounted that the changes seen in the mice are not mediated by microbes.

RESPONSE: We do not state that “changes seen in the mice are not mediated by microbes”. On the contrary, we agree with the reviewer that the microbiome likely contributes significantly, and we hope this is conveyed in the text. We also agree with the references the reviewer pointed out, which we cite (see also our response to point#5 of reviewer 1).

Minor comments

1. It had been shown a long time ago that sams-1 mutants in C. elegans extend lifespan. MTX is likely to influence SAMS levels. This point needs to mentioned. RESPONSE: Thank you. We added the reference.

Page - 6 "folate accelerates worm aging". This statement is not correct and is not what Virk et al 2016 suggests.

RESPONSE: We revised it to the following: “____It has been reported that treating worms with high levels of methotrexate (220μΜ) at the adult stage did not extend their lifespan ____(Virk et al., 2016)____”.

Page 7. "at 100μM, a dose similar to the one used in mice with metabolic syndrome (Asby et al., 2015)." It's not valid to compare the concentration of a drug in the media in a C. elegans experiment to a dose given to mice.

RESPONSE: We appreciate the reviewer's point on comparing drug dosages across species. The intention was to provide a reference point for the concentration used rather than suggesting a direct equivalence with outcomes. We recognize the complexities of cross-species dosage comparisons and have amended the text to clarify that the mention of dosage is for contextual purposes only.

** Referees cross-commenting**

I would like to add that it is important to consider whether there are in fact negative effects of folic acid given in later life and this is one of the only studies that addresses this question in a mammalian model, and thus needs to be reported, once the issues raised have been addressed.

__RESPONSE: As we mentioned in a comment from reviewer 1 and describe in the text (2nd paragraph in the introduction): __

“...analyses ‘did not identify specific risks from existing mandatory folic acid fortification’ in the general population (Field and Stover, 2018). This conclusion neither refutes nor contradicts the idea that a moderate decrease in folic acid intake among older adults may improve healthspan. Merely because high folic acid intake does not harm the health of older adults does not negate the possibility that a lower folic acid intake might enhance health.”

Reviewer #2 (Significance (Required)):

The main strength of this manuscript is that it examines the effect of mice given a folate and choline deficient diet late in life and finds mostly positive effects. This finding challenges the dogma that folate

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

Blank/Polymenis and colleagues explore how reduced folate metabolism impacts aging. While folate supplementation is known to benefit the development and health of young people, little is known about the impact of this substrate at advanced ages. The paper consists of two parts: 1) blocking folate metabolism in yeast and C. elegans while measuring lifespan (reproductive or age of death); 2) measuring a vast array of traits in mice where folate (and choline) is removed from the diet starting at age 1 year. The second approach is most central to the paper's theme, and the authors conclude their 'data raise the exciting possibility that ... reduced folate intake later in life might be beneficial." However, I do accept this conclusion. Instead, the overwhelming fact is that there were no changes in any phenotype due to the absence of F/C in the older animals. Loss of this nutrient is neutral, although perhaps bad for the kidney. In my view, the authors misinterpret their very basic results: loss of dietary folate has no impact on aged mice (one strain, at that). And there is no way to generalize this simple conclusion to humans.

RESPONSE: ____We respectfully disagree with the reviewer's assessment of our study's conclusions and its significance. With the primary focus on evaluating the effects of reduced folate intake in aged mice, we explored a comprehensive range of healthspan markers and molecular analyses. Contrary to the reviewer's assertion, our data demonstrate significant outcomes such as altered body weight and metabolic parameters in mice subjected to folate restriction, along with insights into molecular changes indicative of lower anabolism.

The reviewer's interpretation that folate limitation has no observable impact on aged mice overlooks the nuanced findings presented in our study. While acknowledging the neutral effects observed in some phenotypes, we contend that our results collectively contribute to a deeper understanding of the implications of late-life folate restriction. It is unwarranted to dismiss these findings.

Generalizing findings from model systems to humans is indeed complex, as noted by the reviewer. However, our study, alongside existing literature, provides valuable insights that warrant consideration and further exploration. We stand by the rigor of our methodology, the diversity of data presented, and the significance of our results in enhancing knowledge on the impact of folate metabolism in aging models.

There are other issues throughout the work that need to be addressed but given weakness on its key argument, I will not elaborate these points.

__RESPONSE: Since the reviewer offered no specifics on “other issues,” we cannot respond. We hope, however, that we have addressed them in our response to the other reviewers’ comments. __

Reviewer #3 (Significance (Required)):

Blank/Polymenis and colleagues explore how reduced folate metabolism impacts aging. While folate supplementation is known to benefit the development and health of young people, little is known about the impact of this substrate at advanced ages.

RESPONSE: ____We concur with the reviewer's observation regarding the knowledge gap surrounding the impact of reduced folate metabolism on aging, particularly in advanced stages of life, which ____is why our study significantly contributes to the field. As we mentioned above, not only do we report that some healthspan metrics were improved in folate-limited animals (e.g., body weight, improved metabolic plasticity), but our study also offers for the first time a comprehensive biomarker analysis of folate limitation late in life (e.g., metabolite and mRNAs changes associated with lower anabolism, lower IGF1 levels in females). ____This original contribution enhances our understanding of the complex interplay between folate metabolism and aging.

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Referee #1

Evidence, reproducibility and clarity

Summary: the work presented by the authors detail how pharmacological inhibition of the rate limiting one carbon metabolic enzyme DHFR by the drug methotrexate increases the lifespan of yeast and worms. Furthermore, placing aged mice on dietary folate and choline restriction potentially enhanced metabolic plasticity but did not significantly increase lifespan with sex specific differences observed. The findings in this manuscript are very interesting and important to our understanding of the conserved mechanisms that regulate longevity through one carbon metabolism. This is especially significant in light of the current folate intake and supplementation in the adult human population. The manuscript, however, requires major revisions. Please see comments below for details.

Major comments:

1. The overall tone in this manuscript is colloquial and conversational in nature. A third person academic style and tone, while avoiding the use of subjective descriptive terms would improve the quality of this text. Using terms such as "appeared less diverse", "results are remarkable ...strikingly more pronounced", "possibly positive outcomes" , "appear younger...for unknown reasons", "little Uracil", "tended to be higher", "roughly proportional", "slightly higher", "as a rough readout", and many other examples from the text should not be used in a scientific manuscript. The language should be academic, scientific, precise, and non-ambiguous. A thorough revision of the manuscript with substantial changes to the language and tone is necessary prior to publication.
2. In the results section, we find multiple instances where the results are interpreted and extensively discussed. This should be reserved for the discussion section. The results section should be used to simply report the findings in a detailed manner.
3. The materials and methods section is severely lacking in details in some areas. For example, no details were provided regarding how the worm lifespans were conducted and previous work of collaborators were referenced instead. Important details such as worm numbers, biological and technical replicates, solid agar vs liquid culture, temperature, use of FUdR, antibiotics, transfer frequency, methods of scoring, etc... are lacking. Other details such as the preparation of the plates (Was MTX incorporated into the agar, seeded with the bacterial lawn, or liquid culture was used), storage conditions, age of the plates when lifespan started, how was the UV killing of the lawn verified etc... many other methods subsections lack crucial details. Please carefully review the methodology and include sufficient pertinent details.
4. In the worms, interventions that impact germline proliferation can extend lifespan. Methotrexate is known to impact germline proliferation and can lead to toxic developmental effects and germline arrest. Was fecundity impacted by methotrexate using the dosages found to extend lifespan?
5. The authors stated that UV killed bacteria was used in the worm experiments but did not provide the reasoning for it. Virk had concluded that reduced bacterial pathogenicity is responsible for the lifespan extension and not the worm's OCM. How does your work agree with or refute these previous findings?
6. The authors state that AICAR (100 uM administration to the worms (no experimental details were given) increases their lifespan and concluded that this is proof that manipulation of 1C metabolism promotes longevity. There are 2 concerns here; first, AMPK activation leads to inhibition of TOR and that has been shown to promote longevity in multiple models. While we agree that a significant crosstalk between TOR and OCM exists, this experiment does not necessarily contribute to the argument that the authors are making. Second, it has been established by multiple groups that inhibition (RNAi and pharmacological) of DHFR1, TYMS1, SAMS1 and possibly other OCM enzymes leads to lifespan extension in worms. These findings provide stronger evidence that OCM regulates organismal longevity.
7. In the mouse study, the authors do not provide a rationale on why a folate and choline deficient diet was adopted as opposed to only a folate deficient diet. Additionally, we assume that the diets did not contain antibiotics (succinyl sulfathiazole) to reduce microbiome folate production since it was not mentioned. Where wire bottom cages used to eliminate coprophagy? Were there any significant differences between male and female serum folate levels that could have contributed to the endpoints. Was only a subset of samples assayed for total folate? (fig 2b shows a possible n of 6 per group?). If no antibiotics and no wire bottom cages were used, mice can maintain adequate folate levels from coprophagy without developing signs of anemia. Please discuss these details as it helps clarify the conditions used.
8. There are instances in the results section where statements were made implying that there are differences observed "slightly higher", "negative association" when it is not statistically significant. There can be either statistically significant differences/correlation or not. please be precise in your wording.
9. Graying was observed less significantly in the F/C- group according to the authors. However, no quantitative assessment was made, and it is merely observational. Inference to inhibition of mTOR was made, but mTOR protein and phosphorylation levels were not performed. The authors did perform western blotting on ribosomal S6 protein, however no assessment of the downstream mTOR targets P70S6k1 and 4EBP are shown.
10. Can the change in RER in F/C- mice compared to controls be explained by the increased adiposity in these animals?
11. How was the microbiome normalized between groups prior to the beginning of the experiment? (fecal slurry gavage, bedding exchange, cohabitation, none of the above?). There is no mention of this crucial step in the materials and methods section. Furthermore, additional details regarding the microbiome analysis are required (analysis pipeline, read depth, denoising, software, data processing, PCA analysis, etc...). it is not sufficient to state that Zymo performed the analysis. What is an "easily distinguishable gut microbiome" and "appeared less diverse"? a two-dimensional plot using two principal components would be more suitable for image 5A and allow for better visualization of the clustering of the groups. Since the authors suggest that the microbiome could be a source of 1C metabolites (including natural folate), it is important to clarify if coprophagy is involved.
12. How are inflammatory cytokines and marker levels linked to reduced anabolism and immune function in non-challenged animals?
13. When discussing the epigenetic analysis, the authors state "no changes in the DNA methylation from liver samples.." and "groups appear younger than expected". Please clarify these statements. Additional details are needed regarding the analysis performed and the choice of methylated loci and methods. Please reference the epigenetic clock or model that was used and if was developed for the same strain and sub-strain of mice. Is it using a modified "Hovarth" mouse DNA age epigenetic clock? If so, provide the necessary details and a possible explanation for the discrepancy other than "unknown reasons"
14. Regarding Uracil misincorporation, the liver contains significant stores of folate as it is the main hub for several critical OCM reactions (Phospholipid methylation is a major one). Earlier studies used antibiotics with or without coprophagy prevention measures to induce a state of folate depletion to induce uracil incorporation in various tissues of rodent models. Theres is some controversy whether dietary folic acid restriction/methyl donor restriction alone will lead to uracil misincorporation when there is no apparent depletion or anemia. Please discuss your specific experimental procedures and how it agrees or disagrees with the published literature.
15. The section discussing RPS6 needs to be rewritten and it is difficult to understand. Furthermore, as stated previously, considering phosphorylation of mTOR and its downstream targets 4EBP and S6K1 will give a clear indication of proliferative signaling. Additionally, these pathways are impacted by feeding status, diurnal cycles, and sex. Were these factors controlled prior to sacrifice? Where the animals sacrificed at the same time? In a fed or unfed state?
16. The western blots provided in supplementary files show uneven protein loading across lanes (ponceau stain). No loading control is shown such as B-actin. A separate blot is used for total and phosphorylated proteins as opposed to gently stripping the membrane of the phosphorylated bolt and re-incubating with the antibody for total. While normalizing phosphorylated to total protein levels will eliminate some of the variability in the author's method. The uneven loading may introduce errors in the calculated ratios.
17. While the authors referenced older studies utilizing low dose methotrexate on rodents and provided a composite lifespan based on these findings, why was dietary folate and choline restriction used instead of a low dose methotrexate in mice in the current study? Please provide a rationale for this approach.

Minor comments:

1. While the authors make compelling arguments that lower folate intake later in life may promote healthy aging, an important consideration in the human population that a considerable percentage of older individuals may be consuming an excessive amount of folate due the combination of fortification and voluntary supplementation. An alternate hypothesis that could apply to humans and lab models is that the existing levels of exposure to folate/folic acid may be accelerating the aging process and promoting disease in later life.
2. The common C57BL/6j is being referred to as the "long lived strain". Is this relative to mice in wild conditions? There are many transgenic C57bl/6 strains that live considerably longer. Please clarify if this is meant to describe the aged mice used in the experimental process.
3. While the authors state early in the manuscript that longevity was not a measured outcome in the mouse study, the manuscript contains statements discussing animal survival in the results and survival curves (figure 2). This gives the impression that the study was planned as a survival analysis initially and since no difference was observed between the experimental groups during the earlier stages, the secondary endpoints of health span analysis were adopted. Either approach does not detract from the significance of the study's findings. Further clarity on the approach would be beneficial to the readers.
4. For yeast culture conditions, what are the folate sources and content? Is there added folic acid similar to cell culture conditions where supraphysiological concentrations are used in standard mediums (RPMI and DMEM).
5. In the metabolism section, the authors make statements such as "the differences were minimal" , "probably were due..", "minimal effects", "apparent increase", "tended to be", "little uracil" etc.. please refrain from using subjective language and use precise scientific terms.
6. Figure 2-c, there is a typo, Weeks not months

** Referees cross-commenting**

while we generally agree with the other reviewer's concerns, we find that reviewer 3 rejection of the authors conclusion without considering the evidence presented in the context of what is currently known in the field potentially limiting. Multiple groups have shown that manipulation of OCM enzymes (DHFR, TYMS, SAMS) can extend lifespan in worms. the recent report Antebi's group (Annibal et al. Nature Com, 2021) provides strong evidence that OCM is central to longevity regulation in worms and mice and that folate intake can interact with and modulate organismal longevity. while this manuscript findings are not conclusive, I think it is premature to dismiss it completely. perhaps the alternative is to discuss the limitations of this approach and interpret the results (or the lack of significant differences) in order to help guide future research into this important subject. generalizing rodent results to human is always going to be a limiting factor in this type of work. Mice have significantly higher circulating folate. additionally, DHFR activity (the rate limiting enzyme in folate OCM) in rodents can be up to 100 times higher than its human equivalent. another consideration is that mice, similar to other rodents, engage in coprophagy, thereby recycling and supplementing bacterially produced folate in the absence of antibiotics in the diet. Therefore, mice placed of dietary folate restriction in the absence of antibiotics do not develop signs of anemia or deficiency. Therefore, it could be argued that there is no loss of nutrients in mice in this scenario and that supplementation at the arbitrarily recommended level of synthetic folic acid (2mg/kg day) or higher could impact health and aging. Similarly , in humans excess folate intake has been controversially associated with a number of deleterious health effects. It is important not to dismiss these reports and encourage further research into this subject that impacts a significant percentage of the human population due to the widespread use of supplements.

Significance

A major strength of this study is that the authors show that manipulation of OCM either through pharmacological inhibition or dietary restriction can impact organismal longevity in a conserved manner across species from yeast to worms and mammals. These findings provide compelling evidence that folate intake and metabolism in humans should be rigorously researched as potential regulator of aging. These findings complement and agree with a recent report by Antebi's group (Annibal et al. Nature Com, 2021) highlighting that long-lived worm and mice strains exhibit similar metabolic regulation of one carbon metabolism. In the same report low levels of folate supplementation partially or completely abrogated the lifespan extension in some models. This study provides additional evidence that restricting OCM through drugs or dietary restriction can significantly impact healthspan and lifespan. Additionally, it raises the question whether excessive folate intake in aged adults may have potentially deleterious effects on health and longevity. The limitations of this study can be seen in the overall lack of significant impact of the dietary intervention on the health metrics that were measured in mice. The study does not provide strong evidence that restricting folate and choline intake will produce favorable effects on health. Similarly, no significant impact on mice lifespan was observed based on the partial lifespan analysis. Further clarity is needed regarding the experimental procedures and methods used. The study, nonetheless, is an important step towards investigating the role of folate and OCM in regulating mammalian healthspan and lifespan. Future studies can expand on these findings and investigate whether OCM interventions that are started in early life can produce significant and measurable effects on longevity and health in mammals. The findings here provide a conceptual and incremental advance in our understanding of these complex interactions.

These findings are important to the research communities especially in the areas of longevity, metabolism, and nutrition.

Author response:

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

Reviewer #1 (Recommendations For The Authors):

Will the nanobody be available to the TB research community?

Yes, we will make E11rv available upon request. Please see our materials availability statement.

Reviewer #2 (Recommendations For The Authors):

(1) It would be interesting to test the potential impact of residual ASB-14 contaminant on the biochemical behavior of ESAT-6-CFP10 heterodimer and ESAT-6 homodimer or tetramer and their hemolytic activity in comparison with the ones without ASB-14.

We agree that this is an interesting line of questioning. Based on the study by Refai et al. that we cite in the text, ESAT-6 treated with nonionic detergents ASB-14 or LDAO, but not other common detergents, undergoes a conformational change that increases its cytotoxicity in cell assays, hemolytic activity, and ability to dimerize with CFP-10. What is not known at this point, is how similar the ASB-bound conformation is to anything seen physiologically.

(2) Building on the progress in making anti-ESAT-6 nanobodies and their anti-Mtb effects in the cells, it could have been tested in human or mouse primary macrophages infected with Mtb and a mouse model of Mtb infection for its anti-Mtb efficiency.

We thank the reviewer for this suggestion, and we agree that these would be very informative next steps for determining the therapeutic potential of anti-ESAT-6 nanobodies.

Reviewer #3 (Recommendations For The Authors):

Minor comments:

Line 133: "It is well established that Mm-induced hemolysis is ESX-1 dependent, but our results suggest that Mtb must lack one or more factors necessary for efficient hemolysis.". I would tone this down a bit, as it is also known that M. tuberculosis escapes much later than M. marinum from the phagosome, which could indicate different kinetics.

We thank the reviewer for their insightful comments. We agree that the kinetics of Mtb and Mm infection are quite different and that this may impact the hemolysis assay. As described by Augenstreich et al. some hemolysis by Mtb is observed at 48 hours, though the method of normalization makes it impossible to determine absolute amount of hemolysis that occurred in their experiment. Our findings just show that the absolute amount of Mtb hemolysis in 2 hours is negligible, setting it apart from Mm. We have edited the wording of this statement in the manuscript to avoid any confusion.

Line 155: "Because Mtb often exists in an acidified compartment". First of all, the reference used here does not discuss anything about Mtb, secondly, papers that do measure the acidification of Mtb-loaded phagosomes indicate that this acidification is very mild (typically to pH 6.2).

We agree that this point should be articulated more precisely. We have added additional clarification that the pH of Mtb-containing compartments in macrophages can fall in a broad range depending on the activation state of the macrophages, and that non-activated macrophages are typically only mildly acidic. We have updated our references to better describe the current state of knowledge on this topic.

Line 339: "Whereas most of these functions rely only on the secretion of ESAT-6 into the cytoplasm, the ability of E11rv to access Mtb suggests that this communication is likely two-way." No, not necessary, there are many processes in which ESX-1 substrates affect the macrophage. This nanobody could affect EsxA functioning only once the bacteria reach the cytoplasm. I think checking phagosomal escape in these cells is therefore crucial.

We agree that phagosomal escape and subsequent direct secretion of ESAT-6 into the cytoplasm is a reasonable alternative hypothesis. We have added this point to our discussion, and we agree that looking directly at phagosomal escape is an important next step.

Figure 7 is not mentioned in the text (mistake for Fig 6).

This has been corrected.

Author response:

Public Reviews: 

Reviewer #1 (Public Review): 

As a reviewer for this manuscript, I recognize its significant contribution to understanding the immune response to saprophytic Leptospira exposure and its implications for leptospirosis prevention strategies. The study is well-conceived, addressing an innovative hypothesis with potentially high impact. However, to fully realize its contribution to the field, the manuscript would benefit greatly from a more detailed elucidation of immune mechanisms at play, including specific cytokine profiles, antigen specificity of the antibody responses, and long-term immunity. Additionally, expanding on the methodological details, such as immunophenotyping panels, qPCR normalization methods, and the rationale behind animal model choice, would enhance the manuscript's clarity and reproducibility. Implementing functional assays to characterize effector T-cell responses and possibly investigating the microbiota's role could offer novel insights into the protective immunity mechanisms. These revisions would not only bolster the current findings but also provide a more comprehensive understanding of the potential for saprophytic Leptospira exposure in leptospirosis vaccine development. Given these considerations, I believe that after substantial revisions, this manuscript could represent a valuable addition to the literature and potentially inform future research and vaccine strategy development in the field of infectious diseases. 

We have been interested in understanding how both pathogenic and non-pathogenic Leptospira species affect each other on a mammalian reservoir host. With the current study we continue to elucidate the immune mechanisms engaged by pathogenic Leptospira interrogans versus non-pathogenic L. biflexa, as a follow up to our previous work (Shetty et al, 2021 PMID: 34249775, and Kundu et al 2022 PMID 35392072). We found that both species engaged partially overlapping myeloid immune cells and inflammatory signatures of infection. For example, some chemokines were increased, and macrophage and dendritic cells were engaged at 24h post inoculation with both species of Leptospira (PMID: 34249775). Thus, we questioned whether this robust innate immune response raised to eliminate an immunogenic but rather non-pathogenic bacterium, could also help restrain L. interrogans pathogenesis. In this study we show that L. biflexa pre-exposure to L. interrogans challenge mediates improved kidney homeostasis, mitigates leptospirosis severity and leads to increased shedding of L. interrogans in urine. This suggests an interspecies symbiotic commensalistic process that facilitates survival of the pathogenic species. These findings have high impact on the lives of millions of people in areas endemic for leptospirosis that are naturally exposed to non-pathogenic Leptospira species.

We will expand on the methodological details and will update the introduction and discussion to include answers to questions raised by the three reviewers to further clarify the importance and impact of our study.

Reviewer #2 (Public Review): 

Summary: 

The authors try to achieve a method of protection against pathogenic strains using saprophytic species. It is undeniable that the saprophytic species, despite not causing the disease, activates an immune response. However, based on these results, using the saprophytic species does not significantly impact the animal's infection by a virulent species. 

We separate concepts of exposure to a non-virulent bacterium that establishes a brief infection with engagement of an immune response (L. biflexa), from infection established by a virulent species of Leptospira that leads to pathogenesis (L. interrogans). While trying to understand how both pathogenic and non-pathogenic Leptospira species affect each other on a mammalian reservoir host, we previously found that L. biflexa induces immune responses that should affect immunity of populations naturally exposed to this spirochete. Thus, we designed this study to answer that question.

Strengths: 

Exposure to the saprophytic strain before the virulent strain reduces animal weight loss, reduces tissue kidney damage, and increases cellular response in mice.

Weaknesses: 

Even after the challenge with the saprophyte strain, kidney colonization and the release of bacteria through urine continue. Moreover, the authors need to determine the impact on survival if the experiment ends on the 15th. 

Another novel and unexpected aspect of our findings in the single exposure experiment was that L. biflexa pre-exposure mediated a homeostatic environment in the kidney (lower ColA1, healthier renal physiology) that restrained pathogenesis of L. interrogans after challenge, which resulted in better health outcomes and increased shedding of L. interrogans in urine; in contrast, if the kidney is compromised (high ColA1) by L. interrogans (without L. biflexa pre-exposure) there was lower shedding L. interrogans in urine. Interestingly, this suggests an interspecies symbiotic commensalistic process that facilitates survival of the pathogenic species. Thus, these data suggest that higher shedding of L. interrogans in urine may not be a hallmark of increased disease, but rather it could be the opposite.

We will include these concepts in the updated discussion.

We don’t think that extending this experiment to d21 or d28 would add relevant data to our findings. We provide survival curves for both experiments up to d15 post infection.

Reviewer #3 (Public Review): 

Summary: 

Kundu et al. investigated the effects of pre-exposure to a non-pathogenic Leptospira strain in the prevention of severe disease following subsequent infection by a pathogenic strain. They utilized a single or double exposure method to the non-pathogen prior to challenge with a pathogenic strain. They found that prior exposure to a non-pathogen prevented many of the disease manifestations of the pathogen. Bacteria, however, were able to disseminate, colonize the kidneys, and be shed in the urine. This is an important foundational work to describe a novel method of vaccination against leptospirosis. Numerous studies have attempted to use recombinant proteins to vaccinate against leptospirosis, with limited success. The authors provide a new approach that takes advantage of the homology between a non-pathogen and a pathogen to provide heterologous protection. This will provide a new direction in which we can approach creating vaccines against this re-emerging disease. 

Strengths: 

The major strength of this paper is that it is one of the first studies utilizing a live non-pathogenic strain of Leptospira to immunize against severe disease associated with leptospirosis. They utilize two independent experiments (a single and double vaccination) to define this strategy. This represents a very interesting and novel approach to vaccine development. This is of clear importance to the field. 

The authors use a variety of experiments to show the protection imparted by pre-exposure to the non-pathogen. They look at disease manifestations such as death and weight loss. They define the ability of Leptospira to disseminate and colonize the kidney. They show the effects infection has on kidney architecture and a marker of fibrosis. They also begin to define the immune response in both of these exposure methods. This provides evidence of the numerous advantages this vaccination strategy may have. Thus, this study provides an important foundation for future studies utilizing this method to protect against leptospirosis. 

Weaknesses: 

Although they provide some evidence of the utility of pretreatment with a non-pathogen, there are some areas in which the paper needs to be clarified and expanded. 

The authors draw their conclusions based on the data presented. However, they state the graphs only represent one of two independent experiments. Each experiment utilized 3-4 mice per group. In order to be confident in the conclusions, a power analysis needs to be done to show that there is sufficient power with 3-4 mice per group. In addition, it would be important to show both experiments in one graph which would inherently increase the power by doubling the group size, while also providing evidence that this is a reproducible phenotype between experiments. Overall, this weakens the strength of the conclusions drawn and would require additional statistical analysis or additional replicates to provide confidence in these conclusions. 

We will take these suggestions into consideration and will address as many of these issues as possible in the revised manuscript.

A direct comparison between single and double exposure to the non-pathogen is not able to be determined. The ages of mice infected were different between the single (8 weeks) and double (10 weeks) exposure methods, thus the phenotypes associated with LIC infection are different at these two ages. The authors state that this is expected, but do not provide a reasoning for this drastic difference in phenotypes. It is therefore difficult to compare the two exposure methods, and thus determine if one approach provides advantages over the other. An experiment directly comparing the two exposure methods while infecting mice at the same age would be of great relevance to and strengthen this work. 

Both experiments need to be analyzed as separate but complementary as they provide different hind sights into L. interrogans pathogenesis and potential solutions to the problem. Optimal measurements of disease progression (weight loss, survival curves) require infection of mice at 8 weeks. Based on this, a new L. biflexa double exposure experiment would have to start when mice are 4 weeks old which is just after weaning, and before the mouse immune system is fully developed.

Author response:

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

eLife assessment

This is a valuable contribution to the electric fish community, and to studies of active sensing more generally, in that it provides evidence that a well-studied behavior (chirping) may serve in active sensing rather than communication. For the most part, the evidence is solid. In particular, the evidence showing increased chirping in more cluttered environments and the relationship between chirping and movement are convincing. Nevertheless, evidence to support the argument that chirps are mostly used for navigation rather than communication is incomplete.

Thank you for the comment. In response to what seemed to be a generalized need for more evidence to support our hypothesis, we have extensively reviewed the manuscript, changed the existing figures and added new ones (3 new figures in the main text and 4 in the supplementary information section). Our edits include:

(1) changes to the written text to remove categorical statements ruling out the possible communication function of chirps. When necessary, we have also added details on why we believe a social communication function of chirps could interfere with a role in electrolocation.

(2) new experiments (and related figures) adding details on the behavioral correlates of chirping, on the effects of chirps on electric images (which are a way to represent current flow on the fish skin), and behavioral responses to ramp frequency playback EODs (used to test a continuous range of beat frequencies and fill the sampling gaps left by our experiments using real fish).

Public Reviews:

Reviewer #1 (Public Review):

The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

We thank the Reviewer for the extensive feedback received. Hereby we respond to each of the points raised.

We have better clarified that our intention is not to propose chirps as tools for “conspecific localization” intended as the pinpointing of its particular location. Instead, based on our observation of chirps being employed at very close ranges, we suggest that chirps may serve to assess other parameters related to “conspecific positioning” (which in a wide sense, it is still “electrolocation”), and that could be derived from the beat. These parameters might include size, relative orientation, or subtle changes in position during movement. While the experiments discussed in the manuscript do not provide a conclusive answer in this regard, we prioritize here the presentation of broader evidence for a different use of chirping. We are actively working on another manuscript that explores this aspect more in detail, but, due to space limitations, additional results had to be excluded.

In the abstract we mention a role of chirps in the enhancement of “electrolocation”, but - as above mentioned - it is here meant only in a broad sense. In the introduction (at the very end) we propose chirps as self-directed signals (homeoactive sensing). In the result paragraph dedicated to the novel environment exploration experiment the following lines were added “Most chirps (90%) in fact are produced within a distance corresponding to 1% of the maximum field intensity (i.e. roughly 30 cm; Figure S12B), indicating that chirping occurs way below the threshold range for beat detection (i.e. roughly in the range of 60-120 cm, depending on the study; see appendix 1: Detecting beats at a distance) and likely does not represent a way to improve it”. We conclude this paragraph mentioning “This further corroborates the hypothesized role of chirps in beat processing.”. The last result paragraph (on chirping in cluttered environments) ends with “This supports the notion of chirps as self-referenced probing cues, potentially employed to optimize short-range aspects of conspecific electrolocation, such as conspecific size, orientation, and swimming direction - a hypothesis that will certainly be explored in future studies.”. In the discussion paragraph entitled “probing with chirps”, we do provide hints to possible mechanisms implied in the role of chirps in beat processing. As mentioned, we have planned to add further details in another manuscript, currently in preparation.

The study provides a wealth of interesting observation of behavior and much of this data constitute a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further. However, the data they provide does not support strong conclusion statements arguing that these chirps are used for localization purposes and is even less convincing at rejecting previously established hypotheses on the communication purpose of the chirps.

We intentionally framed our aims a bit provocatively to underscore that, to date, the role of chirps in social communication has been supported solely by correlative evidence. While the evidence we provide to support the role of chirps as probes is also correlative, it opens at the same time critical questions on the long assumed role of chirps in social communication. In fact, chirping is strongly dependent on fish reciprocal positioning, highly constrained by beat frequency, and patterned in such ways that - in our opinion - makes the existence of links between chirp types and internal states less likely, as suggested instead by the current view. Moreover, the use of different chirp types does not appear specific to any of the social contexts analyzed but is primarily explained by DF (beat frequency). This observation, coupled with the analysis of chirp transitions (more self-referenced than reflecting an actual exchange between subjects), leads us to hypothesize with greater confidence that chirp production may be more related to sensing the environment, rather than transmitting information about a specific behavioral state.

Nevertheless, the Reviewer's comment is valid. We've tempered the study's conclusions by introducing the possibility of chirps serving both communication and electrolocation functions, as stated in the conclusion paragraph: "While our results do not completely dismiss the possibility of chirps serving a role in electrocommunication—probing cues could, for instance, function as proximity signals to signal presence, deter approaches, or coordinate behaviors like spawning (Henninger et al., 2018).". Nonetheless, we do emphasize that our hypothesis is more likely to apply - based on our data. We refrain from categorically excluding a communicative function for chirps (between subjects), but we hypothesize that this communication - if occurring - may contain the same type of information as the self-directed signaling implied by the “chirps as probes” idea (i.e. spatial information).

In response to the Reviewer's feedback, we've revised the end of the introduction, removing suggestions of conclusiveness: "Finally, by recording fish in different conditions of electrical 'visibility,' we provide evidence supporting a previously neglected role of chirps: homeoactive sensing." (edit: the word “validating” has been removed to give a less “conclusive” answer to the open functional questions about chirping).

I would suggest thoroughly revising the manuscript to provide a neutral description of the results and leaving any speculations and interpretations for the discussion where the authors should be careful to separate strongly supported hypotheses from more preliminary speculations. I detail below several instances where the argumentation and/or the analysis are flawed.

Following to the reviewer’s comment, we have revised the manuscript to emphasize the following points: 1) the need for a revision of the current view on chirping, 2) our proposal of an alternative hypothesis based on correlations between chirping and behavior, which were previously unexplored, and 3) our acknowledgment that while we offer evidence supporting a probing role of chirps (e.g., lack of behavioral correlation, DF-dependency, stereotypy in repeated trials, modulation by clutter and distance), we do not present here conclusive evidence for chirps detecting specific details of conspecific positioning. Neither do we exclude categorically a role of chirps in social communication.

They analyze chirp patterning and show that, most likely, a chirp by an individual is followed by a chirp in the same individual. They argue that it is rare that a chirp elicits a "response" in the other fish. Even if there are clearly stronger correlations between chirps in the same individual, they provide no statistical analysis that discards the existence of occasional "response" patterns. The fact that these are rare, and that the authors don't do an appropriate analysis of probabilities, leads to this unsupported conclusion.

We employed cross-correlation indices, calculated and assessed with a 3 standard deviation symmetrical boundary (which is a statistically sound and strict criterion). Median values were utilized to depict trends in each group/pair. To support our findings, we added new experiments and new figures: 1) a correlation analysis between chirps and behaviors, providing more convincing evidence of how chirps are employed during "scanning" swimming activity (backward swimming); 2) a text mining approach to underscore chirp-behavior correlations, employing alternative and statistically more robust methods.

One of the main pieces of evidence that chirps can be used to enhance conspecific localization is based on their "interference" measure. The measure is based on an analysis of "inter-peak-intervales". This in itself is a questionable choice. The nervous system encodes all parts of the stimulus, not just the peak, and disruption occurring at other phases of the beat might be as relevant. The interference will be mostly affected by the summed duration of intervals between peaks in the chirp AM. They do not explain why this varies with beat frequency. It is likely that the changes they see are simply an artifact of the simplistic measure. A clear demonstration that this measure is not adequate comes from the observation in Fig7E-H. They show that the interference value changes as the signal is weaker. This measure should be independent of the strength of the signal. The method is based on detecting peaks and quantifying the time between peaks. The only reason this measure could be affected by signal strength is if noisy recordings affect how the peak detection occurs. There is no way to argue that this phenomenon would happen the same way in the nervous system. Furthermore, they qualitatively argue that patterns of chirp production follow patterns of interference strength. No statistical demonstration is done. Even the qualitative appraisal is questionable. For example, they argue that there are relatively few chirps being produced for DFs of 60 or -60 Hz. But these are DF where they have only a very small sample size. The single pair of fish that they recorded at some of these frequencies might not have chirped by chance and a rigorous statistical analysis is necessary. Similarly, in Fig 5C they argue that the position of the chirps fall on areas of the graph where the interferences are strongest (darker blue) but this is far from obvious and, again, not proven.

We would like to clarify that the estimation of the effects of chirps on the beat (referred to as “beat interference”) was not intended to serve as the primary evidence supporting a different use of chirping. In fact, all the experiments conducted prior to that calculation already provide substantial evidence supporting the hypothesis we have proposed. In an attempt to address the Reviewer’s concern and to avoid misleading interpretations, we moved this part now to the Supplementary Information (see now Figures S8 and S9), in agreement with the non crucial relevance of this approach. We also added the following statement to the result paragraph entitled “Chirps significantly interfere with the beat and enhance electric image contrast”: “Obviously, measuring chirp-triggered beat interferences by using an elementary outlier detection algorithm on the distribution of beat cycles does not reflect any physiological process carried out by the electrosensory system and can be therefore used only as an oversimplified estimate.”.

Regarding the meaning of “beat interference” (as here estimated) from a perspective of brain physiology: chirp interference was calculated using the beat cycles as a reference. Beat peaks were used only to estimate beat cycle duration. Regardless of whether or not a beat peak is represented in the brain, beat cycle duration (estimated using the peaks) is the main determinant of p-unit rhythmic response to a beat. Regarding the effect of signal amplitude, this is also not very relevant. It is obvious that a chirp creates more - or less - interference based on the chirp FM and its duration (but also the sign of the DF and the magnitude of the amplitude modulation). If electroreceptor responses are entrained in waves of beat AMs and if “interference” is a measure of how such waves are scrambled, then “interference” is a measure of how chirps scramble waves of electroreceptor activity by affecting beat AMs.

The reason why the interference fades with the signal (previous figure 7, now Figure S12) is because it is weighted on the signal strength (the signals used as carrier for chirps are recalculated based on real measurements of signal strength at different distances). Nonetheless, the Reviewer is right: mathematically speaking interference would not change at all because it is just the result of an outlier detection algorithm. This outlier detection is actually set to have a 1% threshold (percent of beat contrast).

Regarding the comparison “chirps vs interference”, we did not make a statistical analysis because we wanted to just show a qualitative observation. Similar results can be obtained for slightly shorter or longer time windows, within certain limits of course (see added Figure S9, in the Supplementary Information). We hope that moving this analysis to the supplementary information makes it clear that this approach is not central to make our point.

The Reviewer’s point on the DF sampling is correct, we have reconsidered the low chirping at 60Hz as potentially the result of sampling bias and edited the respective result paragraph.

They relate the angle at which one fish produces chirps relative to the orientation of the mesh enclosing. They argue that this is related to the orientation of electric field lines by doing a qualitative comparison with a simplified estimate of field lines. To be convincing this analysis should include a quantitative comparison using the exact same body position of the two fish when the chirps are emitted.

We agree with the Reviewer, this type of experiment would be much better suited to illustrate the correlations between chirping and reciprocal positioning in fish. What we can see is that chirping occurs at certain orientations more often than others. This could have something to do with either field geometry or with locomotion in the particular test environment we have used. As mentioned earlier, we are currently editing a second manuscript which will include the type of analysis/experiment the Reviewer is thinking of. We preferred to focus in this first study on the broader behavioral correlates of chirping. We removed the mention to the field current lines because - we agree - the argument is vague as presented here.

They show that the very vast majority of chirps in Fig 6 occur when the fish are within a few centimeters (e.g. very large first bin in Fig6E-Type2). This is a situation when the other fish signal will be strongest and localization will be the easiest. It is hard to understand why the fish would need a mechanism to enhance localization in these conditions (this is the opposite of difficult conditions e.g. the "cluttered" environment).

Agreed, in fact we do not explicitly propose chirps as means to improve “electrolocation” (this word is used only broadly in the abstract) but instead as probes to extract spatial information (e.g. shape, motion, orientation) from a beat source. In a broader sense, all these spatial parameters contribute to any given instance of "localization." Because we were unable to explore all these aspects in greater detail, we chose to maintain a broader perspective. If chirps contribute to a better resolution of fine spatial attributes of conspecific locations, it is reasonable to expect higher chirping rates in proximity to the target fish.

The argumentation aimed at rejecting the well-established role of chirp in communication is weak at best. First, they ignored some existing data when they argue that there is no correlation between chirping and behavioral interactions. Particularly, Hupe and Lewis (2008) showed a clear temporal correlation between chirps and a decrease in bites during aggressive encounters. It could be argued that this is "causal evidence" (to reuse their wording) that chirps cause a decrease in attacks by the receiver fish (see Fig 8B of the Hupe paper and associated significant statistics). Also, Oboti et al. argue that social interactions involve "higher levels of locomotion" which would explain the use of chirps since they are used to localize. But chirps are frequent in "chirp chamber" paradigms where no movement is involved. They also point out that social context covaries with beat frequency and thus that it is hard to distinguish which one is linked to chirping propensity and then say that it is hard to disentangle this from "biophysical features of EOD fields affecting detection and localization of conspecific fish". But they don't provide any proof that beat frequency affects detection and localization so their argument is not clear. Last, they argue that tests in one species shouldn't be extrapolated to other species. But many of the studies arguing for the role of chirps in communication was done on brown ghost. In conclusion of this point, they do not provide any strong argument that rejects the role of chirps as a communication signal. A perspective that would be better supported by their data and consistent with past research would be to argue that, in addition to a role in communication, chirps could sometimes be used to help localize conspecifics.

We did not intend to disregard the extensive body of literature supporting a role of chirps in social communication. Rather, the primary goal of this study was to present a valid alternative perspective to this prevailing view. The existence of a well-established hypothesis does not imply that new evidence cannot change it; it simply indicates that changing it may be challenging either because it's genuinely difficult or because the idea has not been thoroughly explored. Whatever the case may be, proposing new hypotheses, whether complementary or alternative to established theories, is a challenging undertaking for a single study. We judged that starting from broad correlations would be the most desirable approach.

We did not ignore data from Hupé and Lewis 2008. We cited this study repeatedly and compared their findings to those of others, not only for the correlation chirp-behaviors but also for chirping distance considerations. However, following the Reviewer’s comment, we now cite this study in the context of the behavioral analysis recently added (data from the PSTH plots could possibly confirm the observation of lower chirps during attacks). We also cited the study by Triefenbach and Zakon 2008, which reports something along the same lines. See the statement: “Overall, these results provided mutually reinforcing evidence indicating that chirps are produced more often during locomotion or scanning-related motor activity and confirm previous reports of a lower occurrence of chirping during more direct aggressive contact (as shown also by Triefenbach and Zakon, 2008; Hupé and Lewis, 2008).”, in the result paragraph related to the behavioral correlates of chirping.

In our study we make it clear how we distinguish causal evidence (i.e. providing evidence that A is required for B) from correlation (i.e. evidence for A simply occurring together with B). We also make it clear that we are not going to provide causal evidence but we are going to provide new evidence for correlations that were so far not considered, in order to propose a new unexplored function of chirps.

The Reviewer's point on chirping during motion and while caged in a chirp chamber is valid. Indeed at first we were also puzzled by this finding. However, under the “chirps as probes” paradigm, chirping in a chirp-chamber can be explained by the need to obtain spatial information from an otherwise unreachable beat source (brown ghosts are typically exploring new environmental objects or conspecifics by actively swimming around them - something caged fish can’t do). So, eventually the observation of chirping under conditions of limited movement (such as in a chirp chamber experiment) is not in contradiction with our hypothesis, rather it can be used to support it. Further experiments are required - as rightfully pointed out - to evaluate the effects of beat frequency on beat detection. We added a note about this in the “probing with chirps” discussion paragraph.

The Reviewer's comment regarding generalization is unclear. We acknowledge that most studies are conducted in brown ghosts, as stated in the abstract. Our intention was to highlight that insights gained from this species have been applied to broaden the understanding of chirps in other species. Specifically, the "behavioral meaning idea" of chirping has been extended to other gymnotiform species producing EOD frequency modulations .

Our study's aim is not to dismiss the idea of chirps being used for communication but to present an alternative hypothesis and to provide supporting evidence. While our results may not align well with the communication theory, our intention is not dismissal but rather engaging in a discussion and exploration of alternative perspectives.

The discussion they provide on the possible mechanism by which chirps could help with localization of the conspecific is problematic. They imply that chirps cause a stronger response in the receptors. For most chirps considered here, this is not true. For a large portion of the beat frequencies shown in this paper, chirps will cause a de-synchronization of the receptors with no increase in firing rate. They cannot argue that this represents an enhanced response. They also discuss a role for having a broader frequency spectrum -during the chirp- in localization by making a parallel with pulse fish. There is no evidence that a similar mechanism could even work in wave-type fish.

We have already commented on the “localization” idea in our previous responses. The Reviewer is right in saying that we have provided only vague descriptions of the potential mechanisms implied by our hypothesis. The studies by Benda and others (2005, 2006) demonstrate a clear synchronizing effect of chirps on p-unit firing rates, especially at low DFs (at ranges similar to those considered in this study). This synchronization could lead to an enhanced response at the electroreceptor level, as described in these very studies, which in turn would result in a higher probability of firing in downstream neurons (E-cells in the ELL).

As also reported within the same works, chirps may also exert an opposite effect on p-units (i.e. desynchronization). This is what happens for large chirps at high DFs. Desynchronization may cause temporary lapses of p-unit firing, which in turn may lead to increased activity of I-cells in the ELL (which are indeed specifically tuned to p-unit lack of activity).

So, in general, if we consider both ON and OFF pyramidal cells (in the ELL) and small and large chirps, we could state that chirps can be potentially used to enhance the activity of peripheral electrosensory circuits through different mechanisms, contingent on the chirp type and beat frequency. Unfortunately, space constraints limited our ability to dig into these details in the present study.

However, to address the Reviewer’s rightful point, we now mention this in the manuscript: Since the beat AMs generated by the chirps always trigger reliable responses in primary electrosensory circuits (pyramidal cells in the ELL respond to both increases and decreases in beat AM), any chirp-triggered AM causing a sudden change in p-unit firing could potentially amplify the downstream signal (Marsat and Maler, 2010) and thus enhance EI contrast.” (see result paragraph on beat interference and electric images).

They write the whole paper as if males and females had been identified in their experiments. Although EOD frequency can provide some guess of the sex the method is unreliable. We can expect a non-negligible percentage of error in assigning sex.

We agree and in fact, in the method section we state:

“The limitation of this approach is that females cannot be distinguished from immature males with absolute certainty, since no post-mortem gonadal inspection was carried out.”

to this we added:

“Although a more accurate way to determine the sex of brown ghosts would be to consider other morphological features such as the shape of the snout, the body size, the occurrence of developing eggs, EOD frequency has been extensively used for this purpose.”

Moreover, the consistent behavioral differences observed in low frequency fish, measured with those behavioral experiments aimed at assessing responses to playback stimuli and swimming behavior in novel environments, could also be caused by a younger age (as opposed to femaleness). However, the size ranges of our fish (an admittedly unreliable proxy of age) were all comparable, making this possibility perhaps less likely.

Reviewer #2 (Public Review):

Studying the weakly electric brown ghost knifefish, the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. This is a behavior that has been very well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that could have a great impact on the field. The authors do provide convincing evidence that chirps may function in homeoactive sensing. However, their evidence arguing against a role for chirps in communication is not as strong, and neglects a large body of research. Ultimately, the manuscript has great potential but suffers from framing these two possibilities as mutually exclusive and dismissing evidence in favor of a communicative function.

We thank the Reviewer for the comment. Overall, we have edited the manuscript to soften our conclusions and avoid any strong categorical statement excluding the widely accepted role of chirps in social communication. We have added some new experiments with the aim to add more detail to the behavioral correlates of chirping and to the DF dependency of the production of different types of chirps. Nonetheless, based on our results, we are prone to conclude that the communication idea - although widely accepted - is not as well substantiated as it should be.

Although we do not dismiss the bulk of literature supporting a role of chirps in social communication, we think that our hypothesis (i.e. decoding of spatial parameters from the beat) may be not fully compatible with the social communication hypothesis for the following reasons:

(1) Chirp type dependency on DF makes chirps likely to be adaptive responses to beat frequency. While this idea is compatible with a role of chirps in the detection of beat parameters, their concurrent role in social communication would imply that chirps interacting at given beat frequencies (DFs) would communicate only (or mainly) by delivering a very limited range of “messages”. For instance, assuming type 2 chirps are related to aggression (as widely suggested), are female-male pairs - with larger DFs - interacting less aggressively than same sex pairs? Our experiments often suggested this is not the case. In addition, large DFs are not always indicative of opposite sex interactions, while they are very often characterized by the emission of large chirps. Not to mention that, despite the fact that opposite sex interactions in absence of breeding-like conditions, cannot be considered truly courtship-related, large chirps are often considered courtship signals, regardless of the reproductive state of the emitting fish.

(2) Chirping is highly affected by locomotion (consider female/male pairs with or without mesh divider) and distance (as shown in the novel environment exploration experiment). While the involvement of both parameters is compatible with a role of chirps in active sensing, a role of chirps in social communication implies that such signaling would occur only when fish are in very close proximity to each other. In this case, the beat is therefore heavily distorted not only by fish position/locomotion but also by chirps. Which means that when fish are close to each other, the 2 different types of information relayed by the beat (electrolocation and electrocommunication) would certainly interfere (this idea has been better phrased in the Introduction paragraph).

(3) In our playback experiments we could not see any meaningful matching (e.g. angry-chirp → angry-chirp or sexy-chirp → approach) between playback chirps and evoked chirps, raising doubts on the meaning associated so far with the different types. Considering that playback experiments are typically used to assess signal meaning based on how animals respond to them, this result is suggesting quite strongly that such meaning cannot be assigned to chirps.

(4) In playback experiments in which the same stimulus is provided multiple times, chirp type transitions (i.e. emission of a different chirp type after a given chirp) become predictable (as shown in the added playback experiments using ramping signals). This confirms that the choice to emit a given chirp type has something to do with beat frequency (or a change in this parameter) and not a communication of internal states. It would be otherwise unclear how a fish could change its internal state so quickly - and so reliably - even in the span of a few seconds.

Despite this evidence against a semantic content of chirps in the context of social communication, we conclude the manuscript reminding that we are not providing strong evidence dismissing the communication hypothesis, and that both could coexist (see the example of “proximity signals” in the mating context given in the concluding paragraph).

(1) The specific underlying question of this study is not made clear in the abstract or introduction. It becomes apparent in reading through the manuscript that the authors seek to test the hypothesis that chirps function in active sensing (specifically homeoactive sensing). This should be made explicitly clear in both the abstract and introduction, along with the rationale for this hypothesis.

In the abstract we state “Despite the success of this model in neuroethology over the past seven decades, the underlying logic of their electric communication remains unclear. This study re-evaluates this view, aiming to offer an alternative, and possibly complementary, explanation for why these freshwater bottom dwellers emit electric chirps.”. This statement is meant as a summary of our aims. However, in order to convey a clearer message, we have revised the whole manuscript to more explicitly articulate our objectives. In particular we stress that with our experiments we intend to provide correlative evidence for a different role of chirps (previously unexplored) with the idea to stimulate a discussion and possibly a revision of the current theory about the functional role of chirps.

In the introduction we have added a paragraph explaining our aim and also why we think that communicating through chirps could potentially interfere with efficient electrolocation: “Since both chirps and positional parameters (such as size, orientation or motion) can only be detected as perturbations of the beat (Petzold et al., 2016; Yu et al., 2012; Fotowat et al., 2013), and via the same electroreceptors, the inputs relaying both types of information are inevitably interfering. Moreover, as the majority of chirps are produced within a short range (< 50 cm; Zupanc et al., 2006; Hupé and Lewis 2008; Henninger et al., 2018; see appendix 1) this interference is likely to occur consistently during social interactions.

Under the communication-hypothesis, the assumption that chirps and beats are conveying different types of information (i.e. semantic value as opposed to position and related geometrical parameters) is therefore leaving this issue unresolved.”.

(2) My biggest issue with this manuscript is that it is much too strong in dismissing evidence that chirping correlates with context. This is captured in this sentence in the introduction, "We first show that the choice of different chirp types does not significantly correlate with any particular behavioral or social context." This very strong conclusion comes up repeatedly, and I disagree with it, for the following reasons:

In your behavioral observations, you found sex differences in chirping as well as differences between freely interacting and physically separated fish. Your model of chirp variability found that environmental experience, social experience, and beat frequency (DF) are the most important factors explaining chirp variability. Are these not all considered "behavioral or social context"? Beat frequency (DF) in particular is heavily downplayed as being a part of "context" but it is a crucial part of the context, as it provides information about the identity of the fish you're interacting with.

In your playback experiments, fish responded differently to small vs. large DFs, males chirped more than females, type 2 chirps became more frequent throughout a playback, and rises tended to occur at the end of a playback. These are all examples of context-dependent behavior.

We agree with the Reviewer’s comment and we think that probably we have been unclear in what the meaning of that statement was. We also agree with the Reviewer about what is defined as “context”, and that a given beat frequency (DF) can in the end represent a “behavioral context” as well. In order to make it clearer, we have rephrased this statement and changed it to: “We first show that the relative number of different chirp types in a given recording does not significantly correlate with any particular behavioral or social context.”. This new form refers specifically to the observation that - in all different social conditions examined - the relative amounts of different types of chirps is unchanged (see Figure S2). We thought the Reviewer maybe interpreted our statement as if we suggested that chirp type choice is random or unaffected by any social variable. We agree with the Reviewer that this is not the case. We also reported that sex differences in chirping are present, but we have emphasized they may have something to do with the propensity of the brown ghosts of either sex to swim/explore as opposed to seek refuge and wait (as suggested by our experiments in which FM pairs were either divided or freely interacting and our novel environment exploration experiments).

We agree DF is important, in fact it is the 3rd most important factor explaining chirp variance in our model. In our fish pair recordings, we see a strong correlation of chirp total variance with tank experience (one naïve, one experienced, both fish equally experienced) and social context (novel to each other/familiar to each other, subordinate/dominant, breeding/non breeding, accessible/not accessible) although data clustering seems to better distinguish “divided” vs “freely moving” conditions (and sex may also play a role as well because of the reversal of sexual dimorphism in chirp rates in precisely this case) more than other variables. However, we do not see a specific effect of these variables on the proportion of different types of chirps in any recording (see Figure S2).

We also edited the beginning of the first result paragraph and changed it to “Thus, if behavioral meaning can be attributed to different types of chirps, as posed by the prevailing view (e.g., Hagedorn and Heiligenberg, 1985; Larimer and MacDonald, 1968; Rose, 2004), one should be able to identify clear correlations between behavioral contexts characterizing different internal states and the relative amounts of different types of chirp”, to emphasize we are here assessing the meaning of different types of chirps (not of the total amount of chirping in general).

Further, you only considered the identity of interacting fish or stimulated fish, not their behavior during the interaction or during playback. Such an analysis is likely beyond the scope of this study, but several other studies have shown correlations between social behavior and chirping. In the absence of such data here, it is too strong to claim that chirping is unrelated to context.

We agree with the Reviewer, in fact this analysis was previously carried out but purposely left out in an attempt to limit the manuscript length. We have now made space for this experimental work which is now added (see the new Figure 6).

In summary, it is simply too strong to say that chirping does not correlate with context. Importantly, however, this does not detract from your hypothesis that chirping functions in homeoactive sensing. A given EOD behavior could serve both communication and homeoactive sensing. I actually suspect that this is quite common in electric fish. The two are not mutually exclusive, and there is no reason for you to present them as such. I recommend focusing more on the positive evidence for a homeoactive function and less on the negative evidence against a communication function.

We aimed to clarify that our reference was to the lack of correlation between "chirp type relative numbers" and the analyzed context. Regarding the communication function, we tempered negative statements. However, as this study stems from evidence within the established paradigm of "chirps as communication signals", and aims at proposing an alternative hypothesis, eliminating all references to it could undermine the study's purpose.

(3) The results were generally challenging to follow. In the first 4 sections, it is not made clear what the specific question is, what the approach to addressing that question is, and what specific experiment was carried out (the last two sections of the results were much clearer). The independent variables (contexts) are not clearly established before presenting the results. Instead they are often mentioned in passing when describing the results. They come across as an unbalanced hodgepodge of multiple factors, and it is not made clear why they were chosen. This makes it challenging to understand why you did what you did, the results, and their implications. For each set of major results, I recommend: First, pose a clear question. Then, describe the general approach to answering that question. Next, describe the specifics of the experimental design, with a rationale that appeals to the general approach described. Finally, describe the specific results.

The introductory sentences of the first result paragraphs have been edited, rendering the aim of the experiments more explicit.

(4) Results: "We thus predicted that, if behavioral meaning can be attributed to different types of chirps, as posed by the prevailing view (e.g., Hagedorn and Heiligenberg, 1985; Larimer and MacDonald, 1968; Rose, 2004)..." It should be made clear why this is the prevailing view, and this description should likely be moved to the introduction. There is a large body of evidence supporting this view and it is important to be complete in describing it, especially since the authors seem to seek to refute it.

We understand the Reviewer’s question and we tried to express in the introduction the main reasons for why this is the current view. We state “Different types of chirps are thought to carry different semantic content based on their occurrence during either affiliative or agonistic encounters (Larimer and MacDonald 1968; Bullock 1969; Hopkins 1974; Hagedorn and Heiligenberg 1985; Zupanc and Maler 1993; Engler et al. 2000; Engler and Zupanc 2001; Bastian et al., 2001).”. To this we added: “Although supported mainly by correlative evidence, this idea gained popularity because it is intuitive and because it matches well enough with the numerous behavioral observations of interacting brown ghosts.”.

We believe the prevailing view is based on intuition and a series of basic observed correlations repeated throughout the years. The crystallization of this idea is not due to negligence but mainly to technical limitations existing at the time of the first recordings. In order to assess the role of chirps in behaving fish a tight and precise temporal control over synched video-EOD recordings is most likely necessary, and this is a technical feature probably available only much later than the 50-60ies, when electric communication was first described.

(5) I am not convinced of the conclusion drawn by the analysis of chirp transitions. The transition matrices show plenty of 1-2 and 2-1 transitions occurring. Further, the cross-correlation analysis only shows that chirp timing between individuals is not phase-locked at these small timescales. It is entirely possible that chirp rates are correlated between interacting individuals, even if their precise timing is not.

We agree with the Reviewer: chirp repertoires recorded in different social contexts are not devoid of reciprocal chirp transitions (i.e. fish 1 chirp - to - fish 2 chirp, or vice versa). Yet our point is to emphasize that their abundance is way more limited when compared to the self-referenced ones (i.e. 1-1 and 2-2). This is a fair concern and in order to further address this point, we have added a whole new set of analyses and new experiments (see chirp-behavior correlations, PSTHs and more analysis based on more solid statistical methods; see Figure 6).

Reviewer #3 (Public Review):

Summary:

This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, as well as with playback experiments. It applies state-of-the-art methods for reducing dimensionality and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The exceptional strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that a number of commonly accepted truths about which variable affects chirping must be carefully rewritten or nuanced. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats and objects.

Strengths:

The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry.

The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a communication goal for most chirps. Rather, the key determinants of chirping are the difference frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. These conclusions by themselves will be hugely useful to the field. They will also allow scientists working on other "communication" systems to at least reconsider, and perhaps expand the precise goal of the probes used in those senses. There are a lot of data summarized in this paper, and thorough referencing to past work. For example, the paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-received chirp transitions beyond the known increase in chirp frequency during an interaction.

The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization.

The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water.

Weaknesses:

My main criticism is that the alternative putative role for chirps as probe signals that optimize beat detection could be better developed. The paper could be clearer as to what that means precisely.

We appreciate the Reviewer's kind comments. While we acknowledge that our exploration of chirp function in this study may be limited and not entirely satisfying, we made this decision due to space constraints, opting for a broader and diversified approach. We hope that future studies will build on these data and start filling the gaps. We are also working on another manuscript which is addressing this point more in detail.

Nonetheless, we considered the Reviewer’s criticism and added not only a new figure (to show more explicitly what chirps can do to the perceived electric fields, as simulated by electric images) but also more descriptive parts explaining how we think chirps may act to improve the spatial resolution of beat processing (see the discussion paragraph “probing with chirps”). In this paragraph we rendered more clearly how chirps could improve beat processing by phase shifting EODs and recovering eventual blind-spots on the fish skin caused by disruptive EOD interferences (resulting in lower beat contrast). We also mention that enhancement of electrosensory input triggered by chirps, could be localized not only at the level of electroreceptors (consider the synchronizing effects small chirps have on p-units at low frequency beats) but also at the level of ON and OFF pyramidal cells in the ELL. Looked at from the perspective of these neurons, any chirp would enhance the activity of these input lines, yet in opposite ways.

And there is an egg-and-chicken type issue as well, namely, that one needs a beat in order to "chirp" the beating pattern, but then how does chirping optimize the detection of the said beat? Perhaps the authors mean (as they wrote elsewhere in the paper) that the chirps could enhance electrosensory responses to the beat.

According to the Reviewer’s comment, we have now revised several instances of the misleading phrasing identified.

In the results on novel environment exploration: “If chirps enhance beat processing, for instance, chirping should occur within beat detection range but at a certain distance.”.

“This, in turn, could be used to validate our beat-interference estimates as meaningfully related to beat processing.” and “In all this, rises may represent an exception as their locations are spread over larger distances and even in presence of obstacles potentially occluding the beat source (such as shelters, plants, or walls), all of which are conditions in which beat detection or beat processing could be more difficult (this, could be coherent with the production of rises right at the end of EOD playbacks; Figure S5).”

Last result paragraph (clutter experiment): “Overall, these results indicate that chirping is significantly affected by the presence of environmental clutter partially disrupting - or simply obstructing - the processing of beat related information during locomotion”.

In the probing with chirps discussion paragraph “In theory, chirps could also be used to improve electrolocation of objects as well (as opposed to the processing of the beat).”.

In the conclusions: “optimizing the otherwise passive responses to the beat”.

A second criticism is that the study links the beat detection to underwater object localization. I did not see a sufficiently developed argument in this direction, nor how the data provided support for this argument. It is certainly possible that the image on the fish's body of an object in the environment will be slightly modified by introducing a chirp on the waveform, as this may enhance certain heterogeneities of the object in relation to its environment. The thrust of this argument seems to derive more from the notion of Fourier analysis with pulse type fish (and radar theory more generally) that the higher temporal frequencies in the beat waveform induced by the chirp will enable a better spatial resolution of objects. It remains to be seen whether this is significant.

The Reviewer is correct in noting that this point is not addressed in the manuscript. We introduced it as a speculative discussion point to mention alternative possibilities. These could be subject to further testing in future studies.

I would also have liked to see a proposal for new experiments that could test these possible new roles.

We have added clearer suggestions for future experiments throughout the discussion: these may be aimed at 1) improving playback experiments using more realistic copies of the brown ghost’s EODs (including harmonics), 2) assess fish reciprocal positioning during chirping in better detail and 3) test the use of chirping during target-reaching tasks in order to better assess the probing function of chirps.

The authors should recall for the readers the gist of Bastian's 2001 argument that the chirp "can adjust the beat frequency to levels that are better detectable" in the light of their current. Further, at the beginning of the "Probing with chirps" section, the 3rd way in which chirps could improve conspecific localization mentions the phase-shifting of the EOD. The authors should clarify whether they mean that the tuberous receptors and associated ELL/toral circuitry could deal with that cue, or that the T_unit pathway would be needed?

We thank the Reviewer for identifying this unclear point. We added reference to the p-units “Yet, this does not exclude the possibility that chirps could be used to briefly shift the EOD phase in order to avoid disruptive interferences caused by phase opposition (at the level of p-units)” in the above mentioned paragraph. We would prefer to omit a more detailed reference to t-units in order to avoid lengthy descriptions required to discuss the different electroreceptor types.

On p.17 I don't understand what is meant by most chirps being produced, possibly aligned with the field lines, since field lines are everywhere. And what is one to conclude from the comparison of Fig.6D and 7A? Likewise it was not clear what is meant by chirps having a detectable effect on randomly generated beats.

We agree on the valid point raised by the Reviewer and we have removed reference to current lines from the text.

In the section on Inconsistencies between behaviour and hypothesized signal meaning, the authors could perhaps nuance the interpretation of the results further in the context of the unrealistic copy of natural stimuli using EOD mimics. In particular, Kelly et al. 2008 argued that electrode placement mattered in terms of representation of a mimic fish onto the body of a real fish, and thus, if I properly understand the set up here, the movement would cause the mimic to vary in quality. This may nevertheless be a small confounding issue.

We agree with the Reviewer and added a comment at the beginning of the paragraph mentioned. “Nonetheless, it's plausible that playback stimuli, as employed in our study and others, may not faithfully replicate natural signals, thus potentially influencing the reliability of the observed behaviors. Future studies might consider replicating these findings using either natural signals or improved mimics, which could include harmonic components (excluded in this study).”

Recommendations for the authors:

8Reviewer #2 (Recommendations For The Authors):*

(1) Abstract: "...is probably the most intensely studied species..." is a weak, unsupported, and unnecessary statement. Just state that it has been heavily studied, or is one of the most well-studied,...

rephrased

(2) Abstract: "...are thus used as references to specific internal states during recordings - of either the brain or the electric organ..." This was not clear to me.

rephrased

(3) Abstract: "...the logic underlying this electric communication..." It is not clear to me what the authors mean here by "logic".

rephrased

(4) I strongly recommend clearly defining homeoactive sensing and distinguishing it from allocative sensing when this term is first introduced in the introduction. This is not a commonly used term. Most readers likely think they understand what is meant by the term active sensing, however I recommend first defining it, and then distinguishing amongst these two different types of active sensing.

rephrased

(5) Introduction: "Together with a few other species (Rose, 2004),..." More than a few. There are hundreds of species with electric organs. It is certainly not a "unique" capability.

rephrased

(6) Introduction: "But the real advantage of active electrolocation can be appreciated in the context of social interaction." This is unclear. Why is this the "real advantage" of active electrolocation when an electrically silent fish could detect an electrically communicating fish just fine without interference? Active electrolocation is needed to detect objects that are not actively emitting an electric field. It is not needed to detect signaling individuals.

rephrased

(7) Introduction: why is active sensing using EODs limited to distances of 6-12 cm? Why does it not work at closer range?

Here we meant to give a range based on published data. We rephrased it to “up to 12”.

(8) Introduction: electric fields decay with the cubed of distance, as you show in appendix 1.

rephrased

(9) Introduction: it is not clear what is meant by "blurred EOD amplitude".

rephrased (“noisy”)

(10) Figure 2C is very challenging to interpret. I recommend spending more time in the manuscript walking the reader through this analysis and its presentation.

We are grateful for the comment as we probably overlooked this point. We now added a small paragraph to explain these data in better detail.

(11) Results: "This was done by calculating the ratio between the duration of the beat cycles affected by the chirp (beat interpeak intervals) and the total duration of the beat cycles detected within a fixed time window (roughly double the size of the maximum chirp duration, 700 ms)." This was not clear to me.

We now rephrased to “Estimates of beat interference were made by calculating the ratio between the cumulative duration of the beat cycles affected by a given chirp (1 beat cycle corresponding to the beat comprised by two consecutive beat peaks, or - more simply - the beat inter-peak interval) over the cumulative duration of all the beat cycles within the time window used as a reference (700 ms; other analysis windows were tested Figure S9)” to clarify this method.

(12) Results: "For each chirp, the interference values obtained for 4 different phases (90{degree sign} steps) were averaged." Why was this done?

To consider an average effect across phases. Although it is true that chirp parameters may have a different impact on the beat, depending on EOD phase, including this parameter in our figure/s would have considerably increased the volume of data reported giving too much emphasis to an analysis we judged not crucially important. In addition, since we did not consider EOD phase in our recordings, we opted for an average estimate encompassing different phase values.

(13) Discussion: "Third, observations in a few species are generalized to all other gymnotiforms without testing for species differences (Turner et al., 2007; Smith et al., 2013; Petzold et al., 2016)." I strongly disagree with this statement. First, the studies referenced here do explicitly compare chirps across species. Second, you only studied one species here, so it is not clear to me how this is a relevant concern in interpreting your findings.

Here we have probably been unclear in the writing: the point we wanted to make is that the idea of chirps having semantic content has been generalized to other species without investigating the nature of their chirping with as much detail as done for brown ghosts.

We have now rephrased the statement and changed it to: “Second, observations in a few species are generalized to all other gymnotiforms without testing whether chirping may have similar functions in other species (Turner et al., 2007; Smith et al., 2013; Petzold et al., 2016)”

(14) Discussion: "The two beats could be indistinguishable (assuming that the mechanism underlying the discrimination of the sign of DF at low DFs, and thought to be the basis of the so called jamming avoidance response (JAR; Metzner, 1999), is not functional at higher DFs)." Why would you assume this?

What we meant here is that it is unlikely that the two DFs are not discriminated by the same mechanisms implied in the JAR, even if the DF is higher than the levels at which usually JARs are detected (i.e. DF = 1-10 Hz?). To improve clarity, we rephrased this statement. “The two beats could be indistinguishable (assuming - perhaps not realistically - that the same mechanism involved in DF discrimination at lower DF values would not work in this case; Metzner, 1999)”.

(15) Discussion: "...an idea which seems congruent with published electrophysiological studies..." How so?

Rephrased to “Based on our beat interference estimates, we propose that the occurrence of the different types of chirps at more positive DFs (such as in male-to-female chirping) may be explained by their different effect on the beat (Figure 5D; Benda et al., 2006; Walz et al., 2013).”

Reviewer #3 (Recommendations For The Authors):

On p.2 there is a discrepancy between the quoted ranges for active sensing of objects, first 10-12 cm, and then 6-12 cm further down. And in the following paragraph right below this passage, electric fields are said to decay with the squared distance (appendix 1). That expression has a cos(theta) which is inversely proportional to the distance, and so one is really dealing, as expected for dipolar fields, with a drop-off that decays with the distance cubed.

We thank the Reviewer for the comment, we have now corrected the mistake and added “cubed”. We also removed the imprecise reference to the range 6-12 cm, rephrased to “up to 12 cm”.

At the end of the section on Inconsistencies..., it is not clear what "activity levels" refers to. It should also be made clearer at the outset, and reminded in this section too, that for the authors, behavioural context does not include social experience, which is somewhat counter-intuitive.

We now specified we meant “locomotor activity levels”. Regarding the social experience we included it as “behavioral context”, we now made it clearer in the first result paragraph. We hope we resolved the confusion.

The caption of Fig.8 could use more clarity in terms of what is being compared in (C) (and is "1*2p" a typo?)

We corrected the typo and edited the figure to make the references more clear.

The concept of "high self-correlation of chirp time series" is presented only in the Conclusion using those words. The word self-correlation is not used beforehand. This needs to be fixed so the reader knows clearly what is being referred to.

Thank you for noting this. We have now changed the wording using the term “auto-correlation” and changed a statement at the beginning of the “interference” result paragraph accordingly, removing references to self-correlation.

Author response:

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

We thank the reviewers for their thorough re-evaluation of our revised manuscript. Addressing final issues they raised has improved the manuscript further. We sincerely appreciate the detailed explanations that the reviewers provided in the "recommendations for authors" section. This comprehensive feedback helped us identify the sources of ambiguity within the analysis descriptions and in the discussion where we interpreted the results. Below, you will find our responses to the specific comments and recommendations.

Reviewer #1 (Recommendations):

(1) I find that the manuscript has improved significantly from the last version, especially in terms of making explicit the assumptions of this work and competing models. I think the response letter makes a good case that the existence of other research makes it more likely that oscillators are at play in the study at hand (though the authors might consider incorporating this argumentation a bit more into the paper too). Furthermore, the authors' response that the harmonic analysis is valid even when including x=y because standard correlation analysis were not significant is a helpful response. The key issue that remains for me is that I have confusions about the additional analyses prompted by my review to a point where I find it hard to evaluate how and whether they demonstrate entrainment or not. 

First, I don't fully understand Figure 2B and how it confirms the Arnold tongue slice prediction. In the response letter the authors write: "...indicating that accuracy increased towards the preferred rate at fast rates and decreased as the stimulus rate diverged from the preferred rate at slow rates". The figure shows that, but also more. The green line (IOI < preferred rate) indeed increases toward the preferred rate (which is IOI = 0 on the x-axis; as I get it), but then it continues to go up in accuracy even after the preferred rate. And for the blue line, performance also continues to go up beyond preferred rate. Wouldn't the Arnold tongue and thus entrainment prediction be that accuracy goes down again after the preferred rate has passed? That is to say, shouldn't the pattern look like this (https://i.imgur.com/GPlt38F.png) which with linear regression should turn to a line with a slope of 0?

This was my confusion at first, but then I thought longer about how e.g. the blue line is predicted only using trials with IOI larger than the preferred rate. If that is so, then shouldn't the plot look like this? (https://i.imgur.com/SmU6X73.png). But if those are the only data and the rest of the regression line is extrapolation, why does the regression error vary in the extrapolated region? It would be helpful if the authors could clarify this plot a bit better. Ideally, they might want to include the average datapoints so it becomes easier to understand what is being fitted. As a side note, colours blue/green have a different meaning in 2B than 2D and E, which might be confusing. 

We thank the reviewer for their recommendation to clarify the additional analyses we ran in the previous revision to assess whether accuracy systematically increased toward the preferred rate estimate. We realized that the description of the regression analysis led to misunderstandings. In particular, we think that the reviewer interpreted (1) our analysis as linear regression (based on the request to plot raw data rather than fits), whereas, in fact, we used logistic regression, and (2) the regression lines in Figure 2B as raw IOI values, while, in fact, they were the z-scored IOI values (from trials where stimulus IOI were faster than an individual’s preferred rate, IOI < preferred rate, in green; and from trials stimulus IOI were slower than an individual’s preferred rate, IOI > preferred rate, in blue), as the x axis label depicted. We are happy to have the opportunity to clarify these points in the manuscript. We have also revised Figure 2B, which was admittedly maybe a bit opaque, to more clearly show the “Arnold tongue slice”.  

The logic for using (1) logistic regression with (2) Z-scored IOI values as the predictor is as follows. Since the response variable in this analysis, accuracy, was binary (correct response = 1, incorrect response = 0), we used a logistic regression. The goal was to quantify an acrosssubjects effect (increase in accuracy toward preferred rate), so we aggregated datasets across all participants into the model. The crucial point here is that each participant had a different preferred rate estimate. Let’s say participant A had the estimate at IOI = 400 ms, and participant B had an estimate at IOI = 600 ms. The trials where IOI was faster than participant A’s estimate would then be those ranging from 200 ms to 398 ms, and those that were slower would range from 402 ms to 998 ms. For Participant B, the situation would be different:  trials where IOI was faster than their estimate would range from 200 ms to 598 ms, and slower trials would range between 602 ms to 998 ms. For a fair analysis that assesses the accuracy increase, regardless of a participant’s actual preferred rate, we normalized these IOI values (faster or slower than the preferred rate). Zscore normalization is a common method of normalizing predictors in regression models, and was especially important here since we were aggregating predictors across participants, and the predictors ranges varied across participants. Z-scoring ensured that the scale of the sample (that differs between participant A and B, in this example) was comparable across the datasets. This is also important for the interpretation of Figure 2B. Since Z-scoring involves mean subtraction, the zero point on the Z-scaled IOI axis corresponds to the mean of the sample prior to normalization (for Participant A: 299 ms, for Participant B: 399 ms) and not the preferred rate estimate. We have now revised Figure 2B in a way that we think makes this much clearer.  

The manuscript text includes clarification that the analyses included logistic regression and stimulus IOI was z-scored: 

“In addition to estimating the preferred rate as stimulus rates with peak performance, we investigated whether accuracy increased as a function of detuning, namely, the difference between stimulus rate and preferred rate, as predicted by the entrainment models (Large, 1994; McAuley, 1995; Jones, 2018). We tested this prediction by assessing the slopes of mixed-effects logistic regression models, where accuracy was regressed on the IOI condition, separately for stimulus rates that were faster or slower than an individual’s preferred rate estimate. To do so, we first z-scored IOIs that were faster and slower than the participant’s preferred rate estimates, separately to render IOI scales comparable across participants.” (p. 7)

While thinking through the reviewer’s comment, we realized we could improve this analysis by fitting mixed effects models separately to sessions’ data. In these models, fixed effects were z-scored IOI and ‘detuning direction’ (i.e., whether IOI was faster or slower than the participant’s preferred rate estimate). To control for variability across participants in the predicted interaction between z-scored IOI and direction, this interaction was added as a random effect. 

“Ideally, they might want to include the average datapoints so it becomes easier to understand what is being fitted.”

Although we agree with the reviewer that including average datapoints in a figure in addition to model predictions usually better illustrates what is being fitted than the fits alone, this doesn’t work super well for logistic regression, since the dependent variable is binary. To try to do a better job illustrating single-participant data though, we instead  fitted logistic models to each participant’s single session datasets, separately to conditions where z-scored IOI from fasterthan-preferred rate trials, and those from slower-than-preferred rate trials, predicted accuracy. From these single-participant models, we obtained slope values, we referred to as ‘relative detuning slope’, for each condition and session type. This analysis allowed us to illustrate the effect of relative detuning on accuracy for each participant. Figure 2B now shows each participant’s best-fit lines from each detuning direction condition and session.

Since we now had relative detuning slopes for each individual (which we did not before), we took advantage of this to assess the relationship between oscillator flexibility and the oscillator’s behavior in different detuning situations (how strongly leaving the preferred rate hurt accuracy, as a proxy for the width of the Arnold tongue slice). Theoretically, flexible oscillators should be able to synchronize to wide range of rates, not suffering in conditions where detuning is large (Pikovsky et al., 2003). Conversely, synchronization of inflexible oscillators should depend strongly on detuning. To test whether our flexibility measure predicted this dependence on detuning, which is a different angle on oscillator flexibility, we first averaged each participant’s detuning slopes across detuning directions (after sign-flipping one of them). Then, we assessed the correlation between the average detuning slopes and flexibility estimates, separately from conditions where |-𝚫IOI| or |+𝚫IOI| predicted accuracy. The results revealed significant negative correlations (Fig. 2F), suggesting that performance of individuals with less flexible oscillators suffered more as detuning increased. Note that flexibility estimates quantified how much accuracy decreased as a function of trial-to-trial changes in stimulus rate (±𝚫IOI). Thus, these results show that oscillators that were robust to changes in stimulus rate were also less dependent on detuning to be able to synchronize across a wide range of stimulus rates. We are excited to be able to provide this extra validation of predictions made by entrainment models. 

To revise the manuscript with the updated analysis on detuning:

• We added the descriptions of the analyses to the Experiment 1 Methods section.

Calculation of detuning slopes and their averaging procedure are in Preferred rate estimates:

“In addition to estimating the preferred rate as stimulus rates with peak performance, we investigated whether accuracy increased as a function of detuning, namely, the difference between stimulus rate and preferred rate, as predicted by the entrainment models (Large, 1994; McAuley, 1995; Jones, 2018). We tested this prediction by assessing the slopes of mixed-effects logistic regression models, where accuracy was regressed on the IOI condition, separately for stimulus rates that were faster or slower than an individual’s preferred rate estimate. To do so, we first z-scored IOIs that were faster and slower than the participant’s preferred rate estimates, separately to render IOI scales comparable across participants. The detuning direction (i.e., whether stimulus IOI was faster or slower than the preferred rate estimate) was coded categorically. Accuracy (binary) was predicted by these variables (zscored IOI, detuning direction), and their interaction. The model was fitted separately to datasets from random-order and linear-order sessions, using the fitglme function in MATLAB. Fixed effects were z-scored IOI and detuning direction and random effect was their interaction. We expected a systematic increase in performance toward the preferred rate, which would result in a significant interaction between stimulus rate and detuning direction. To decompose the significant interaction and to visualize the effects of detuning, we fitted separate models to each participant’s single-session datasets, and obtained slopes from each direction condition, hereafter denoted as the ‘relative-detuning slope’. We treated relative-detuning slope as an index of the magnitude of relative detuning effects on accuracy. We then evaluated these models, using the glmval function in MATLAB to obtain predicted accuracy values for each participant and session. To visualize the relative-detuning curves, we averaged the predicted accuracies across participants within each session, separately for each direction condition (faster or slower than the preferred rate). To obtain a single value of relative-detuning magnitude for each participant, we averaged relative detuning slopes across direction conditions. However, since slopes from IOI > preferred rate conditions quantified an accuracy decrease as a function of detuning, we sign-flipped these slopes before averaging. The resulting average relative detuning slopes, obtained from each participant’s single-session datasets, quantified how much the accuracy increase towards preferred rate was dependent on, in other words, sensitive to, relative detuning.” (p. 7-8)

• We added the information on the correlation analyses between average detuning slopes in Flexibility estimates.

“We further tested the relationship between the flexibility estimates (𝛽 from models where |𝚫IOI| or |+𝚫IOI| predicted accuracy) and average detuning slopes (see Preferred rate estimates) from random-order sessions. We predicted that flexible oscillators (larger 𝛽) would be less severely affected by detuning, and thus have smaller detuning slopes. Conversely, inflexible oscillators (smaller 𝛽) should have more difficulty in adapting to a large range of stimulus rates, and their adaptive abilities should be constrained around the preferred rate, as indexed by steeper relative detuning slopes.” (p. 8)

• We provided the results in Experiment 1 Results section.

“Logistic models assessing a systematic increase in accuracy toward the preferred rate estimate in each session type revealed significant main effects of IOI (linear-order session: 𝛽 = 0.264, p < .001; random-order session: 𝛽 = 0.175, p < .001), and significant interactions between IOI and direction (linear-order session: 𝛽 = -0.444, p < .001; random-order session: 𝛽 = -0.364, p < .001), indicating that accuracy increased as fast rates slowed toward the preferred rate (positive slopes) and decreased again as slow rates slowed further past the preferred rate (negative slopes), regardless of the session type. Fig. 2B illustrates the preferred rate estimation method for an example participant’s dataset and shows the predicted accuracy values from models fitted to each participant’s single-session datasets. Note that the main effect and interaction were obtained from mixed effects models that included aggregated datasets from all participants, whereas the slopes quantifying the accuracy increase as a function of detuning (i.e., relative detuning slopes) were from models fitted to single-participant datasets.” (p. 9-10)

“We tested the relationship between the flexibility estimates and single-participant relative detuning slopes from random-order sessions (Fig. 2B). The results revealed negative correlations between the relative detuning slopes and flexibility estimates, both with 𝛽 (r(23) =0.529, p = 0.007) from models where |-𝚫IOI| predicted accuracy (adapting to speeding-up trials), and 𝛽 (r(23) =-0.580, p = 0.002) from models where |+𝚫IOI| predicted accuracy (adapting to slowing-down trials). That is, the performance of individuals with less flexible oscillators suffered more as detuning increased. These results are shown in Fig. 2F.” (p. 10)

• We modified Figure 2. In Figure 2B, there are now separate subfigures with the z-scored IOI faster (left) or slower (right) than the preferred rate predicting accuracy. We illustrated the correlations between average relative detuning slopes and flexibility estimates in Figure 2F. 

Author response image 1.

Main findings of Experiment 1. A Left: Each circle represents a single participant’s preferred rate estimate from the random-order session (x axis) and linear-order session (y axis). The histograms along the top and right of the plot show the distributions of estimates for each session type. The dotted and dashed lines respectively represent 1:2 and 2:1 ratio between the axes, and the solid line represents one-to-one correspondence. Right: permutation test results. The distribution of summed residuals (distance of data points to the closest y=x, y=2*x and y=x/2 lines) of shuffled data over 1000 iterations, and the summed residual from original data (dashed line) that fell below .008 of the permutation distribution. B Top: Illustration of the preferred rate estimation method from an example participant’s linear-order session dataset. Estimates were the stimulus rates (IOI) where smoothed accuracy (orange line) was maximum (arrow). The dotted lines originating from the IOI axis delineate the stimulus rates that were faster (left, IOI < preferred rate) and slower (right, IOI > preferred rate) than the preferred rate estimate and expand those separate axes, the values of which were Z-scored for the relative-detuning analysis. Bottom: Predicted accuracy, calculated from single-participant models where accuracy in random-order (purple) and linear-order (orange) sessions was predicted by z-scored IOIs that were faster than a participant’s preferred rate estimate (left), and by those that were slower (right). Thin lines show predicted accuracy from single-participant models, solid lines show the averages across participants and the shaded areas represent standard error of the mean. Predicted accuracy is maximal at the preferred rate and decreases as a function of detuning. C Average accuracy from random-order (left, purple) and linear-order (right, orange) sessions. Each circle represents a participant’s average accuracy. D Flexibility estimates. Each circle represents an individuals’ slope (𝛽) obtained from logistic models, fitted separately to conditions where |𝚫IOI| (left, green) or |+𝚫IOI| (right blue) predicted accuracy, with greater values (arrow’s direction) indicating better oscillator flexibility. The means of the distributions of 𝛽 from both conditions were smaller than zero (dashed line), indicating a negative effect of between-trial absolute rate change on accuracy. E Participants’ average bias from |𝚫IOI| (green), and |+𝚫IOI| (blue) conditions in random-order (left) and linear-order (right) sessions. Negative bias indicates underestimation of the comparison intervals, positive bias indicates the opposite. Box plots in C-E show median (black vertical line), 25th and 75th percentiles (box edges) and extreme datapoints (whiskers). In C and E, empty circles show outlier values that remained after data cleaning procedures. F Correlations between participants’ average relative detuning slopes, indexing the steepness of the increase in accuracy towards the preferred rate estimate (from panel B), and flexibility estimates from |-𝚫IOI| (top, green), and |+𝚫IOI| (bottom, blue) conditions (from panel C). Solid black lines represent the best-fit line, dashed lines represent 95% confidence intervals.

• We discussed the results in General Discussion and emphasized that only entrainment models, compared to timekeeper models, predict a relationship between detuning and accuracy that is amplified by oscillator’s inflexibility: “we observed systematic increases in task accuracy (Experiment 1) toward the best-performance rates (i.e., preferred rate estimates), with the steepness of this increase being closely related to the effects of rate change (i.e., oscillator flexibility). Two interdependent properties of an underlying system together modulating an individual’s timing responses show strong support for the entrainment approach” (p. 24)

“As a side note, colours blue/green have a different meaning in 2B than 2D and E, which might be confusing.” 

Upon the reviewer’s recommendation, we changed the color scale across Figure 2, such that colors refer to the same set of conditions across all panels. 

(2) Second, I don't understand the additional harmonic relationship analyses in the appendix, and I suspect other readers will not either. As with the previous point, it is not my view that the analyses are faulty or inadequate, it is rather that the lack of clarity makes it challenging to evaluate whether they support an entrainment model or not. 

We decided to remove the analysis that was based on a circular approach, and we have clarified the analysis that was based on a modular approach by giving example cases: 

“We first calculated how much the slower estimate (larger IOI value) diverts, proportionally from the faster estimate (smaller IOI value) or its multiples (i.e., harmonics) by normalizing the estimates from both sessions by the faster estimate. The outcome measure was the modulus of the slower, with respect to the faster estimate, divided by the faster estimate, described as mod(max(X), min(X))/min(X) where X = [session1_estimate session2_estimate]. An example case would be a preferred rate estimate of IOI = 603 ms from the linear-order session and an estimate of IOI = 295 ms from the random-order session. In this case, the slower estimate (603 ms) diverts from the multiple of the faster estimate (295*2 = 590 ms) by 13 ms, a proportional deviation of 4% of the faster estimate (295 ms). The outcome measure in this example is calculated as mod(603,295)/295 = 0.04.” (Supplementary Information, p. 2)

Crucially, the ability of oscillators to respond to harmonically-related stimulus rates is a main distinction between entrainment and interval (timekeeper) models. In the current study, we found that each participant’s best-performance rates, the preferred rate estimates, had harmonic relationships. The additional analyses further showed that these harmonic relationships were not due to chance. This finding speaks against the interval (timekeeper) approaches and is maximally compatible with the entrainment framework. 

Here are a number of questions I would like to list to sketch my confusion: 

• The authors write: "We first normalized each participant's estimates by rescaling the slower estimate with respect to the faster one and converting the values to radians". Does slower estimate mean: "task accuracy in those trials in which IOI was slower than a participant's preferred frequency"? 

Preferred rate estimates were stimulus rates (IOI) with best performance, as described in Experiment 1 Methods section. 

“We conceptualized individuals' preferred rates as the stimulus rates where durationdiscrimination accuracy was highest. To estimate preferred rate on an individual basis, we smoothed response accuracy across the stimulus-rate (IOI) dimension for each session type, using the smoothdata function in Matlab. Estimates of preferred rate were taken as the smoothed IOI that yielded maximum accuracy” (p. 7). 

The estimation method and the resulting estimate for an example participant was provided in Figure 2B. The updated figure in the current revision has this illustration only for linear-order session. 

“Estimates were the stimulus rates (IOI) where smoothed accuracy (orange line) was maximum (arrow)” (Figure caption, p. 9).

• "We reasoned that values with integer-ratio relationships should correspond to the same phase on a unit circle". What is values here; IOI, or accuracy values for certain IOIs? And why should this correspond to the same phase? 

We removed the analysis on integer-ratio relationships that was based on a circular approach that the reviewer is referring to here. We clarified the analysis that was based on a modular approach and avoided using the term ‘values’ without specifying what values corresponded to.

• Des "integer-ratio relationships" have to do with the y=x, y=x*2 and y=x/2 relationships of the other analyses?  

Integer-ratio relationships indeed refer to y=x, y=x*2 and y=x/2 relationships. For example, if a number y is double of another number x (y = x*2), these values have an integer-ratio relationship, since 2 is an integer. This holds true also for the case where y = x/2 since x = y*2. 

• Supplementary Figure S2c shows a distribution of median divergences resulting from the modular approach. The p-value is 0.004 but the dashed line appears to be at a much higher percentile of the distribution. I find this hard to understand. 

We thank the reviewer for a detailed inspection of all figures and information in the manuscript. The reviewer’s comment led us to realize that this figure had an error. We updated the figure in Supplementary Information (Supplementary Figure S2). 

Reviewer #2 (Public Review):

To get a better understanding of the mechanisms underlying the behavioral observations, it would have been useful to compare the observed pattern of results with simulations done with existing biophysical models. However, this point is addressed if the current study is read along with this other publication of the same research group: Kaya, E., & Henry, M. J. (2024, February 5). Modeling rhythm perception and temporal adaptation: top-down influences on a gradually decaying oscillator.       https://doi.org/10.31234/osf.io/q9uvr 

We agree with the reviewer that the mechanisms underlying behavioral responses can be better understood by modeling approaches. We thank the reviewer for acknowledging our computational modeling study that addressed this concern. 

Reviewer #2 (Recommendations):

I very much appreciate the thorough work done by the authors in assessing all reviewers' concerns. In this new version they clearly state the assumptions to be tested by their experiments, added extra analyses further strengthening the conclusions and point the reader to a neurocomputational model compatible with the current observations. 

I only regret that the authors misunderstood the take home message of our Essay (Doelling & Assaneo 2021). Despite this being obviously out of the scope of the current work, I would like to take this opportunity to clarify this point. In that paper, we adopted a Stuart-Landau model not to determine how an oscillator should behave, but as an example to show that some behaviors usually used to prove or refute an underlying "oscillator like" mechanism can be falsified. We obviously acknowledge that some of the examples presented in that work are attainable by specific biophysical models, as explicitly stated in the essay: "There may well be certain conditions, equations, or parameters under which some of these commonly held beliefs are true. In that case, the authors who put forth these claims must clearly state what these conditions are to clarify exactly what hypotheses are being tested." 

This work did not mean to delineate what oscillator is (or in not), but to stress the importance of explicitly introducing biophysical models to be tested instead of relying on vague definitions sometimes reflecting the researchers' own beliefs. The take home message that we wanted to deliver to the reader appears explicitly in the last paragraph of that essay: "We believe that rather than concerning ourselves with supporting or refuting neural oscillators, a more useful framework would be to focus our attention on the specific neural dynamics we hope to explain and to develop candidate quantitative models that are constrained by these dynamics. Furthermore, such models should be able to predict future recordings or be falsified by them. That is to say that it should no longer be sufficient to claim that a particular mechanism is or is not an oscillator but instead to choose specific dynamical systems to test. In so doing, we expect to overcome our looping debate and to ultimately develop-by means of testing many model types in many different experimental conditions-a fundamental understanding of cognitive processes and the general organization of neural behavior." 

We appreciate the reviewer’s clarification of the take-home message from Doelling and Assaneo (2021). We concur with the assertions made in this essay, particularly regarding the benefits of employing computational modeling approaches. Such methodologies provide a nuanced and wellstructured foundation for theoretical predictions, thereby minimizing the potential for reductionist interpretations of behavioral or neural data.

In addition, we would like to underscore the significance of delineating the level of analysis when investigating the mechanisms underlying behavioral or neural observations. The current study or Kaya & Henry (2024) involved no electrophysiological measures. Thus, we would argue that the appropriate level of analysis across our studies concerns the theoretical mechanisms rather than how these mechanisms are implemented on the neural (physical) level. In both studies, we aimed to explore or approximate the theoretical oscillator that guides dynamic attention rather than the neural dynamics underlying these theoretical processes. That is, theoretical (attentional) entrainment may not necessarily correspond to neural entrainment, and differentiating these levels could be informative about the parallels and differences between these levels. 

References

Doelling, K. B., & Assaneo, M. F. (2021). Neural oscillations are a start toward understanding brain activity rather than the end. PLoS Biol, 19(5), e3001234. https://doi.org/10.1371/journal.pbio.3001234  Jones, M. R. (2018). Time will tell: A theory of dynamic attending. Oxford University Press. 

Kaya, E., & Henry, M. J. (2024). Modeling rhythm perception and temporal adaptation: top-down influences on a gradually decaying oscillator. PsyArxiv. https://doi.org/https://doi.org/10.31234/osf.io/q9uvr 

Large, E. W. (1994). Dynamic representation of musical structure. The Ohio State University. 

McAuley, J. D. (1995). Perception of time as phase: Toward an adaptive-oscillator model of rhythmic pattern processing Indiana University Bloomington]. 

Pikovsky, A., Rosenblum, M., & Kurths, J. (2003). Synchronization: A Universal Concept in Nonlinear Sciences. Cambridge University Press.

Author response:

eLife assessment

The authors present an algorithm and workflow for the inference of developmental trajectories from single-cell data, including a mathematical approach to increase computational efficiency. While such efforts are in principle useful, the absence of benchmarking against synthetic data and a wide range of different single-cell data sets make this study incomplete. Based on what is presented, one can neither ultimately judge if this will be an advance over previous work nor whether the approach will be of general applicability.

We thank the eLife editor for the valuable feedback. We wish to emphasize that both, benchmarking against other methods and validation on a synthetic dataset (“dyntoy”) are indeed presented in Supplementary Note, although we failed to sufficiently emphasize it in the main text. 

We will extend the benchmarking to more TI methods and we will improve the results and discussion sections to present those facts more clearly to the reader.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors present tviblindi, a computational workflow for trajectory inference from molecular data at single-cell resolution. The method is based on (i) pseudo-time inference via expecting hitting time, (ii) sampling of random walks in a directed acyclic k-NN where edges are oriented away from a cell of origin w.r.t. the involved nodes' expected hitting times, and (iii) clustering of the random walks via persistent homology. An extended use case on mass cytometry data shows that tviblindi can be used elucidate the biology of T cell development.

Strengths:

- Overall, the paper is very well written and most (but not all, see below) steps of the tviblindi algorithm are explained well.

- The T cell biology use case is convincing (at least to me: I'm not an immunologist, only a bioinformatician with a strong interest in immunology).

We thank the reviewer for feedback and suggestions that we will accommodate, we respond point-by-point below

Weaknesses:

- The main weakness of the paper is that a systematic comparison of tviblindi against other tools for trajectory inference (there are many) is entirely missing. Even though I really like the algorithmic approach underlying tviblindi, I would therefore not recommend to our wet-lab collaborators that they should use tviblindi to analyze their data. The only validation in the manuscript is the T cell development use case. Although this use case is convincing, it does not suffice for showing that the algorithms's results are systematically trustworthy and more meaningful (at least in some dimension) than trajectories inferred with one of the many existing methods.

We have compared tviblindi to several trajectory inference methods (Supplementary note section 8.2: Comparison to state-of-the-art methods, namely Monocle3 (v1.3.1) Cao et al. (2019), Stream (v1.1) Chen et al. (2019), Palantir (v1.0.0) Setty et al. (2019), VIA (v0.1.89) Stassen et al. (2021) and PAGA (scanpy==1.9.3) Wolf et al. (2019).) We will add thorough and systematic comparisons to the other algorithms mentioned by reviewers. We will include extended evaluation on publically available datasets.

Also, we have successfully used tviblindi to investigate human B-cell development in primary immunodeficiency (manuscript in revisions), double negative T-cells development in ALPS (Autoimmune Lymphoproliferative Syndrome) by mass cytometry (project in progress).

- The authors' explanation of the random walk clustering via persistent homology in the Results (subsection "Real-time topological interactive clustering") is not detailed enough, essentially only concept dropping. What does "sparse regions" mean here and what does it mean that "persistent homology" is used? The authors should try to better describe this step such that the reader has a chance to get an intuition how the random walk clustering actually works. This is especially important because the selection of sparse regions is done interactively. Therefore, it's crucial that the users understand how this selection affects the results. For this, the authors must manage to provide a better intuition of the maths behind clustering of random walks via persistent homology.

In order to satisfy both reader types: the biologist and the mathematician, we explain the mathematics in detail in the Supplementary Note, section 4. We will improve the Results text to better point the reader to the mathematical foundations in the Supplementary Note.

- To motivate their work, the authors write in the introduction that "TI methods often use multiple steps of dimensionality reduction and/or clustering, inadvertently introducing bias. The choice of hyperparameters also fixes the a priori resolution in a way that is difficult to predict." They claim that tviblindi is better than the original methods because "analysis is performed in the original high-dimensional space, avoiding artifacts of dimensionality reduction." However, in the manuscript, tviblindi is tested only on mass cytometry data which has a much lower dimensionality than scRNA-seq data for which most existing trajectory inference methods are designed. Since tviblindi works on a k-NN graph representation of the input data, it is unclear if it could be run on scRNA-seq data without prior dimensionality reduction. For this, cell-cell distances would have to be computed in the original high-dimensional space, which is problematic due to the very high dimensionality of scRNA-seq data. Of course, the authors could explicitly reduce the scope of tviblindi to data of lower dimensionality, but this would have to be stated explicitly.

In the manuscript we tested the framework on the scRNA-seq data from Park et al 2020 (DOI: 10.1126/science.aay3224). To illustrate that tviblindi can work directly in the high-dimensional space, we applied the framework successfully on imputed 2000 dimensional data.

The idea behind tviblindi is to be able to work without the necessity to use non-linear dimensionality reduction techniques, which reduce the dimensionality to a very low number of dimensions and whose effects on the data distribution are difficult to predict. On the other hand the use of (linear) dimensionality reduction techniques which effectively suppress noise in the data such as PCA is a good practice (see also response to reviewer 2). We will emphasize this in the revised version and add the results of the corresponding analysis.

- Also tviblindi has at least one hyper-parameter, the number k used to construct the k-NN graphs (there are probably more hidden in the algorithm's subroutines). I did not find a systematic evaluation of the effect of this hyper-parameter.

Detailed discussion of the topic is presented in the Supplementary Note, section 8.1, where Spearman correlation coefficient between pseudotime estimated using k=10 and k=50 nearest neighbors was 0.997.   The number k however does affect the number of candidate endpoints. But even when larger k causes spurious connection between unrelated cell fates, the topological clustering of random walks allows for the separation of different trajectories. We will expand the “sensitivity to hyperparameters section” also in response to reviewer 2.

Reviewer #2 (Public Review):

Summary:

In Deconstructing Complexity: A Computational Topology Approach to Trajectory Inference in the Human Thymus with tviblindi, Stuchly et al. propose a new trajectory inference algorithm called tviblindi and a visualization algorithm called vaevictis for single-cell data. The paper utilizes novel and exciting ideas from computational topology coupled with random walk simulations to align single cells onto a continuum. The authors validate the utility of their approach largely using simulated data and establish known protein expression dynamics along CD4/CD8 T cell development in thymus using mass cytometry data. The authors also apply their method to track Treg development in single-cell RNA-sequencing data of human thymus.

The technical crux of the method is as follows: The authors provide an interactive tool to align single cells along a continuum axis. The method uses expected hitting time (given a user input start cell) to obtain a pseudotime alignment of cells. The pseudotime gives an orientation/direction for each cell, which is then used to simulate random walks. The random walks are then arranged/clustered based on the sparse region in the data they navigate using persistent homology.

We thank the reviewer for feedback and suggestions that we will accommodate, we respond point-by-point below.

Strengths:

The notion of using persistent homology to group random walks to identify trajectories in the data is novel.

The strength of the method lies in the implementation details that make computationally demanding ideas such as persistent homology more tractable for large scale single-cell data.

This enables the authors to make the method more user friendly and interactive allowing real-time user query with the data.

Weaknesses:

The interactive nature of the tool is also a weakness, by allowing for user bias leading to possible overfitting for a specific data.

tviblindi is not designed as a fully automated TI tool (although it implements a fully automated module), but as a data driven framework for exploratory analysis of unknown data. There is always a risk of possible bias in this type of analysis - starting with experimental design, choice of hyperparameters in the downstream analysis, and an expert interpretation of the results. The successful analysis of new biological data involves a great deal of expert knowledge which is difficult to a priori include in the computational models.

tvilblindi tries to solve this challenge by intentionally overfitting the data and keeping the level of resolution on a single random walk. In this way we aim to capture all putative local relationships in the data. The on-demand aggregation of the walks using the global topology of the data allows researchers to use their expert knowledge to choose the right level of detail (as demonstrated in the Figure 4 of the manuscript) while relying on the topological structure of the high dimensional point cloud. At all times tviblindi allows to inspect the composition of the trajectory to assess the variance in the development, possible hubs on the KNN-graph etc.

The main weakness of the method is lack of benchmarking the method on real data and comparison to other methods. Trajectory inference is a very crowded field with many highly successful and widely used algorithms, the two most relevant ones (closest to this manuscript) are not only not benchmarked against, but also not sited. Including those that specifically use persistent homology to discover trajectories (Rizvi et.al. published Nat Biotech 2017). Including those that specifically implement the idea of simulating random walks to identify stable states in single-cell data (e.g. CellRank published in Lange et.al Nat Meth 2022), as well as many trajectory algorithms that take alternative approaches. The paper has much less benchmarking, demonstration on real data and comparison to the very many other previous trajectory algorithms published before it. Generally speaking, in a crowded field of previously published trajectory methods, I do not think this one approach will compete well against prior work (especially due to its inability to handle the noise typical in real world data (as was even demonstrated in the little bit of application to real world data provided).

We provide comparisons of tviblindi and vaevictis in the Supplementary Note, section 8.2, where we compare it to Monocle3 (v1.3.1) Cao et al. (2019), Stream (v1.1) Chen et al. (2019), Palantir (v1.0.0) Setty et al. (2019), VIA (v0.1.89) Stassen et al. (2021) and PAGA (scanpy==1.9.3) Wolf et al. (2019). We use two datasets: artificial Dyntoy and real mass cytometry thymus+peripheral blood dataset. We thank the reviewer for suggesting specific methods.  CellRank was excluded from the benchmarking as it was originally designed for RNA-velocity data (not available in mass cytometry data), but will include recent upgrade CellRank2 (preprint at doi.org/10.1101/2023.07.19.549685) which offers more flexibility.

We will add further benchmarking as suggested by the reviewer in the course of revisions.

Beyond general lack of benchmarking there are two issues that give me particular concern. As previously mentioned, the algorithm is highly susceptible to user bias and overfitting. The paper gives the example (Figure 4) of a trajectory which mistakenly shows that cells may pass from an apoptotic phase to a different developmental stage. To circumvent this mistake, the authors propose the interactive version of tviblindi that allows users to zoom in (increase resolution) and identify that there are in fact two trajectories in one. In this case, the authors show how the author can fix a mistake when the answer is known. However, the point of trajectory inference is to discover the unknown. With so much interactive options for the user to guide the result, the method is more user/bias driven than data-driven. So a rigorous and quantitative discussion of robustness of the method, as well as how to ensure data-driven inference and avoid over-fitting would be useful.

Local directionality in expression data is a challenge which is not, to our knowledge, solved. And we are not sure it can be solved entirely, even theoretically. The random walks passing “through” the apoptotic phase are biologically infeasible, but it is an (unbiased) representation of what the data look like based on the diffusion model. It is a property of the data (or of the panel design), which has to be interpreted properly rather than a mistake. Of note, except for Monocle3 (which does not provide the directionality) other tested methods did not discover this trajectory at all.

The “zoom in” has in fact nothing to do with “passing through the apoptosis”. We show how the researcher can investigate the suggested trajectory to see if there is an additional structure of interest and/or relevance. This investigation is still data driven (although not fully automated). Anecdotally in this particular case this branching was discovered by an bioinformatician, who knew nothing about the presence of beta-selection in the data. 

We show that the trajectory of apoptosis of cortical thymocytes consists of 2 trajectories corresponding to 2 different checkpoints (beta-selection and positive/negative selection). This type of structure, where 2 (or more) trajectories share the same path for most of the time, then diverge only to be connected at a later moment (immediately from the point of view of the beta-selection failure trajectory) is a challenge for TI algorithms and none of tested methods gave a correct result. More importantly there seems to be no clear way to focus on these kinds of structures (common origin and common fate) in TI methods.

Of note, the “zoom in” is a recommended and convenient method to look for an inner structure, but it does not necessarily mean addition of further homological classes. Indeed, in this case the reason that the structure is not visible directly is the limitation of the dendrogram complexity (only branches containing at least 10% of simulated random walks are shown by default).

In summary, tviblindi effectively handled all noise in the data that obscured biologically valid trajectories for other methods. We will improve the discussion of the robustness in the reviewed version. 

Second, the paper discusses the benefit of tviblindi operating in the original high dimensions of the data. This is perhaps adequate for mass cytometry data where there is less of an issue of dropouts and the proteins may be chosen to be large independent. But in the context of single-cell RNA-sequencing data, the massive undersampling of mRNA, as well as high degree of noise (e.g. ambient RNA), introduces very large degree of noise so that modeling data in the original high dimensions leads to methods being fit to the noise. Therefore ALL other methods for trajectory inference work in a lower dimension, for very good reason, otherwise one is learning noise rather than signal. It would be great to have a discussion on the feasibility of the method as is for such noisy data and provide users with guidance. We note that the example scRNA-seq data included in the paper is denoised using imputation, which will likely result in the trajectory inference being oversmoothed as well.

We agree with the reviewer. In our manuscript we wanted to showcase that tviblindi can directly operate in high-dimensional space (thousands of dimensions) and we used MAGIC imputation for this purpose. This was not ideal. More standard approach, which uses 30-50 PCs as input to the algorithm resulted in equivalent trajectories. We will add this analysis to the study.

In summary, the fact that tviblindi scales well with dimensionality of the data and is able to work in the original space does not mean that it is always the best option. We will emphasize in the revised paper that we aim to avoid the non-linear dimensional reduction techniques as a data preprocessing tool, as the effect of the reduction is difficult to predict. We will also discuss the preprocessing of scRNA-seq data in greater detail.

Reviewer #3 (Public Review):

Summary:

Stuchly et al. proposed a single-cell trajectory inference tool, tviblindi, which was built on a sequential implementation of the k-nearest neighbor graph, random walk, persistent homology and clustering, and interactive visualization. The paper was organized around the detailed illustration of the usage and interpretation of results through the human thymus system.

Strengths:

Overall, I found the paper and method to be practical and needed in the field. Especially the in-depth, step-by-step demonstration of the application of tviblindi in numerous T cell development trajectories and how to interpret and validate the findings can be a template for many basic science and disease-related studies. The videos are also very helpful in showcasing how the tool works.

Weaknesses:

I only have a few minor suggestions that hopefully can make the paper easier to follow and the advantage of the method to be more convincing.

(1) The "Computational method for the TI and interrogation - tviblindi" subsection under the Results is a little hard to follow without having a thorough understanding of the tviblindi algorithm procedures. I would suggest that the authors discuss the uniqueness and advantages of the tool after the detailed introduction of the method (moving it after the "Connectome - a fully automated pipeline".

We thank the reviewer for the suggestion and we will accommodate it to improve readability of the text.

Also, considering it is a computational tool paper, inevitably, readers are curious about how it functions compared to other popular trajectory inference approaches. I did not find any formal discussion until almost the end of the supplementary note (even that is not cited anywhere in the main text). Authors may consider improving the summary of the advantages of tviblindi by incorporating concrete quantitative comparisons with other trajectory tools.

We provide comparisons of tviblindi and vaevictis in the Supplementary Note, section 8.2, where we compare it to Monocle3 (v1.3.1) Cao et al. (2019), Stream (v1.1) Chen et al. (2019), Palantir (v1.0.0) Setty et al. (2019), VIA (v0.1.89) Stassen et al. (2021) and PAGA (scanpy==1.9.3) Wolf et al. (2019). We use two datasets: artificial Dyntoy and real mass cytometry thymus+peripheral blood dataset. We will also add CellRank2 into comparisons and we will strengthen the message of the benchmarking results in the Discussion section.

(2) Regarding the discussion in Figure 4 the trajectory goes through the apoptotic stage and reconnects back to the canonical trajectory with counterintuitive directionality, it can be a checkpoint as authors interpret using their expert knowledge, or maybe a false discovery of the tool. Maybe authors can consider running other algorithms on those cells and see which tracks they identify and if the directionality matches with the tviblindi.

We have indeed used the thymus dataset for comparison of all TI algorithms listed above. Except for Monocle 3 they failed to discover the negative selection branch (Monocle 3 does not offer directionality information). Therefore, a valid topological trajectory with incorrect (expert-corrected) directionality was partly or entirely missed by other algorithms.

(3) The paper mainly focused on mass cytometry data and had a brief discussion on scRNA-seq. Can the tool be applied to multimodality data such as CITE-seq data that have both protein markers and gene expression? Any suggestions if users want to adapt to scATAC-seq or other epigenomic data?

The analysis of multimodal data is the logical next step and is the topic of our current research. At this moment tviblindi cannot be applied directly to multimodal data. It is possible to use the KNN-graph based on multimodal data (such as weighted nearest neighbor graph implemented in Seurat) for pseudotime calculation and random walk simulation. However, we do not have a fully developed triangulation for the multimodal case yet.

Reviewer #2 (Public Review):

Summary: In Deconstructing Complexity: A Computational Topology Approach to Trajectory Inference in the Human Thymus with tviblindi, Stuchly et al. propose a new trajectory inference algorithm called tviblindi and a visualization algorithm called vaevictis for single-cell data. The paper utilizes novel and exciting ideas from computational topology coupled with random walk simulations to align single cells onto a continuum. The authors validate the utility of their approach largely using simulated data and establish known protein expression dynamics along CD4/CD8 T cell development in thymus using mass cytometry data. The authors also apply their method to track Treg development in single-cell RNA-sequencing data of human thymus.

The technical crux of the method is as follows: The authors provide an interactive tool to align single cells along a continuum axis. The method uses expected hitting time (given a user input start cell) to obtain a pseudotime alignment of cells. The pseudotime gives an orientation/direction for each cell, which is then used to simulate random walks. The random walks are then arranged/clustered based on the sparse region in the data they navigate using persistent homology.

Strengths:<br /> The notion of using persistent homology to group random walks to identify trajectories in the data is novel.<br /> The strength of the method lies in the implementation details that make computationally demanding ideas such as persistent homology more tractable for large scale single-cell data. This enables the authors to make the method more user friendly and interactive allowing real-time user query with the data.

Weaknesses:<br /> The interactive nature of the tool is also a weakness, by allowing for user bias leading to possible overfitting for a specific data.

The main weakness of the method is lack of benchmarking the method on real data and comparison to other methods. Trajectory inference is a very crowded field with many highly successful and widely used algorithms, the two most relevant ones (closest to this manuscript) are not only not benchmarked against, but also not sited. Including those that specifically use persistent homology to discover trajectories (Rizvi et.al. published Nat Biotech 2017). Including those that specifically implement the idea of simulating random walks to identify stable states in single-cell data (e.g. CellRank published in Lange et.al Nat Meth 2022), as well as many trajectory algorithms that take alternative approaches. The paper has much less benchmarking, demonstration on real data and comparison to the very many other previous trajectory algorithms published before it. Generally speaking, in a crowded field of previously published trajectory methods, I do not think this one approach will compete well against prior work (especially due to its inability to handle the noise typical in real world data (as was even demonstrated in the little bit of application to real world data provided).

Beyond general lack of benchmarking there are two issues that give me particular concern. As previously mentioned, the algorithm is highly susceptible to user bias and overfitting. The paper gives the example (Figure 4) of a trajectory which mistakenly shows that cells may pass from an apoptotic phase to a different developmental stage. To circumvent this mistake, the authors propose the interactive version of tviblindi that allows users to zoom in (increase resolution) and identify that there are in fact two trajectories in one. In this case, the authors show how the author can fix a mistake when the answer is known. However, the point of trajectory inference is to discover the unknown. With so much interactive options for the user to guide the result, the method is more user/bias driven than data-driven. So a rigorous and quantitative discussion of robustness of the method, as well as how to ensure data-driven inference and avoid over-fitting would be useful.

Second, the paper discusses the benefit of tviblindi operating in the original high dimensions of the data. This is perhaps adequate for mass cytometry data where there is less of an issue of dropouts and the proteins may be chosen to be large independent. But in the context of single-cell RNA-sequencing data, the massive undersampling of mRNA, as well as high degree of noise (e.g. ambient RNA), introduces very large degree of noise so that modeling data in the original high dimensions leads to methods being fit to the noise. Therefore ALL other methods for trajectory inference work in a lower dimension, for very good reason, otherwise one is learning noise rather than signal. It would be great to have a discussion on the feasibility of the method as is for such noisy data and provide users with guidance. We note that the example scRNA-seq data included in the paper is denoised using imputation, which will likely result in the trajectory inference being oversmoothed as well.

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.

39:00 Vanevar Bush misses out on a whole swath of history regarding commonplace books and indexing. In As We May Think he presents these older methods to the computer. "Why not imitate?" Aldrich says, instead of trying to reinvent the wheel (or thinking you are doing so).

Author response:

We would like to thank all the reviewers and editors for their thoughtful and detailed comments, critiques and suggestions. We will revise our manuscript in accordance with all the points raised by the reviewers. Here we summarize some of the main points that we intend to address in our revised manuscript.

The reviewers noted that we were not sufficiently careful in identifying possible exogenous cues that the mice might be using to locate the cues and that we did not consider why such cues might be ineffective. As the reviewers point out, the mice may be ignoring the visual landmarks (and floor scratches) because they are not reliable cues and their relation to the food varies with the entrance the mice have used. In particular, a reviewer refers to papers that show that “in environments with 'unreliable' landmarks, place cells are not controlled by landmarks”. These papers were known to the authors but failed to make final cut of our extensive discussion. This important point will be thoroughly addressed.

Another critical point was the mice were often doing thigmotaxis. The literature on thigmotaxis was known to us and we will now directly refer to this point. We do note that the final average start to food trajectory (TEV) is directly to the food. In other words, the thigmotaxic trajectories and “towards the center” trajectories effectively average out.

There was a very cogent point about the difficulty of totally eliminating odor cues that we will now address. Finally, based on studies using a virtual reality environment, one reviewer questioned the use of “path integration” as a signal that encodes goal location. The relevance of path integration to spatial learning and performance is a very difficult issue that, to our knowledge, has never been entirely settled in the vast spatial learning literature. We do not think that our data can “settle’ this issue but will try to at least be explicit re the complexity of the path integration hypothesis as it applies to both our own data and the virtual reality literature. In particular, we will discuss the potential roles of optic flow versus proprioceptive and vestibular inputs to a putative path integration mechanism.

Finally, the reviewers raised many important technical points re statistics reporting and how the figures are presented. In our revision, we will completely comply with all these helpful critiques.

Author response:

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

eLife assessment

Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments provide compelling evidence that conditional deletion of Vglut2 in noradrenergic neurons does not impact steadystate breathing or metabolic activity in room air, hypercapnia, or hypoxia. This study provides an important contribution to our understanding of how noradrenergic neurons regulate respiratory homeostasis in conscious adult mice.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments show that conditional deletion of Vglut2 in NA neurons does not impact steady-state breathing or metabolic activity in room air, hypercapnia, or hypoxia. Their observations challenge the importance of glutamatergic signaling from Vglut2 expressing NA neurons in normal respiratory homeostasis in conscious adult mice.

Strengths:

The comprehensive Vglut1, Vglut2, and Vglut3 co-expression profiles in the central noradrenergic system and the combined measurements of breathing and oxygen consumption are two major strengths of this study. Observations from these experiments provide previously undescribed insights into (1) expression patterns for subtypes of the vesicular glutamate transporter protein in the noradrenergic system and (2) the dispensable nature of Vglut2-dependent glutamate signaling from noradrenergic neurons to breathing responses to physiologically relevant gas challenges in adult conscious mice.

Weaknesses:

Although the cellular expression profiles for the vesicular glutamate transporters are provided, the study fails to document that glutamatergic-based signaling originating from noradrenergic neurons is evident at the cellular level under normal, hypoxic, and/or hypercapnic conditions. This limits the reader's understanding of why conditional Vglut2 knockdown is dispensable for breathing under the conditions tested.

We thank the reviewers for their positive evaluation of our work. First, we would like to highlight that multiple studies have provided anatomical evidence of innervation of multiple cardio-respiratory nuclei by Vglut2+ noradrenergic fibers. Thus, the anatomical substrates are present for noradrenergic based Vglut2 signaling to either play a direct role in breathing control or, upon perturbation, to indirectly affect breathing through disrupted metabolic or cardiovascular control. We have included supplemental table 1 that summarizes central noradrenergic Vglut2+ innervations of respiratory and autonomic nuclei. Additionally, Ultrastructural evidence shows asymmetric synaptic contacts assuming glutamatergic transmission between C1 neurons and LC, A1, A2 and the dorsal motor nucleus of the vagus (DMV) (Milner et al., 1989; Abbott et al., 2012; Holloway et al., 2013; DePuy et al., 2013).

Functionally, electrophysiological evidence showed that photostimulating C1 neurons activate LC, A1, A2 noradrenergic neurons monosynaptically by releasing glutamate (Holloway et al., 2013; DePuy et al., 2013) and optogenetic stimulation of LC neurons excite the downstream parabrachial nucleus (PBN) neurons by releasing glutamate. Thus, at least the glutamatergic signaling from C1 and LC noradrenergic neurons (two noradrenergic nuclei that have been shown to play a role in breathing control) is evident at the cellular level under normal conditions. Other evidence, highlighted in our manuscript, is more circumstantial.

Reviewer #2 (Public Review):

The authors characterized the recombinase-based cumulative fate maps for vesicular glutamate transporters (Vglut1, Vglut2 and Vglut3) expression and compared those maps to their real-time expression profiles in central NA neurons by RNA in situ hybridization in adult mice. Authors have revealed a new and intriguing expression pattern for Vglut2, along with an entirely uncharted co-expression domain for Vglut3 within central noradrenergic neurons. Interestingly, and in contrast to previous studies, the authors demonstrated that glutamatergic signaling in central noradrenergic neurons does not exert any influence on breathing and metabolic control either under normoxic/normocapnic conditions or after chemoreflex stimulation. Also, they showed for the first-time the Vglut3-expressing NA population in C2/A2 nuclei. In addition, they were also able to demonstrate Vglut2 expression in anterior NA populations, such as LC neurons, by using more refined techniques, unlike previous studies.

A major strength of the study is the use of a set of techniques to investigate the participation of NA-based glutamatergic signaling in breathing and metabolic control. The authors provided a full characterization of the recombinase-based cumulative fate maps for Vglut transporters. They performed real-time mRNA expression of Vglut transporters in central NA neurons of adult mice. Further, they evaluated the effect of knocking down Vglut2 expression in NA neurons using a DBH-Cre; Vglut2cKO mice on breathing and control in unanesthetized mice. Finally, they injected the AAV virus containing Cre-dependent Td tomato into LC of v-Glut2 Cre mice to verify the VGlut2 expression in LC-NA neurons. A very positive aspect of the article is that the authors combined ventilation with metabolic measurements. This integration holds particular significance, especially when delving into the exploration of respiratory chemosensitivity. Furthermore, the sample size of the experiments is excellent.

Despite the clear strengths of the paper, some weaknesses exist. It is not clear in the manuscript if the experiments were performed in males and females and if the data were combined. I believe that the study would have benefited from a more comprehensive analysis exploring the sex specific differences. The reason I think this is particularly relevant is the developmental disorders mentioned by the authors, such as SIDS and Rett syndrome, which could potentially arise from disruptions in central noradrenergic (NA) function, exhibit varying degrees of sex predominance. Moreover, some of the noradrenergic cell groups are sexually dimorphic. For instance, female Wistar rats exhibit a larger LC size and more LC-NA neurons than male subjects (Pinos et al., 2001; Garcia-Falgueras et al., 2005). More recently, a detailed transcriptional profiling investigation has unveiled the identities of over 3,000 genes in the LC. This revelation has highlighted significant sexual dimorphisms, with more than 100 genes exhibiting differential expression within LC-NA neurons at the transcript level. Furthermore, this investigation has convincingly showcased that these distinct gene expression patterns have the capacity to elicit disparate behavioral responses between sexes (Mulvey et al., 2018). Therefore, the authors should compare the fate maps, Vglut transporters in males and females, at least considering LC-NA neurons. Even in the absence of identified sex differences, this information retains significant importance.

All experiments contained both males and females as described in the original submission. In our analysis of breathing and metabolism, sex was included in the analysis and no significant phenotypic difference was observed. For the fate map and in situ experiments, we did not see obvious differences in the expression patterns in the three glutamate transporters between females and males, though the group size is small. Though all the anatomical and phenotypic data in this manuscript are presented as combined graphs, we have differentially labeled our data points by sex. The reviewer does raise important questions regarding possible sexual dimorphisms in the central noradrenergic system and whether such dimorphisms may extend to glutamate transporter co-expression. Our thorough interrogation of respiratory-metabolic parameters fails to reveal any sex specific differences in control or experimental mice. Thus, it is unclear if any of the previously described and cited dimorphisms are functionally relevant in this setting. Given the large differences in the real time expression and cumulative fate maps of Vglut2, a worthwhile interrogation of differential glutamate transporter expression would be best served by longitudinal studies with large group sizes across age as it is not clear what underlies the dynamic VGlut2 expression changes. Such changes may at times be greater in males and other times in females, driven by experience or physiological challenges etc., but resulting in averaged cumulative fatemaps that are similar between sexes. Such a longitudinal quantitative study of real-time and fatemapped cell populations across the central NA system would be of a scale that is beyond the scope of this report, especially when no phenotypic changes have been observed in our respiratory data.

An important point well raised by the authors is that although suggestive, these experiments do not definitively rule out that NA-Vglut2 based glutamatergic signaling has a role in breathing control. Subsequent experiments will be necessary to validate this hypothesis.

As noted, we discuss that we only address requirement, not sufficiency, of NA Vglut2 in breathing. Functional sufficiency experiments usually involve increasing the relevant output. However, these experiments can lead to non-specific, pleiotropic effects that would be difficult to disambiguate, even if done with high cellular specificity. Viral or genetic overexpression of Vglut2 in NA neurons may be a feasible approach. Conditional ablation of TH or DBH with concurrent chemo or optogenetic stimulation may also be informative. These approaches would require significant investments in mouse model generation and suffer additional experimental limitations.

An improvement could be made in terms of measuring body temperature. Opting for implanted sensors over rectal probes would circumvent the need to open the chamber, thereby preventing alterations in gas composition during respiratory measurements. Further, what happens to body temperature phenotype in these animals under different gas exposures? These data should be included in the Tables.

While surgical implantation of sensors would provide a more direct assessment of temperature, it requires components that were not available at the time of the study and addresses a question (temperature changes during a time course of gas exposure) that go beyond the scope of the current work focused on respiratory response. As we have done for prior experiments (Martinez et al., 2019; Ray et al., 2011), the body temperature was measured immediately before and after measuring breathing only. Our flow through system using inline gas sensors (AEI P-61B CO2 sensor and AEI N-22M O2 sensor) ensure that gas challenges were constant and consistent across all measurements. Any disruption in gas composition would have been noted by our software analysis system, Breathe Easy, and the data rejected. We did not observe any such perturbations.

Is it plausible that another neurotransmitter within NA neurons might be released in higher amounts in DBH-Cre; Vglut2 cKO mice to compensate for the deficiency in glutamate and prevent changes in ventilation?

We agree that compensation is always a possibility at the synaptic, cellular, and circuit levels that may involve a variety of transcriptional, translational, cellular, and circuit mechanisms (i.e., synaptic strength). This could be interrogated by combining multiple conditional alleles and recombinase drivers for various transmitters and receptors, but would, in our experience, take multiple years for the requisite breeding to be completed.

Continuing along the same line of inquiry is there a possibility that Vglut2 cKO from NA neurons not only eliminates glutamate release but also reduces NA release? A similar mechanism was previously found in VGLUT2 cKO from DA neurons in previous studies (Alsio et al., 2011; Fortin et al., 2012; Hnasko et al., 2010). Additionally, does glutamate play a role in the vesicular loading of NA? Therefore, could the lack of effect on breathing be explained by the lack of noradrenaline and not glutamate?

These are all excellent points, but prior studies suggest that reductions in NA signaling would itself have an apparent effect (Zanella et al., 2006; Kuo et al., 2016). Although several studies showed that LC and C1 NA neurons co-release noradrenaline and glutamate, no direct evidence yet makes clear that glutamate facilitates NA release or vice versa. However, it would be of great interest to test if reduced or lack of NA compensated for loss of glutamate in the future. We do fully acknowledge that compensation in the manuscript that any number of compensatory events could be at play in these findings.

Reviewer #3 (Public Review):

Summary:

The authors, Y Chang and colleagues, have performed elegant studies in transgenic mouse models that were designed to examine glutamatergic transmission in noradrenergic neurons, with a focus on respiratory regulation. They generated 3 different transgenic lines, in which a red fluorophore was expressed in dopamine-B-hydroxylase (DBH; noradrenergic and adrenergic neurons) neurons that did not express a vesicular glutamate transporter (Vglut) and a green fluorophore in DBH neurons that did express one of either Vglut1, Vglut2 or Vglut3.

Further experiments generated a transgenic mouse with knockout of Vglut2 in DBH neurons. The authors used plethysmography to measure respiratory parameters in conscious, unrestrained mice in response to various challenges.

Strengths:

The distribution of the Vglut expression is broadly in agreement with other studies, but with the addition of some novel Vglut3 expression. Validation of the transgenic results, using in situ hybridization histochemistry to examine mRNA expression, revealed potential modulation of Vglut2 expression during phases of development. This dataset is comprehensive, wellpresented and very useful.

In the physiological studies the authors observed that neither baseline respiratory parameters, nor respiratory responses to hypercapnea (5, 7, 10% CO2) or hypoxia (10% O2) were different between knockout mice and littermate controls. The studies are well-designed and comprehensive. They provide observations that are supportive of previous reports using similar methodology.

Weaknesses:

In relation to the expression of Vglut2, the authors conclude that modulation of expression occurs, such that in adulthood there are differences in expression patterns in some (nor)adrenergic cell groups. Altered sensitivity is provided as an explanation for different results between studies examining mRNA expression. These are likely explanations; however, the conclusion would really be definitive with inclusion of a conditional cre expressing mouse. Given the effort taken to generate this dataset, it seems to me that taking that extra step would be of value for the overall understanding of glutamatergic expression in these catecholaminergic neurons

The seemingly dynamic Vglut2 expression pattern across the NA system is intriguing. As noted in our comments to reviewer 2, a robust age dependent interrogation would require a large magnitude study. The reviewer correctly points out that a temporally controlled recombinase fate mapping experiment would offer greater insight into the dynamic expression of Vglut2. We strongly agree with that idea and did work to develop a Vglut2-CreER targeted allele that, despite our many other successes in mouse genetic engineering (Lusk et al., 2022; Sun and Ray, 2016), did not succeed on the first attempt. We aim to complete the line in the near future so that we may better understand the Vglut2 expression pattern in central noradrenergic neurons in a time-specific manner and sex specific manner.

The respiratory physiology is very convincing and provides clear support for the view that Vglut2 is not required for modulation of the respiratory parameters measured and the reflex responses tested. It is stated that this is surprising. However, comparison with the data from Abbott et al., Eur J Neurosci (2014) in which the same transgenic approach was used, shows that they also observed no change in baseline breathing frequency. Differences were observed with strong, coordinated optogenetic stimulation, but, as discussed in this manuscript, it is not clear what physiological function this is relevant to. It just shows that some C1 neurons can use glutamate as a signaling molecule. Further, Holloway et al., Eur J Neurosci (2015), using the same transgenic mouse approach, showed that the respiratory response to optogenetic activation of Phox2 expressing neurons is not altered in DBH-Vglut2 KO mice. The conclusion seems to be that some C1 neuron effects are reliant upon glutamatergic transmission (C1DMV for example), and some not.

We agree that activation of C1 neurons may be sufficient to modulate breathing when artificially stimulated and that such stimulation relies on glutamatergic transmission for its effect. This is why we find our results surprising and important in clarifying for the field that glutamatergic signaling in noradrenergic cells is dispensable for breathing and hypoxic and hypercapnic responses under physiological conditions.

Further contrast is made in this manuscript to the work of Malheiros-Lima and colleagues (eLife 2020) who showed that the activation of abdominal expiratory nerve activity in response to peripheral chemoreceptor activation with cyanide was dependent upon C1 neurons and could be attenuated by blockade of glutamate receptors in the pFRG - i.e. the supposition that glutamate release from C1 neurons was responsible for the function. However, it is interesting to observe that diaphragm EMG responses to hypercapnia (10% CO2) or cyanide, and the expiratory activation to hypercapnia, were not affected by the glutamate receptor blockade. Thus, a very specific response is affected and one that was not measured in the current study.

As we mention above, we do not dispute that glutamate signaling can be manipulated to create a response in non-physiological conditions – we suggest that framing the interpretation around the glutamatergic role in a model that better matches physiological conditions should inform our interpretation. Furthermore, we do include an examination of expiratory flow – which was not impacted by loss of glutamatergic activity in NA neurons – which would be likely to have been impacted if abdominal expiratory nerve activity was modified.

These previous published observations are consistent with the current study which provides a more comprehensive analysis of the role of glutamatergic contributions respiratory physiology. A more nuanced discussion of the data and acknowledgement of the differences, which are not actually at odds, would improve the paper and place the information within a more comprehensive model.

Thank you for the comments. As noted in the original and extended discussion, we respectfully disagree with the perspective that our results align with prior results.

Recommendations for the authors:

The three reviewers believe this is an important study. They have numerous suggestions for improvement of the manuscript (outlined below), but no new experiments are required. The Editor requests some nomenclature changes as indicated in attachment 1.

Reviewer #1 (Recommendations For The Authors):

Abstract/Introduction: Although the need for this study is obvious, it is important that the authors explicitly communicate their working hypothesis < before the start of the work> to the reader. In the current form, it is unclear whether the authors aimed to test the hypothesis that glutamatergic signaling from noradrenergic neurons is important to breathing or whether to test the hypothesis that glutamatergic signaling from noradrenergic neurons is not important to breathing. If it is the latter-it is not important-then the study (related to the breathing measurements) is poorly justified and designed, as additional orthogonal approaches (e.g., actual measurements of glutamatergic signaling at the cellular level) are almost requisite. If the authors' hypothesis was originally based on existing literature suggesting that glutamatergic signaling from noradrenergic neurons is important to breathing, then the experimental design appropriate.

Thank you for the suggestion. The working hypothesis has been added in the abstract (line 2425) and the introduction (line 92-94)), making clear that we initially hypothesized that glutamatergic signaling from noradrenergic neurons is important in breathing.

Results: While the steady state measurements for breathing metrics are clearly important in defining how glutamatergic signaling may contribute to be pulmonary function, the role of glutamatergic signaling may have a greater role in the dynamics of patterns (i.e., regularity of the breathing rhythms) such traits can be described using SD1 and SD2 from Poincare maps, and/or entropy measurements. Such an analysis should be performed.

Thank you for the suggestion. The dynamic patterns of respiratory rate (Vf), tidal volume (VT), minute ventilation (VE), inspiratory duration (TI), expiratory duration (TE), breath cycle duration (TTOT), inspiratory flow rate (VT/TI), expiratory flow rate (VT/TE) have been shown as Poincaré plots and quantified and tested using the SD1 and SD2 statistics in the supplemental figures of Figure 4-7.

Results: Analyses of Inspiratory time (Ti) and flow rate (i.e., Tidal Volume / Ti) should be assessed and included.

Thank you for the suggestion. Inspiratory duration (Ti), expiratory duration (TE), breath cycle duration (TTOT), inspiratory flow rate (VT/Ti), and expiratory flow rate (VT/TE) have been included in the Figures 4-7.

Results/Methods: If similar analytical approaches were used in the current study as to that in Lusk et al. 2022, it appears that data was discontinuously sampled, rejecting periods of movement and only including periods of quiescent breathing. Were the periods of quiescent breathing different? Information should be provided to describe the total sampling duration included.

For room air, the entire gas condition was used for data analysis. For hypercapnia (5% CO2, 7% CO2, 10% CO2), only the last 5 minutes of the gas challenge period was used for data analysis. For hypoxia (10% O2), we analyzed the breathing trace of three 5-minute epochs following initiation of the gas exposure separately, e.g., epoch 1 = 5-10min, epoch 2 = 10-15min, and epoch 3 = 15-20min. All breaths included as quiescent breathing were analyzed in the aggregate for each group and experimental condition, we did not compare individual periods of quiescent breathing within or across an animal(s)/group(s)/experimental condition(s). We have added the details in the Materials and Methods (line 637-642).

Results: As mice were conscious in this study, were sniff periods (transient periods of fast breathing, i.e.,>8Hz) included in the analysis?

No, only regular quiescent breathing periods were included in the analysis.

Discussion: The authors need to discuss the limitations of their findings.

• How should the reader interpret the findings? Concluding that glutamatergic signaling is dispensable implies that it occurs in room air, hypoxia, and hypercapnia.

We have edited our discussion for clarity to highlight our conclusions that Vglut2-based glutamatergic signaling from noradrenergic neurons is ultimately dispensable for baseline breathing and hypercapnia and hypoxic chemoreflex in unanesthetized and unrestrained mice.

• Assuming that glutamatergic signaling is active during the conditions tested, then the authors should discuss what may be the potential compensations.

We have provided additional discussion surrounding potential compensatory events that may have taken place and could result in the unchanged phenotype in the experimental group.

• The authors need to discuss how age and state of consciousness may play a role in their finds. The current discussion gives the impression that their findings are broadly applicable in all cases, but the lack of differences in this study may not hold true under different conditions.

The study was done in adult (6–8-week-old) unanesthetized and unrestrained mice. In the discussion (line 472-474), we highlight that in our unpublished results, loss of NA-expressed Vglut2 does not change the survival curve in P7 neonate mice undergoing repeated bouts of autoresuscitation until death. Thus, we believed that Vglut2-based glutamatergic signaling in central NA neurons is dispensable for baseline breathing and the hypercapnic and hypoxic chemoreflexes in unanesthetized and unrestrained mice across different ages. Otherwise, we do not imply that we have interrogated any other aspects of breathing in our discussion.

Methods: Further description of the analysis window for the respiratory metrics should be provided. Were breath values for each condition taken throughout the entire condition? This is particularly important for hypoxia, where the stereotypical respiratory response is biphasic.

For room air, the entire gas condition was used for data analysis. For hypercapnia (5% CO2, 7% CO2, 10% CO2), only the last 5min of the gas challenge period was used for data analysis. For hypoxia (10% O2), we analyzed the breathing trace of three 5min time periods separately including 5-10min, 10-15min, and 15-20min during the hypoxic challenge as noted in our original manuscript, we graph and assess three 5min epochs during hypoxic exposure to capture the dynamic nature of the hypoxic ventilatory response. We have added the details in the Materials and Methods (line 637-642).

Methods: How was consciousness determined?

The conscious mice mentioned in the manuscript refer to the mice without anesthesia. We have replaced “awake” and “conscious” with “unanesthetized” in the text.

Reviewer #2 (Recommendations For The Authors):

Since no EEG/EMG recording was performed it would be more appropriate to remove "awake" and "conscious" throughout the manuscript and include the term "unanesthetized".

Thank you for the suggestion. “Awake” and “conscious” have been replaced by “unanesthetized” in the text.

Line 545: Why 32C? Isn't this temperature too high for animals?

30-32°C is the thermoneutral zone for mice. It is the range of ambient temperature where mice can maintain a stable core temperature with their minimal metabolic rate (Gordon, 1985). Whole-body plethysmography uses the barometric technique to detect pressure oscillations caused by changes in temperature and humidity with each breathing act when an animal sits in a sealed chamber (Mortola et al., 2013). Thus, maintaining the chamber temperature near the thermoneutral zone during the plethysmography assay is required to maintain constancy in respiratory and metabolic parameters from trial to trial as well as to maintain linearity of ventilatory pressure changes due to humidification, rarefaction, and thermal expansion and contraction during inspiration and expiration (Ray et al., 2011). The chamber temperature that has been used for adult plethysmography has been set across a range 30-34°C (Hodges et al., 2008; Ray et al., 2011; Hennessy et al., 2017). We use 32°C in this manuscript which is consistent with previously published literature from other groups and our own work (Sun et al., 2017; Lusk et al., 2022).

I would include the units of the physiological variables in the tables.

Thank you for the suggestion. The units of the physiological variables have been added in all the tables.

Reviewer #3 (Recommendations For The Authors):

Why is the C3 group not considered in this study?

The C3 adrenergic group, best characterized in rat, is only seen in rodents but not in many other species including primates (including human) (Kitahama et al., 1994). Thus, the C3 group is not the focus of this study where we aim to discuss if glutamate derived from noradrenergic neurons could be the potential therapeutic target of human respiratory disorders. The C3 adrenergic group is typically described as a population containing only about 30 neurons. We have added the fate map data and the adult expression pattern for the three vesicular glutamate transporters for the C3 group in the figure 1 and 2 supplements for reference.

Sub CD/CV does not appear to be defined in the manuscript.

Thank you for the point. The definition of sub CD/CV has been added in the text (line 126).

The data on line 131-133 is interesting but could be described more effectively and clearly.

Thank you for the suggestion. The text has been modified accordingly.

The end of the paragraph at lines 140 onwards is rather repeated in the paragraph that starts at line 146.

The repeated text has been removed accordingly.

Whilst anterior and posterior are correct anatomical terms, for a quadraped, rostral and caudal are more widely used - particularly in the brainstem field. Is there a particular reason for using anterior/posterior?

We followed the anatomical terminations in the Robertson et al. (2013) where they used anterior/posterior to describe C2/A2 and C1/A1.

On the protocol lines include in Figure 4-7 it would be worth adding the test day. This seems a little strange. Why wait up to one week after the habituation to perform the stimulation. How many mice were left for each day between habituation and experimentation, and does this timing affect responses? Do mice forget the habituation after a period?

Thank you for the point. We have added the test day for plethysmography in figures 4-7. After the 5 days of habituation, we began the plethysmography recordings on the sixth day. A maximum of 6 mice can be assayed for plethysmography per day due to the limited number of barometric flow through plethysmography and metabolic measurement systems we have. Thus, all animals were finished with plethysmography “within” one week of the last day of habituation. This protocol is consistent with our previous published work (Martinez et al., 2019; Lusk et al., 2022; Lusk et al., 2023). For the experiments in this manuscript, mice were assayed within 3 days after habituation. As noted in our methods and figures, each mouse is given as much as 40 mins to acclimate to the chamber (determined by directly observed quiet breathing) before data acquisition. We have no reason or evidence that indicates testing order and thus timing was a factor. The detailed explanation for the plethysmography protocol has been added in the material and methods section (line 606-625).

Please state clearly that each mouse is only exposed to one gas mixture (what I interpret is the case), or could one mouse be exposed to several different stimuli?

Each mouse is only exposed to one gas challenge (5% CO2, 7% CO2, 10% CO2, or 10% O2) in a testing period. Each testing period for an individual mouse was separated by 24hs to allow for a full recovery. The protocol is to put the mouse under room air for 45mins, switch to one gas challenge for 20mins, and switch back to room air for 20mins.

With apologies if I missed this, but did each of the respiratory stimuli produce a statistically significant response in the control mice? For example, the response to 10%O2?

Yes, each respiratory stimuli including 5/7/10% CO2 and 10% O2 produced a statistically significant response in both mutant and control mice. We have labeled the statistical significance in the Figures 4-7. Thank you for pointing this out.

Line 312: Optogenetic stimulation induced an increase from 130 to 180 breaths per min (Abbott et al., EJN 2014). It is surprising that this is called "modest". Baseline respiratory frequency was presented.

Thank you for the point. The word “modest” has been removed and the discussion has been changed accordingly (line 355-360).

Line 338: This discussion is not sufficiently nuanced. It is the increased Dia amplitude (to KCN only, not 10%CO2 ) and the stimulation of active expiration, to both stimuli, that is blocked by kyn in pFRG. There is no effect of breathing frequency. The current study would not detect such differences in active expiration.

Thank you for the suggestion. The discussion has been modified accordingly (line 382-388).

Author response:

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

eLife assessment

In this important paper, Blin and colleagues develop a high-throughput behavioral assay to test spontaneous swimming and olfactory preference in individual Mexican cavefish larvae. The authors present compelling evidence that the surface and cave morphs of the fish show different olfactory preferences and odor sensitivities and that individual fish show substantial variability in their spontaneous activity that is relevant for olfactory behaviour. The paper will be of interest to neurobiologists working on the evolution of behaviour, olfaction, and the individuality of behaviour.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors posed a research question about how an animal integrates sensory information to optimize its behavioral outputs and how this process evolved. Their data (behavioral output analysis with detailed categories in response to the different odors in different concentrations by comparing surface and cave populations and their hybrid) partially answer this tough question. They built a new low-disturbance system to answer the question. They also found that the personality of individual fish is a good predictor of behavioral outputs against odor response. They concluded that cavefish evolved to specialize their response to alanine and histidine while surface fish are more general responders, which was supported by their data.

Strengths:

With their new system, the authors could generate clearer results without mechanical disturbances. The authors characterize multiple measurements to score the odor response behaviors, and also brought a new personality analysis. Their conclusion that cavefish evolved as a specialist to sense alanine and histidine among 6 tested amino acids was well supported by their data.

Weaknesses:

The authors posed a big research question: How do animals evolve the processes of sensory integration to optimize their behavioral outputs? I personally feel that, to answer the questions about how sensory integration generates proper (evolved) behavior, the authors at least need to show the ecological relevance of their response. For the alanine/histidine preference in cavefish, they need data for the alanine and other amino acid concentrations in the local cave water and compare them with those of surface water.

We agree with the reviewer. This is why, in the Discussion section, we had written: “…Such significant variations in odor preferences or value may be adaptive and relate to the differences in the environmental and ecological conditions in which these different animals live. However, the reason why Pachón cavefish have become “alanine specialists” remains a mystery and prompts analysis of the chemical ecology of their natural habitat. Of note, we have not found an odor that would be repulsive for Astyanax so far, and this may relate to their opportunist, omnivorous and detritivore regime (Espinasa et al., 2017; Marandel et al., 2020).” This is also why we currently develop field work projects aimed at clarifying this question. However, such experiments and analyses are challenging, practically and technically. We hope we can reach some conclusions in the future.

To complete the discussion we have also added an important hypothesis: “Alternatively, specialization for alanine may not need to be specific for an olfactory cue present only, or frequently, or in high amounts in caves. Bat guano for example, which is probably the main source of food in the Pachón cave, must contain many amino acids. Enhanced recognition of one of them - in the present case alanine but evolution may have randomly acted for enhanced recognition of another amino acid – should suffice to confer cavefish with augmented sensitivity to their main source of nutriment.”

Also, as for "personality matters", I read that personality explains a large variation in surface fish. Also, thigmotaxis or wall-following cavefish individuals are exceeded to respond well to odorants compared with circling and random swimming cavefish individuals. However, I failed to understand the authors' point about how much percentages of the odorant-response variations are explained (PVE) by personality. Association (= correlation) was good to show as the authors presented, but showing proper PVE or the effect size of personality to predict the behavioral outputs is important to conclude "personality is matter"; otherwise, the conclusion is not so supported.

From the above, I recommend the authors reconsider the title also their research questions well. At this moment, I feel that the authors' conclusions and their research questions are a little too exaggerated, with less supportive evidence.

Thank you for this interesting suggestion, which we have fully taken into consideration. We have therefore now calculated and plotted PVE (the percentage of variation explained on the olfactory score) as a function of swimming speed or as a function of swimming pattern. The results are shown in modified Figure 8 of our revised ms and they suggest that the personality (here, swimming patterns or swimming speed) indeed predicts the olfactory response skills. Therefore, we would like to keep our title as we provide support for the fact that “personality matters”.

Also, for the statistical method, Fisher's exact test is not appropriate for the compositional data (such as Figure 2B). The authors may quickly check it at https://en.wikipedia.org/wiki/Compositional_data or https://www.annualreviews.org/doi/pdf/10.1146/annurev-statistics-042720-124436.

The authors may want to use centered log transformation or other appropriate transformations (Rpackage could be: https://doi.org/10.1016/j.cageo.2006.11.017). According to changing the statistical tests, the authors' conclusion may not be supported.

Actually, in most cases, the distributions are so different (as seen by the completely different colors in the distribution graphs) that there is little doubt that swimming behaviors are indeed different between surface and cavefish, or between ‘before’ and ‘after’ odor stimulation. However, it is true that Fisher’s exact test is not fully appropriate because data can be considered as compositional type. For this kind of data, centered log transformation have been suggested. However, our dataset contains many zeros, and this is a case where log transformations have difficulty handling.

To help us dealing with our data, the reviewer proposed to consider the paper by Greenacre (2021) (https://www.annualreviews.org/doi/pdf/10.1146/annurev-statistics-042720-124436). In his paper, Greenacre clearly wrote: "Zeros in compositional data are the Achilles heel of the logratio approach (LRA)."

Therefore, we have now tested our data using CA (Correspondence Analysis), that can deal with table containing many zeros and is a trustable alternative to LRA (Cook-Thibeau, 2021; Greenacre, 2011).

The results of CA analysis are shown in Supplemental figure 8 and they fully confirm the difference in baseline swimming patterns between morphs as well as changes (or absence of changes) in behavioral patterns after odor stimulation suggested by the colored bar plots in main figures, with confidence ellipses overlapping or not overlapping, depending on cases. Therefore, the CA method fully confirms and even strengthens our initial interpretations.

Finally, we have kept our initial graphical representation in the ms (color-coded bar plots; the complete color code is now given in Suppl. Fig7), and CA results are shown in Suppl. Figure 8 and added in text.

Reviewer #2 (Public Review):

In their submitted manuscript, Blin et al. describe differences in the olfactory-driven behaviors of river-dwelling surface forms and cave-dwelling blind forms of the Mexican tetra, Astyanax mexicanus. They provide a dataset of unprecedented detail, that compares not only the behaviors of the two morphs but also that of a significant number of F2 hybrids, therefore also demonstrating that many of the differences observed between the two populations have a clear (and probably relatively simple) genetic underpinning.

To complete the monumental task of behaviorally testing 425 six-week-old Astyanax larvae, the authors created a setup that allows for the simultaneous behavioral monitoring of multiple larvae and the infusion of different odorants without introducing physical perturbations into the system, thus biasing the responses of cavefish that are particularly fine-tuned for this sensory modality. During the optimization of their protocol, the authors also found that for cave-dwelling forms one hour of habituation was insufficient and a full 24 hours were necessary to allow them to revert to their natural behavior. It is also noteworthy that this extremely large dataset can help us see that population averages of different morphs can mask quite significant variations in individual behaviors.

Testing with different amino-acids (applied as relevant food-related odorant cues) shows that cavefish are alanine- and histidine-specialists, while surface fish elicit the strongest behavioral responses to cysteine. It is interesting that the two forms also react differently after odor detection: while cave-dwelling fish decrease their locomotory activity, surface fish increase it. These differences are probably related to different foraging strategies used by the two populations, although, as the observations were made in the dark, it would be also interesting to see if surface fish elicit the same changes in light as well.

Thank you for these nice comments.

Further work will be needed to pinpoint the exact nature of the genetic changes that underlie the differences between the two forms. Such experimental work will also reveal how natural selection acted on existing behavioral variations already present in the SF population.

Yes. Searching for genetic underpinnings of the sensory-driven behavioral differences is our current endeavor through a QTL study and we should be able to report it in the near future.

It will be equally interesting, however, to understand what lies behind the large individual variation of behaviors observed both in the case surface and cave populations. Are these differences purely genetic, or perhaps environmental cues also contribute to their development? Does stochasticity provided by the developmental process has also a role in this? Answering these questions will reveal if the evolvability of Astyanax behavior was an important factor in the repeated successful colonization of underground caves.

Yes. We will also access (at least partially) responses to most of these questions in our current QTL study.

Reviewer #3 (Public Review):

Summary:

The paper explores chemosensory behaviour in surface and cave morphs and F2 hybrids in the Mexican cavefish Astyanax mexicanus. The authors develop a new behavioural assay for the longterm imaging of individual fish in a parallel high-throughput setup. The authors first demonstrate that the different morphs show different basal exploratory swimming patterns and that these patterns are stable for individual fish. Next, the authors test the attraction of fish to various concentrations of alanine and other amino acids. They find that the cave morph is a lot more sensitive to chemicals and shows directional chemotaxis along a diffusion gradient of amino acids. For surface fish, although they can detect the chemicals, they do not show marked chemotaxis behaviour and have an overall lower sensitivity. These differences have been reported previously but the authors report longer-term observations on many individual fish of both morphs and their F2 hybrids. The data also indicate that the observed behavior is a quantitative genetic trait. The approach presented will allow the mapping of genes' contribution to these traits. The work will be of general interest to behavioural neuroscientists and those interested in olfactory behaviours and the individual variability in behavioural patterns.

Strengths:

A particular strength of this paper is the development of a new and improved setup for the behavioural imaging of individual fish for extended periods and under chemosensory stimulation. The authors show that cavefish need up to 24 h of habituation to display a behavioural pattern that is consistent and unlikely to be due to the stressed state of the animals. The setup also uses relatively large tanks that allow the build-up of chemical gradients that are apparently present for at least 30 min.

The paper is well written, and the presentation of the data and the analyses are clear and to a high standard.

Thank you for these nice comments.

Weaknesses:

One point that would benefit from some clarification or additional experiments is the diffusion of chemicals within the behavioural chamber. The behavioural data suggest that the chemical gradient is stable for up to 30 min, which is quite surprising. It would be great if the authors could quantify e.g. by the use of a dye the diffusion and stability of chemical gradients.

OK. We had tested the diffusion of dyes in our previous setup and we also did in the present one (not shown). We think that, due to differences of molecular weight and hydrophobicity between the tested dyes and the amino acid molecules we are using, their diffusion does not constitute a proper read-out of actual amino acid diffusion. We anticipate that amino acid diffusion is extremely complex in the test box, possibly with odor plumes diffusing and evolving in non-gradient patterns, in the 3 dimensions of the box, and potentially further modified by the fish swimming through it, the flow coming from the opposite water injection side and the borders of the box. This is the reason why we have designed the assay with contrasting “odor side” and “water control side”. Moreover, our question here is not to determine the exact concentration of amino acid to which the fish respond, but to compare the responses in cavefish, surface fish and F2 hybrids. Finally and importantly, we have performed dose/response experiments whereby varying concentrations have been presented for 3 of the 6 amino acids tested, and these experiments clearly show a difference in the threshold of response of the different morphs.

The paper starts with a statement that reflects a simplified input-output (sensory-motor) view of the organisation of nervous systems. "Their brains perceive the external world via their sensory systems, compute information and generate appropriate behavioral outputs." The authors' data also clearly show that this is a biased perspective. There is a lot of spontaneous organised activity even in fish that are not exposed to sensory stimulation. This sentence should be reworded, e.g. "The nervous system generates autonomous activity that is modified by sensory systems to adapt the behavioural pattern to the external world." or something along these lines.

Done

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

In addition to my comments in the "weakness" section above, here are my other comments.

How many times fish were repeatedly assayed and what the order (alanine followed by cysteine, etc) was, is not clear (Pg 24, Materials and Methods). I am afraid that fish memorize the prior experience to get better/worse their response to the higher conc of alanine, etc. Please clarify this point.

Many fish were tested in different conditions on consecutive days, indeed. Most often, control experiments (eg, water/nothing; water/water; nothing/nothing) were followed by odor testing. In such cases, there is no risk that fish memorize prior experience and that such previous experience interferes with response to odor. In other instances, fish were tested with a low concentration of one amino acid, followed by a high concentration of another amino acid, which is also on the safe side. Of note, on consecutive days, the odors were always perfused on alternate sides of the test box, to avoid possibility of spatial memory. Finally, in the few cases where increasing concentrations of the same amino acids were perfused consecutively, 1) they were perfused on alternate sides, 2) if the fish does not detect a low concentration below threshold / does not respond, then prior experience should not interfere for responding to higher concentrations, and 3) we have evidence (unpublished, current studies) that when a fish is given increasing concentrations of the same amino acid above detection threshold, then the behavioral response is stable and reproducible (eg does not decrease or increase).

Minor points:

Thygmotaxis and wall following.

Classically, thigmotaxis and wall following are treated as the same (sharma et al., 2009; https://pubmed.ncbi.nlm.nih.gov/19093125/) but the authors discriminate it in thigmotaxis at X-axis and Y-axis because fish repeatedly swam back and forth on x-axis wall or y-axis wall. I understand the authors' point to discriminate WF and T but present them with more explanations (what the differences between them) in the introduction and result sections.

Done

Pg5 "genetic architecture" in the introduction.

"Genetic architecture" analysis needs a more genomic survey, such as GWAS, QTL mapping, and Hi-C. Phenotype differences in F2 generation can be stated as "genetic factor(s)" "genetic component(s)", etc. please revise.

Done

Pg10 At the serine treatment, the authors concluded that "...suggesting that their detection threshold for serine is lower than for alanine." I believe that the 'threshold for serine is higher' according to the authors' data. Their threshold-related statement is correct in Pg21 "as SF olfactory concentration detection threshold are higher than CF,..." So the statement on page 10 is a just mistake, I think. Please revise.

Done (mistake indeed)

Pg11 After explaining Fig5, the statement "In sum, the responses of the different fish types to different concentrations of different amino acids were diverse and may reflect complex, case-bycase, behavioral outputs" does not convey any information. Please revise.

OK. Done : “In sum, the different fish types show diverse responses to different concentrations of different amino acids.”

For the personality analysis (Fig 7)

The index value needs more explanation. I read the materials and methods three times but am still confused. From the equation, the index does not seem to exceed 1.0, unless the "before score" was a negative value, and the "after score" value was positive. I could not get why the authors set a score of 1.5 as the threshold for the cumulative score of these different behavior index values (= individual score). Please provide more description. Currently, I am skeptical about this index value in Fig 7.

Done, in results and methods.

Pg15 the discussion section

Please discuss well the difference between the authors' finding (cavefish respond 10^-4M for position and surface fish responded 10^-4 for thig-Y; Fig 4AB), and those in Hinaux et al. 2016 (cavefish responded 10^-10M alanine but surface fish responded 10^-5M or higher). It seems that surface fish could respond to the low conc of alanine as cavefish do, which is opposed to the finding in Hinaux 2016.

The increase in NbrtY at population level for surface fish with 10-4M alanine (~10-6M in box) was most probably due to only a few individuals. Contrarily to cavefish, all other parameters were unchanged in surface fish for this concentration. Moreover, at individual level, only 3.2% of surface fish had significant olfactory scores (to be compared to 81.3% for cavefish). Thus, we think that globally this result does not contradict our previous findings in Hinaux et al (2016), and solely represent the natural, unexplained variations inherent to the analysis of complex animal behaviors – even when we attempt to use the highest standards of controlled conditions.

Of note, in the revised version, we have now included a full dose/response analysis for alanine concentration ranging from 10-2M to 10-10M, on cavefish. Alanine 10-5M has significant effects (now shown in Suppl Fig2 and indicated in text; a column has been added for 10-5M in Summary Table 1). Lower concentrations have milder effects (described in text) but confirm the very low detection threshold of cavefish for this amino acid.

Pg19, "In sum, CF foraging strategy has evolved in response to the serious challenge of finding food in the dark"

My point is the same as explained in the 'weakness' section above: how this behavior is effective in the cave life, if they conclude so? Please explain or revise this statement.

The present manuscript reports on experiments performed in “artificial” and controlled laboratory conditions. We are fully aware that these conditions are probably distantly related to conditions encountered in the wild. Note that we had written in original version (page 20) “…for 6-week old juveniles in a rectangular box - but the link may be more elusive when considering a fish swimming in a natural, complex environment.” As the reviewer may know, we also perform field studies in a more ethological approach of animal behaviors, thus we may be able to discuss this point more accurately in the future.

Pg20 "To our knowledge, this is the first time individual variations are taken into consideration in Astyanax behavioral studies."

This is wrong. Please see Fernandes et al., 2022. (https://pubmed.ncbi.nlm.nih.gov/36575431/).

OK. The sentence is wrong if taken in its absolute sense, i.e., considering inter-individual variations of a given parameter (e.g., number of neuromasts per individual or number of approaches to vibrating rod in Fernandez et al, 2022). In this same sense, Astyanax QTL studies on behaviors in the past also took into account variations among F2 individuals. Here, we wanted to stress that personality was taken into consideration. The sentence has been changed: “To our knowledge, this is the first time individual temperament is taken into consideration in Astyanax behavioral studies.”

Figure 2B and others.

The order of categories (R, R-TX, etc) should match in all columns (SF, F2, and CF). Currently, the category orders seem random or the larger ratio categories at the bottom, which is quite difficult to compare between SF, F2, and CF. Also, the writings in Fig 2A (times, Y-axis labels, etc), and the bargraphs' writings are quite difficult to read in Fig 2B, Fig 3B 4H, 5GN, 6EFG. Also, no need to show fish ID in Fig 2C in the current way, but identify the fish data points of the fish in Fig 2D (SF#40, CF#65, and F2#26) in Fig 2C if the authors want to show fish ID numbers in the boxplots. Fish ID numbers in other boxplot figures are recommended to be removed too.

We have thought a lot on how to best represent the distributions of swimming patterns in graphs such as Fig 2B and others. The difficulty is due to the existence of many combinations (33 possibilities in total, see new Suppl Fig7), which are never the same in different plots/conditions because individual tested fish are different. We decided that that the best way was to represent, from bottom to top, the most used to the less used swimming patterns, and to use a color code that matches at best the different combinations. It was impossible to give the full color code on each figure, therefore it was simplified, and we believe that the results are well conveyed on the graphs. We would like to keep it as it is. To respond (partially) to the reviewer’s concern, we have now added a full color code description in a new Supplemental Figure 7 (associated to Methods).

Size of lettering has been modified in all pattern graphs like Fig2A. Thanks for the suggestion, it reads better now.

Finally, we would like to keep the fish ID numbers because this contributes to conveying the message of the paper, that individuality matters.

Raw data files were not easy to read in Excel or LibreOffice. Please convert them into the csv format to support the rigor in the authors' conclusion.

We do not understand this request. Our very large dataset must be analysed with R, not excel for stats or for plotting and pattern analysis. However, raw data files can be opened in excel with format conversion.

Reviewer #2 (Recommendations For The Authors):

I think most of the experimental procedures (with few exceptions, see below) are well-defined and nicely described, so the majority of my suggestions will be related to the visualization of the data. I think the authors have done a great job in presenting this complex dataset, but there are still some smaller tweaks that could be used to increase the legibility of the presented data.

First and perhaps foremost, a better definition of the swimming pattern subsets is needed. I have no problem understanding the main behavioral types, but whereas the color codes for these suggest that there is continuous variance within each pattern, it is not clear (at least to me), what particular aspect(s) of the behaviors vary. Also, whereas the sidebars/legends suggest a continuum within these behaviors, the bar charts themselves clearly present binned data. I did not find a detailed description of how the binning was done. As this has been - according the Methods section - a manual process, more clarity about the details of the binning would be welcome. I would also suggest using binned color codes for the legends as well.

Done, in Results and Methods. We hope it is now clear that there is no “continuum”, rather multiple combinations of discrete swimming patterns. The gradient aspect in color code in figures has been removed to avoid the idea of continuum. According to the chosen color code, WF is in red, R in blue, T in yellow and C in green. Then, combination are represented by colors in between, for example, R+WF is purple. We have now added a full color code description for the swimming patterns and their combinations in a new Supplemental Figure 7 (associated to Methods).

Also, to better explain the definition of the swimming patterns and the graphical representation, it now reads (in Methods):

“The determination of baseline swimming patterns and swimming patterns after odor injection was performed manually based on graphical representations such as in Figure 2A or Figure 3A. Four distinctive baseline behaviors clearly emerged: random swim (R; defined as haphazard swimming with no clear pattern, covering entirely or partly the surface of the arena), wall following (WF; defined as the fish continuously following along the 4 sides of the box and turning around it, in a clockwise or counterclockwise fashion), large or small circles (C; self explanatory), and thigmotactism (T, along the X- or the Y-axis of the box; defined as the fish swimming back and forth along one of the 4 sides of the box). On graphical representations of swimming pattern distributions, we used the following color code: R in blue, WF in red, C in green, T in yellow. Of note, many fish swam according to combination(s) of these four elementary swimming patterns (see descriptions in the legends of Supplemental figures, showing many examples). To fully represent the diversity and the combinations of swimming patterns used by individual fish, we used an additional color code derived from the “basic” color code described above and where, for example R+WF is purple. The complete combinatorial color code is shown in Suppl. Fig7.”

It would be also easier to comprehend the stacked bar charts, presenting the particular swimming patterns in each population, if the order of different swimming patterns was the same for all the plots (e.g. the frequency of WF always presented at the bottom, R on the top, and C and T in the middle). This would bring consistency and would highlight existing differences between SF, CF, and F2s. Furthermore, such a change would also make it much easier to see (and compare) shifts in behaviors.

We have thought a lot on how to best represent the distributions of swimming patterns in graphs such as Fig 2B and others. The difficulty is due to the existence of many combinations, which are never the same in different plots/conditions because the individual fish tested are different. We decided to keep it as it currently stands, because we think re-doing all the graphs and figures would not significantly improve the representation. In fact, we think that the differences between morphs (dominant blue in SF, dominant red in CF) and between conditions (bar charts next to each other) are easy to interpret at first glance in the vast majority of cases. Moreover, they are now completed by CA analyses (Suppl Figure 8).

While the color coding of the timeline in the "3D" plots presented for individual animals is a nice feature, at the moment it is slightly confusing, as the authors use the same color palette as for the stacked bar charts, representing the proportionality of the particular swimming patterns. As the y-axis is already representing "time" here, the color coding is not even really necessary. If the authors would like to use a color scheme for aesthetic reasons, I would suggest using another palette, such as "grey" or "viridis".

We would like to keep the graphical aspect of our figures as they are, for aesthetic reasons. To avoid confusion with stacked bar chart color code, we have added a sentence in Methods and in the legend of Figure 2, where the colors first appear:

“The complete combinatorial color code is shown in Suppl. Figure 7. Of note, in all figures, the swimming pattern color code does not relate whatsoever with the time color code used in the 2D plus time representation of swimming tracks such as in Figure 2A”.

I would also suggest changing the boxplots to violin-plots. Figure 7 clearly shows bimodality for F2 scores (something, as the authors themselves note, not entirely surprising given the probably poligenic nature of the trait), but looking at SF and CF scores I think there are also clear hints for non-normal distributions. If non-normal distribution of traits is the norm, violin-plots would capture the variance in the data in a more digestible way. (The existence of differently behaving cohorts within the population of both SF and CF forms would also help to highlight the large pre-existing variance, something that was probably exploited by natural selection as well, as mentioned briefly in the Discussion by the authors, too.)

The bimodal distribution of scores shown by F2s in Figure 7B is indeed probably due to the polygenic nature of the trait. However, such distribution is rather the exception than the norm. Moreover, the boxplot representations we have used throughout figures include all the individual points, and outliers can be identified as they have the fish ID number next to them. This allows the reader to grasp the variance of the data. Again, redoing all graphs and figures would constitute a lot of work, for little gain in term of conveying the results. Therefore, we choose not to change the boxplot for violin plots.

The summary data of individual scores in Table 1B shows some intriguing patterns, that warrant a bit further discussion, in my opinion. For example, we can see opposite trends in scores of SF and CF forms with increasing alanine concentration. Is there an easy explanation for this? Also, in the case of serine, the CF scores do not seem to respond in a dose-dependent manner and puzzlingly at 10^(-3)M serine concentration F2 scores are above those of both grandparental populations.

That is true. However, we have no simple explanation for this. To begin responding to this question, we have now performed full dose/responses expts for alanine (concentrations tested from 10-2M to 10-10M on cavefish; confirm that CF are bona fide “alanine specialists”) and for serine (10-2M to 104M tested on both morphs; confirm that both morphs respond well to this amino acid). These complementary results are now included in text and figures (partially) and in the summary table 1.

If anything is known about this, I would also welcome some discussion on how thigmotactic behavior, a marker of stress in SF, could have evolved to become the normal behavior of CF forms, with lower cortisol levels and, therefore lower anxiety.

We actually think thigmotactism is a marker of stress in both morphs. See Pierre et al, JEB 2020, Figure S3A: in both SF and CF thigmotaxis behavior decreases after long habituation times. In our hands, the only difference between the two morphs is that surface fish (at 5 month of age) express stress by thigmotactism but also freezing and rapid erratic movements, while cavefish have a more restricted stress repertoire.

This is why in the present paper we have carefully made the distinction between thigmotactism (= possible stress readout) and wall following (= exploratory behavior). Our finding that WF and large circles confers better olfactory response scores to cavefish is in strong support of the different nature of these two swimming patterns. Then, why is swimming along the 4 walls of a tank fundamentally different from swimming along one wall? The question is open, although the number of changes of direction is probably an important parameter: in WF the fish always swims forward in the same direction, while in T the fish constantly changes direction when reaching the corner of the tank – which is similar to erratic swim in stressed surface fish.

Finally two smaller suggestions:

• When referring to multiple panels on the same figure it would be better to format the reference as "Figure 4D-G" instead of "Figure 4DEFG";

Done

• On page 4, where the introduction reads as "although adults have a similar olfactory rosette with 2025 lamellae", in my opinion, it would be better to state that "while adults of the two forms have a similar olfactory rosette with 20-25 lamellae".

Done

Reviewer #3 (Recommendations For The Authors):

Consider moving Figure 3 to be a supplement of Figure 4. This figure shows a water control and therefore best supplements the alanine experiment.

We would like to keep this figure as a main figure: we consider it very important to establish the validity of our behavioral setup at the beginning of the ms, and to establish that in all the following figures we are recording bona fide olfactory responses.

"sensory changes in mecano-sensory and gustatory systems " - mechano-sensory.

Done

Figure 2 legend: "(3) the right track is the 3D plus time (color-coded)" - shouldn't it be 2D plus time or 3D (x,y, time).

True! Thanks for noting this, corrected.

Figure 4 legend "E, Change in swimming patterns" should be H.

Done

"suggesting that their detection threshold for serine is lower than for alanine" - higher?

Done

In the behavioural plots, I assume that the "mean position" value represents the mean position along the X-axis of the chamber - this should be clarified and the axis label updated accordingly.

That is correct and has been updated in Methods and Figures and legends.

"speed, back and forth trips in X and Y, position and pattern changes (see Methods; Figure 7A)." - here it would be helpful to add an explanation like "to define an olfactory score for individual fish."

This has been changed in Results and more detailed explanations on score calculations are now given in Methods.

"possess enhanced mecanosensory lateral line" - mechanosensory.

Done

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript "comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation" aims to provide insights into the mediators and mechanisms underlying tardigrade radiation tolerance. The authors start by assessing the effect of ionizing radiation (IR) on the tardigrade lab species, H. exemplaris, as well as the ability of this organism to recover from this stress - specifically, they look at DNA double and single-strand breaks. They go on to characterize the response of H. exemplaris and two other tardigrade species to IR at the transcriptomic level. Excitingly, the authors identify a novel gene/protein called TDR1 (tardigrade DNA damage response protein 1). They carefully assess the induction of expression/enrichment of this gene/protein using a combination of transcriptomics and biochemistry - even going so far as to use a translational inhibitor to confirm the de novo production of this protein. TDR1 binds DNA in vitro and co-localizes with DNA in tardigrades.

Reverse genetics in tardigrades is difficult, thus the authors use a heterologous system (human cells) to express TDR1 in. They find that when transiently expressed TDR1 helps improve human cell resistance to IR.

This work is a masterclass in integrative biology incorporating a holistic set of approaches spanning next-gen sequencing, organismal biology, biochemistry, and cell biology. I find very little to critique in their experimental approaches.

Strengths:

(1) Use of trans/interdisciplinary approaches ('omics, molecular biology, biochemistry, organismal biology)

(2) Careful probing of TDR1 expression/enrichment

(3) Identification of a completely novel protein seemingly involved in tardigrade radio-tolerance.

(4) Use of multiple, diverse, tardigrade species of 'omics comparison.

Weaknesses:

(1) No reverse genetics in tardigrades - all insights into TDR1 function from heterologous cell culture system.

(2) Weak discussion of Dsup's role in preventing DNA damage in light of DNA damage levels measured in this manuscript.

(3) Missing sequence data which is essential for making a complete review of the work.

Overall, I find this to be one of the more compelling papers on tardigrade stress-tolerance I have read. I believe there are points still that the authors should address, but I think the editor would do well to give the authors a chance to address these points as I find this manuscript highly insightful and novel.

We thank the reviewer for his comments.

We agree that it will be important to further investigate the role of Dsup in radio-tolerance. We briefly mentioned this point in the discussion (p14). Our findings show that tardigrades undergo DNA damage at levels roughly similar to radio-sensitive organisms and therefore support a major role for DNA repair in the maintenance of genome integrity after exposure to IR. Nevertheless, we believe that more precise quantification of DNA damage may still reveal a contribution of genome protection to radio-tolerance of tardigrades compared to radio-sensitive organisms. Dsup loss of function experiments in tardigrades would clearly be the best way to assess this possibility. In the absence of experiments directly addressing the function of Dsup, we prefer to refrain from drawing any firm conclusion on prevention of DNA damage by Dsup and thus to keep a more open position. In any case, as discussed in the text, we note that Dsup has only been reported in Hypsibioidea and other molecular players, such as TDR1, are likely involved in radio-tolerance in other tardigrade species.

The sequence data can be accessed at the NCBI SRA database with Bioproject ID PRJNA997229.

Reviewer #3 (Public Review):

Summary:

This paper describes transcriptomes from three tardigrade species with or without treatment with ionizing radiation (IR). The authors show that IR produces numerous single-strand and double-strand breaks as expected and that these are substantially repaired within 4-8 hours. Treatment with IR induces strong upregulation of transcripts from numerous DNA repair proteins including Dsup specific to the Hypsobioidea superfamily. Transcripts from the newly described protein TDR1 with homologs in both Hypsibioidea and Macrobiotoidea supefamilies are also strongly upregulated. They show that TDR1 transcription produces newly translated TDR1 protein, which can bind DNA and co-localizes with DNA in the nucleus. At higher concentrations, TDR appears to form aggregates with DNA, which might be relevant to a possible function in DNA damage repair. When introduced into human U2OS cells treated with bleomycin, TDR1 reduces the number of double-strand breaks as detected by gamma H2A spots. This paper will be of interest to the DNA repair field and to radiobiologists.

Strengths:

The paper is well-written and provides solid evidence of the upregulation of DNA repair enzymes after irradiation of tardigrades, as well as upregulation of the TRD1 protein. The reduction of gamma-H2A.X spots in U2OS cells after expression of TRD1 supports a role in DNA damage.

Weaknesses:

Genetic tools are still being developed in tardigrades, so there is no mutant phenotype to support a DNA repair function for TRD1, but this may be available soon.

We thank the reviewer for his comments.

Reviewer #4 (Public Review):

The manuscript brings convincing results regarding genes involved in the radio-resistance of tardigrades. It is nicely written and the authors used different techniques to study these genes. There are sometimes problems with the structure of the manuscript but these could be easily solved. According to me, there are also some points which should be clarified in the result sections. The discussion section is clear but could be more detailed, although some results were actually discussed in the results section. I wish that the authors would go deeper in the comparison with other IR-resistant eucaryotes. Overall, this is a very nice study and of interest to researchers studying molecular mechanisms of ionizing radiation resistance.

I have two small suggestions regarding the content of the study itself.

(1) I think the study would benefit from the analyses of a gene tree (if feasible) in order to verify if TDR1 is indeed tardigrade-specific.

(2) It would be appreciated to indicate the expression level of the different genes discussed in the study, using, for example, transcript per millions (TPMs).Recommendations for the authors: please note that you control which revisions to undertake from the public reviews and recommendations for the authors

We thank the reviewer for his comments.

(1) To identify TDR1 homologous sequences in non-tardigrade species, we conducted extensive homology searches using multiple homology-based approaches (Blastp and Diamond against the NCBI non-redundant protein sequences (nr) database and hmmsearch against the EBI reference proteomes), which failed to identify TDR1 homologs in non-tardigrade ecdysozoans, thus strongly supporting that TDR1 is indeed tardigrade-specific.

To be clearer in the manuscript, we now state the absence of hits for TDR1 in non-tardigrade ecdysozoans. Given the absence of homologs in non-tardigrade species, it is not possible to make a gene tree with non-tardigrade species.

(2) To further document expression levels (which were already available from the Tables in the initial submission), we added MAplots (representing log2foldchange and logNormalized read counts) in the supplementary materials (Supp Figure 3 and Supp Figure 8). These additional figures clearly document that the DNA repair genes discussed in the main text and TDR1 are highly expressed genes after IR and after Bleomycin treatment.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

We thank the reviewer for his comments.

(1) It has always seemed strange to me that tardigrades accumulate just as much DNA damage as any other organism when irradiated and yet their Dsup protein is supposed to shield and protect their DNA from damage. Perhaps this is an appropriate time for this idea to be reconsidered given the Dsup was NOT induced by IR in this study and the authors found that their animals incurred just as much damage as other biological systems. While Dsup is clearly not the focus of this manuscript, it is the protein most associated with tardigrade radio-tolerance and I would argue this new paper would call into question previous conclusions made about Dsup.

We agree that it will be important to further investigate the role of Dsup in radio-tolerance. We briefly mentioned this point in the discussion (p14). Our findings show that tardigrades undergo DNA damage at levels roughly similar to radio-sensitive organisms and therefore support a major role for DNA repair in the maintenance of genome integrity after exposure to IR. Nevertheless, we believe that more precise quantification of DNA damage may still reveal a contribution of genome protection to radio-tolerance of tardigrades compared to radio-sensitive organisms. Dsup loss of function experiments in tardigrades would clearly be the best way to assess this possibility. In the absence of experiments directly addressing the function of Dsup, we prefer to refrain from drawing any firm conclusion on prevention of DNA damage by Dsup and thus to keep a more open position. In any case, as discussed in the text, we note that Dsup has only been reported in Hypsibioidea and other molecular players, such as TDR1, are likely involved in radio-tolerance in other tardigrade species.

(2) While reverse genetics are difficult in tardigrades, they are not impossible, and RNAi can be used to good effect in these animals. In fact several authors on this manuscript have used RNAi to examine the necessity of genes in tardigrade stress tolerance in the past. Was an attempt made to RNAi TDR1? If not, why? With the large amount of work that the authors put into showing the sufficiency of TDR1 for increasing radiotolerance in cell culture, one would think looking at necessity in tardigrades would be of great interest. If RNAi was performed, what were the results? Even a negative result here is informative since a protein can be sufficient but not necessary for a function - if this were the case it would mean tardigrades have some redundant mechanism(s) for surviving radiation exposure beyond TDR1.

We have attempted RNAi experiments targeting TDR1 or a mix of DNA repair genes (including XRCC5) and examined response to a bleomycin treatment of 2 weeks. Unfortunately, we could not distinguish any difference between uninjected animals and animals injected with TDR1 dsRNAs , or the mix of DNA repair genes dsRNAs. We concluded that, bleomycin treatment, that we used because it is much easier to perform than irradiation, was perhaps not the best way to assay a potential impact of RNAi on survival since it required long term treatment for several days during which the effect of RNAi may have waned. Another attempt was therefore made injecting with TDR1 or control GFP dsRNAs and exposing animals to a 2000Gy IR treatment. We noticed that the viability was lower after injection with GFP dsRNAs than with TDR1 dsRNAs (likely due to problems we had with the injection needle during injections). The next day, animals were irradiated and we observed after 24h that animals injected with GFP dsRNAs exhibited higher lethality rates than animals injected with TDR1 dsRNAs or uninjected animals. We found that this set of experiments were not conclusive. Our current experimental set up will make it difficult to distinguish lethality due to injections from lethality due to potentially decreased resistance to IR. In particular, many key controls are difficult to make (in particular, we could not confirm the efficiency of target gene knockdown, as it is very challenging given the low amount of biological material available and the poor expression of these genes without irradiation). From a practical point of view, performing these experiments is thus very challenging. We nevertheless agree that, in future work, further experimentation is needed to examine the impact of knock-down by RNAi of TDR1 or of other genes such as DNA repair genes or Dsup, in tardigrade DNA repair and survival after IR. Gene knock-out with CRISPR-Cas9 is a very promising alternative to RNAi given that studies in mutant lines will eliminate the confounding effect of lethality due to injections.

(3) Regarding the U2OS experiments. I have several questions/points of clarification:

a. Were survival/proliferation levels tested or only H2AX foci? I think that showing decreased H2AX foci (fewer double-stranded breaks) correlates with higher survival rates would be important.

In the experiments reported in Figure 6, cells were transiently transfected with expression vectors and we did not examine the impact on survival rates. U2OS cells are resistant to high doses of Bleomycin and testing survival would require longer exposure at much higher concentrations (Buscemi et al, 2014, PMID: 25486478). In order to try and better address an impact on cell survival, we therefore generated populations of cells stably expressing the candidate tardigrade proteins fused to GFP. Despite trying different experiment conditions for treatment with Bleomycin, we could not detect a reproducibly significant benefit on cell survival for any of the tardigrade proteins tested, including RvDsup which was used as a positive control (since it was previously reported to improve cell survival in response to X-rays). One possibility is that the analysis should be performed in clones and not in populations of cells with heterogeneous expression levels of the tardigrade protein tested. For example, expression levels of the tardigrade protein needed to reduce the number of phospho-H2AX foci in response to DNA damage may interfere with cell division. We note that in the original Dsup paper, the benefit of RvDsup on cell survival was reported in specific transgenic clones. Experiments in different biological systems have also started to document toxic effects of RvDsup expression, illustrating the challenge, when performing experiments in heterologous systems, to achieve suitable expression levels of the tested protein. Trying to perform such a finer analysis, in our opinion, would go beyond the scope of our manuscript and will be best addressed in future studies. We are therefore careful in the text not to make any claim on the benefit of TDR1 expression on cell survival in response to Bleomycin in human cultured cells.

(b) From the methods I am a bit confused as to how the images were treated/foci quantified. With the automatic segmentation and foci identification, is this done through the entire Z-series or a single layer? If the latter then I am not sure the results are meaningful, since we do not know how many foci might be present in other layers of the nuclei analyzed. If the former, please clarify this in the method since it is a very important consideration.

We have acquired images throughout the entire Z-series and edited the text to make it more clear ; We now write: “ Z-stacks were maximum projected and analyzed with Zen Blue software (v2.3)...”. To limit the time needed for image analysis, we have generated an artificial image by projecting the entire Z-series into a single image and counted foci in that single maximum projection image. Although there are potential drawbacks, such as potentially only counting one focus when two foci are superposed along the Z axis, this approach overcomes the limitations of quantification from a single layer. We further ensured statistical robustness of the analysis by performing quantification from several independent fields of the labelled cells and several independent biological replicates (n>=3 as now specified in the legend of figure 6a).

(c) RvDsup reduced levels of HXA1 foci in these experiments, however, HeDsup was not found to be enriched in the transcriptomic analysis performed here. Was there a reason HeDsup was not used in the cell-based experiments? One could argue that RvDsup is from a different species of tardigrade, but it is a bit concerning that an ortholog of a protein found NOT to be induced by radiation exposure seems to perform as well (if not better) than some versions of TDR1.

RvDsup is the protein initially shown to increase survival of human HEK293 cells treated with X-rays and reduce the number of phospho-H2AX foci induced: it was therefore used as a positive control in our experiments. The sequence of HeDsup is only poorly similar to RvDsup (with 26% identity) and activity of HeDsup in cultured cells has not been reported before. We therefore believe that HeDsup is not well suited to provide a positive control for the experiments performed in our manuscript.

(d) From the methods, it seems that cells were treated with Bleomycin and then immediately fixed without any sort of recovery time. In this short timeframe, the presence of TDR1 appears to be enough to deal with a substantial amount of double-stranded breaks (as evidenced by the reduced number of HXA1 foci). Does this make sense? How quickly could one expect DNA repair machinery to make significant progress in resolving damaged DNA? This response seems much faster than what was observed in tardigrades. Perhaps the authors to comment on this.

Kinetic studies in human cells show extremely rapid repair of DNA double-strand breaks. Sensing of DNA double strand breaks by PARP proteins takes place within seconds after irradiation by IR (Pandey and Black, 2021, PMID: 33674152). NHEJ is then observed to take place by formation of 53BP1 foci within 15 minutes (Schultz et al, 2000, PMID: 11134068). The number of phospho-H2AX and 53BP1 foci peaks at 30 minutes and starts declining thereafter, showing that at a significant number of sites, DNA repair is proceeding very rapidly (by NHEJ). Although we are not aware of any studies of DNA repair kinetics in U2OS cells after addition of Bleomycin, DNA damage must be instantaneous and further take place during exposure to the drug in parallel to DNA repair, which would be expected to have similar kinetics than after irradiation with IR.

In our experiments, several mechanisms may be involved in reducing the number of phospho-H2AX foci induced by Bleomycin, such as DNA protection (for Dsup expression) or stimulation of DNA repair (for RNF146 expression). For TDR1, the molecular mechanism involved remains to be determined. Given our finding that TDR1 can form aggregates with DNA, an additional possibility is that clustering of phospho-H2AX foci is induced.

(4) I could not find the sequences of the TDR1 proteins studied here. I did find the cDNA sequence of HeTDR1 in the final supplementary file, but not the other TDR1 orthologs. In the place where it appeared the TDR1 sequences from other tardigrades should be there were very short segments of the HETDR1 sequence. All sequences of proteins used in this study should be easily accessible to the reader and reviewers as it is not possible to review this work without accessing the sequences.

Our apologies for the inappropriate documentation of TDR1 sequences in the original manuscript. As requested, we have now included the TDR1 sequences in the Supplementary Table 4.

(5) Likewise, the RNA sequence data is said to be deposited in NCBI under PRJNA997229, but I do not find this available on NCBI.

The RNA sequence data was deposited in NCBI under the indicated reference before submission of the manuscript. The data has now been released and is fully available on NCBI.

(6) A few typographical errors: e.g., Page 10 - sentence 4 has two periods ". ." or page 14 which has an open parenthesis that is not closed.

These typos have been corrected in the revised manuscript.

Reviewer #3 (Recommendations For The Authors):

We thank the reviewer for his comments.

In Figure 4C, what fraction of the 50 genes upregulated in all species and treatments are DNA repair genes? Is there any other notable commonality between these 50 genes? The bulk of upregulated genes are specific to a species and to treatment with IR or bleomycin. What fraction of DNA repair genes are specific to a species or treatment?

The results in Figure 4C on the 50 putative orthologous genes upregulated in all species and treatments are further detailed in supp Figure 10. The legend to supp Figure 10 now provides the requested information: 14/50 genes are DNA repair genes and the other notable commonality is that 21/50 are “stress response genes”. We did not further breakdown the analysis to evaluate the fraction of DNA repair genes specific to a species or treatment. It will be interesting to gather data in more species to hed light on the evolutionary history of DNA repair gene regulation in response to IR.

How does the suite of upregulated tardigrade DNA repair proteins after IR or bleomycin compare with DNA or repair proteins upregulated under similar treatments in human cells? Are they quantitatively or qualitatively different, or both?

There is a great wealth of studies documenting genes differentially expressed in human cells in response to IR (e.g. Borras-Fresneda et al, 2016, PMID: 27245205; Rieger and Chu, 2004, PMID: 15356296; Budwoeth et al, 2012, PMID: 23144912 ; Rashi-Elkeles et al, 2011, PMID: 21795128; Jen and Cheung, 2003, PMID: 12915489...). Upregulation of DNA repair and cell cycle genes is commonly found. However, the number of DNA repair genes induced is always very limited and fold stimulation very modest compared to the massive upregulation observed in tardigrades.

On page 14, please explain the acronym BER. Do the authors mean Base Excision Repair? Or something else?

As assumed by the reviewer, the acronym BER stands for Base Excision Repair. The acronym has been removed from the main text and replaced by the full name.

Reviewer #4 (Recommendations For The Authors):

We thank the reviewer for his comments.

Abstract:

The abstract is fine. What was hard to grasp at the beginning is why TDR1 gene was named that way. It should be clearer that this study decided to further focus on that gene, one of the most overexpressed gene after IR, with an unknown function. Then maybe introduce that it was found to be unique to tardigrade and to interact with DNA. Therefore, it was named TDR1.

Introduction:

The introduction has been modified according to the suggestions of Reviewer#4 below. One of the suggested references, Nicolas et al 2023 from the Van Doninck lab, was published while our manuscript was under review and cannot be considered as background information for our study.

1st paragraph:

The study is on tardigrades, I found it strange that the first paragraph is on D. radiodurans. I think it is fine to mention what is known in bacteria and eucaryotes but we should already know what will be the main topic in the first paragraph of the introduction. Some details about D. radiodurans seem less important and distracting from the main topic (3D conformation).

2nd paragraph:

When mentioning radio-resistant eurcaryotes the authors do not mention the larvae of the anhydrobiotic insect Polypedilum vanderplanki. Stating that the mechanisms of resistance are poorly characterized should perhaps be nuanced. There are some recent studies on D. radiodurans (Ujaoney et al., 2017) the insect P. vanderplanki (Ryabova et al., 2017), tardigrades (Kamilari et al., 2019), and rotifers (Nicolas et al., 2023, Moris et al., 2023). Perhaps these papers are worth indicating that if mechanisms are not elucidated yet, recent studies suggest some actors involved in their resistance. Regarding the sentence stating that DNA repair rather than DNA protection plays a predominant role in the radio-resistance of bdelloid rotifers should also be nuanced. Indeed, many chaperones, antioxidants were mentioned to play a role in the radio-resistance of bdelloid rotifers (Moris et al., 2023). The authors mentioned the reference Hespeels et al., 2023 which is not found in their list of references, I am not sure which paper they refer to. The last sentence of the second paragraph does not mean much. I am not sure what the authors want to state with this. Perhaps they should specify if they mean that the function of many other genes overexpressed after IR remains unknown.

Still, in the second paragraph, the authors focus on rotifers. They also do not mention what is known in the insect P. vanderplanki, which should be added. They still do not mention tardigrades. I think it is nice to first start with eucaryotes and then focus on tardigrades but as I mentioned before it would help to understand the aim of the paper if the first paragraph mentioned briefly the tardigrades and then could go into detail in the third paragraph.

3rd paragraph:

The sentence starting "with over 1400 species" best to remove from it "but they can differ in their resistance" and start the next sentence with that.

4th paragraph:

Very clear, we finally understand what is the focus of the manuscript.

5th paragraph:

Very clear. The authors should mention the names of the three studied species. Here, A. antarcticus is missing. The sentence "Further analyses in H. exemplaris... showed that TDR1 protein is present and upregulated". The authors should mention in which conditions the protein is upregulated. In that paragraph the authors mention phospho-H2AX: it might be good to introduce its functions before in the introduction (it is mentioned in the second sentence of the results: best to move it to the introduction).

Results:

There are a few sentences in this section which rather discuss the results than describe them. I think the manuscript might gain in quality if these interpretations of the results are moved into the discussion section. That would make the result section more concise and the discussion enriched.

For instance, I suggest to move these sentences into the discussion:

• "the finding of persistent DSBs in gonads at 72h.... likely explains...".

• "suggesting that (i) DNA synthesis..."

• " Phospho-H2AX....also suggested"

• "Moreover, expression of TDR1-GFP..., supporting the potential role of TDR1 proteins..."

• "our results suggest that RNF146 upreguation could contribute..."

• "AMNP gene g12777 was shown to increase...Based on our results, it is possible that..."

Interpretations mentioned here above were always introduced cautiously (-"suggesting that (i) DNA synthesis..." ; -" Phospho-H2AX....also suggested" ; -"Moreover, expression of TDR1-GFP..., supporting the potential role of TDR1 proteins..." ; -"our results suggest that RNF146 upreguation could contribute..." ). These cautious interpretations were usually important in deciding next steps of the work. We therefore believe it is important to mention these interpretations in the results section to clearly expose the milestones marking the progression of the study.

For some results, they were directly discussed in the results section for the sake of concision (for example -"the finding of persistent DSBs in gonads at 72h.... likely explains..."; -"AMNP gene g12777 was shown to increase...Based on our results, it is possible that..." ) since, in our opinion, there was no need to mention them again in the main discussion.

Some other parts could be good to be moved into the introduction:

• "Previous studies have indicated that irradiation with IR increases expression of Rad51,..." none of the actors involved in DNA repair are mentioned in the introduction. Also, change resistant into resistance

• "A. antarcticus ..., known for its resistant to high doses of UV....

We have moved these parts to the introduction as recommended.

It was in O. areolatus.... that the first demonstration..."

This piece of information is somewhat anecdotical. We choose to keep it it here in the results section. This information on the radio-resistance of the species P. areolatus is only relevant at this specific step of the study because it encouraged us to consider that P. fairbanksi, which we isolated fortuitously, would be a good model species for studying radio-resistance of tardigrades.

Here are some additional comments/suggestions on the result section:

1st section

• Remove the Gross et al., 2018 from the sentence "using confocal microscopy", it looks otherwise that these results are from their study, not yours.

We have changed the text to make it clear that this is indeed a finding of Gross et al which was previously made in non-irradiated tardigrades. We replicated this finding, which showed that the protocol was working appropriately, and that we could use this control result for comparison with irradiated animals. We apologize for this confusion.

The text now states: “Using confocal microscopy, we could detect DNA synthesis in replicating intestinal cells of control animals, as previously shown by (Gross et al. 2018).”

2nd section

• It is confusing what has been found induced by IR and/or by Bleomycin.

• I think it might help if the authors first present what is induced after IR, then write if it is similar after Bleomycin. Especially since they start to do it in the first paragraph of that section. However, they only mention TDR1 in the second paragraph dedicated to Bleomycin treatment which is confusing as it is also overexpressed after IR. It is also not clear if RNF146 is also induced by Bleomycin.

As recommended, the text presents first what is induced after IR and then what is induced by Bleomycin in the following paragraph. When reporting results with Bleomycin, we have provided a global assessment of what is common to both treatments in Supp Figure 3 and in Supp Table 3. In this figure, we also specifically highlighted several key genes of DNA repair induced by both treatments. These are also mentioned in the text (p8) to illustrate the point that many key DNA repair genes are common to both treatments. We have now added RNF146 to that list as recommended.

• Regarding TDR1, it is not clear when introduced in the text as "promising candidate" why it is the case. It is clear in the figures but perhaps the authors should explain why they chose these genes for further analyses: high log2foldchange and expression level for instance. Regarding that last comment, it would be interesting to have an idea about the expression level of the genes with high log2foldchange. In Figures 2, 3, and 4 the pvalue and log2foldchange are represented but not the expression level (ideally Transcript per Millions). These values would give an additional idea on the importance of that gene. While looking at the figures, it is unclear why you did not further characterize other genes with high log2foldchange (some with even hints of their function): the mentioned RNF146, macroH2A1 (not even mentioned in the results), some genes unannotated in the figures with likely unknown functions,

When selecting genes of interest, we did indeed take into account high expression levels. To more clearly document expression levels (which were already available from the Tables), we added MAplots (representing log2foldchange and logNormalized read counts) in the supplementary materials (Supp Figure 3 and Supp Figure 8).

• It is also unclear at that stage why you named it "Tardigrade DNA damage response protein", as it is characterized as DNA repair/damage proteins by specific GO id or is it based on your downstream analyses, I think it might be worth to quickly mention the reason of that name.

The name illustrates two points which were already characteristic at this point in time of the study i.e. 1) it is a tardigrade specific protein and 2) it is induced in response to DNA damage.

• Regarding the BLAST analyses the protein was searched in C. elegans, D. melanogaster and H. sapiens. Why only these three species? What were the threshold evalues used for these analyses. As mentioned in the main comment, it would be worth searching species phylogenetically close to tardigrades to verify if it is well-tardigrade specific. Did you try to make a gene tree, after looking for a conserved domain (using hmmersearch)?

As indicated in the methods section, the “Tardigrade-specific" annotation was determined by absence of hits after high-throughput alignment (with diamond using –ultrasensitive-option) on the NCBI nr database and absence of hits after blast search on C. elegans, D. melanogaster and H. sapiens proteomes as a complementary criterion (the latter blast search was primarily performed to enrich for functional annotations). Based on these criteria, TDR1 was annotated as “Tardigrade-specific”. As stated in the text, we also searched for TDR1 related sequences with 1) blastp (which is more sensitive than diamond) on the NCBI nr database and 2) HMMER on Reference Proteomes, and no hits were found among non-tardigrade ecdysozoans organisms, confirming TDR1 is specific to tardigrades. For Blast search for example, there were five hits in non-ecdysozoans organisms (two cephalochordates, one mollusc and two echinoderma). The blastp and HMMER results are now included in the revised supplementary material (Supp Table 5). These very few hits in species phylogenetically distant from tardigrades cannot be taken to support the existence of TDR1 genes outside tardigrades.

To be clearer in the manuscript, we now state the absence of hits for TDR1 in non-tardigrade ecdysozoans. Given the absence of homologs in non-tardigrade species, it is not possible to make a gene tree with non-tardigrade species.

• Page 9: "Proteins extracts from H. exemplaris... at 4h and 24h..." I think this sentence can be removed as this is mentioned again 2 paragraphs after: "...we conducted an unbiased proteome analysis... at 4h..." The log2foldchange threshold mentioned for the proteomic analyses is 0.3: why this threshold, was it chosen randomly?

This is threshold is commonly used when considering log2foldchange with the technology used in our study, an isobaric multiplexed quantitative proteomic strategy which is known to compress ratios (Hogrebe et al. 2018).

• Page 10:

It would be good for more clarity to indicate at the beginning of the new section which species were investigated after IR or Bleomycin treatment.

TDR1 homologs in the other tardigrade species were identified based on what? Best reciprocal hit?

As indicated in the methods section of the manuscript, we searched for homologs in other tardigrade species by BLAST. A best reciprocal hit approach was not performed to try to determine which homologs might be orthologs. In particular, most TDR1 homologs identified are known from transcriptome assemblies and high-contiguity genome assemblies are needed to more confidently identify orthology (using synteny). The results of the BLASTP search are now provided as supplementary material (Supp Table 5).

Preliminary experiments indicated that A. antarcticus and P. fairbanski survived exposure to 1000 Gy: is there a supplementary graph showing this?

We have corrected the text to avoid any confusion. We have not rigorously examined the dose-dependent survival of P. fairbanksi in response to irradiation. Text was changed to: “We found by visual inspection of animals after IR that A. antarcticus and P. fairbanksi readily survived exposure to 1000 Gy.”

• Page 11:

"A set of 50 genes was upregulated in the three species": please be precise if only after IR.

Done

These genes cannot be the same as they are from different species. Did the author mean that they are coding for similar proteins? It might be good to give some more details even if the supplementary figure is mentioned.

Obviously, these genes are putative orthologs. We have changed the text to:

” a set of 50 putative orthologous genes was upregulated in response to IR in all three species”

Discussion:

• General comment: the discussion is focused mainly on TDR1, it would be nice to also discuss the other results: DNA repair genes, RNF146.

A whole paragraph is devoted to discussion of results on DNA repair genes and RNF146. We have extended that discussion following on the suggestion of the reviewer. In particular, we have explicitly mentioned the apparent paradox that XRCC5 and XRCC6, which are among the most highly stimulated genes at the mRNA level, only display modest upregulation at the protein level. Although further studies would be needed to examine the mechanisms involved, we propose that upregulation of RNF146, whose human homolog has been shown to drive degradation of PARylated XRCC5 and XRCC6 proteins in response to IR (Kang et al. 2011), may be responsible for higher degradation rates and may thus counterbalance increased levels of protein synthesis.

• Pulse field electrophoresis would be nice to be performed. It has been used to assess DSBs in bdelloid rotifers, is it possible in tardigrades?

As stated in the discussion, we believe that it would be challenging to perform pulse field electrophoresis in tardigrades. However, if possible, these experiments would certainly bring invaluable information to complement our analysis of DNA damage induced by IR.

• "By comparative transcriptomics": please rephrase that sentence.

• Proteins acting early in DNA repair: I am not sure I understand this sentence. Actors as ligases act not at the beginning of the repair pathways.

Well noted. We have removed ligases from the list.

• It is confusing that the authors mention NHEJ and double-strand break repair pathways as different pathways. There are 2 main pathways to repair DBSs: NHEJ and HR. It would be nice to add a reference to the sentence "PARP proteins act as sensors of DNA damage etc."

A typo in the sentence gave rise to the misleading suggestion that NHEJ is not a double strand repair pathway. It has been corrected.

A reference has been added for PARP proteins.

• It would be nice if the authors can explain deeper their suggestion that degradation of DNA repair actors is essential for tardigrade IR resistance.

We have expanded this part of the discussion and hope that it is clearer.

“For XRCC5 and XRCC6, our studyestablished, by two independent methods, proteomics and Western blot analysies, that the stimulation at the protein level could be much more modest (6 and 20-fold at most (Supp Figure 6) than at the RNA level (420 and 90 fold respectively). This finding suggests that the abundance of DNA repair proteins does not simply increase massively to quantitatively match high numbers of DNA damages. Interestingly, in response to IR, the RNF146 ubiquitin ligase was also found to be strongly upregulated. RNF146 was previously shown to interact with PARylated XRCC5 and XRCC6 and to target them for degradation by the ubiquitin-proteasome system (Kang et al. 2011). To explain the lower fold stimulation of XRCC5 and XRCC6 at the protein levels, it is therefore tempting to speculate that, XRCC5 and XRCC6 protein levels (and perhaps that of other scaffolding complexes of DNA repair as well) are regulated by a dynamic balance of synthesis, promoted by gene overexpression, and degradation, made possible by RNF146 upregulation. Consistent with this hypothesis, we found that, similar to human RNF146 (Kang et al. 2011), He-RNF146 expression in human cells reduced the number of phospho-H2AX foci detected in response to Bleomycin (Figure 6).”

• Page 15: Please add a reference for the sentence "Functional analysis of promotor sequences in transgenic tardigrades etc."

The reference has been added to fix this omission.

Material and Methods:

Small comments:

• 40 μm mesh: space missing

• 100 μm mesh: space missing

• (for Bleomycin)): parenthesis missing

• remove "as indicated in the text"

• The investigated time points after radiation need to be clearly stated in the method section. It is also unclear in the IR and Bleomycin section which tardigrades were treated with what. Not all were treated with Bleomycin.

The small comments above have been fixed in the revised version of the manuscript.

• Page 21: please precise the coverage of the RNA sequencing

Statistics on mapping of RNAseq reads are now provided in Supp Table 10.

• Page 22: Was any read trimming performed? Anything about the quality check of the reads?

Trimming was conducted using trimmomatic (v0.39) and quality check using FastQC (v. ?) This information has been added to the Methods section.

• Were the analyses confirmed by a second approach: for instance, EdgeR? Deseq2 and EdgeR do not always have the same results. For more robust analyses it is advised to use both.

Differential transcriptome analyses were conducted with DESeq2 only. The robustness of our identification of differentially expressed genes in response to IR stems from performing comparative analyses in three different species, rather than from using two bioinformatics pipelines in a single species. We also note that benchmarking reported in the initial DEseq2 paper showed that identification of differentially expressed genes with large log fold changes (which, as reported in our manuscript, is characteristic of many DNA repair genes in response to IR) is very consistent between DEseq2 and EdgeR.

Figures:

• Figure 2: Legend vertical dotted line does not indicate log2foldchange value of 4 in all panels: it would be good to indicate for panels a and c as well.

Figure 2has been improved following on the suggestions of the reviewer. Dotted lines now show log2foldchange value of 2 in all panels (ie Fold Change of 4 as mentioned in the main text).

• Figure 2C: There are a few points with high log2foldchange which are not annotated: was it because nothing was found in the blast research? If yes, it would be good to indicate their functions. If not, it would be good to mention in the discussion that there are some genes with still unknown functions which might play an important role in the resistance of tardigrades to IR.

The few points which are not annotated in figure 2c can now be found in Supp Table 3 Some of them have no hit in Blast search, some others such as BV898_09662 or BV898_07145 have hits on DNA repair genes as RBBP8/CtIP or XRCC6 respectively but are not annnotated as such by eggnog in KEGG pathway.

• Figure 4C: Why not have included the response of P. fairbanski to bleomycin? I guess it was not done, but it is unclear in the results and methods sections.

P.fairbanksi response to bleomycin wasn’t assessed as we didn’t get enough animals to run the study. The method section has been modified to precise this point.

Author response:

Reviewer #1 (Public Review):

“… it remains unclear how ninein reduction causes bone defects …”

We have added several control experiments that permit us to conclude that osteoblast numbers remain unaltered in the ninein-knockout embryos, and that bone abnormalities in vivo are caused by fusion defects of osteoclast precursor cells, whereas the proliferation, viability, or the adhesion of these precursor cells remain unaffected. For details, please see our comments below.

“Discussion includes several unfounded potential mechanisms that really need to be thoroughly analyzed to gain a mechanistic understanding of the bone defects…”

The new data back up our claim of fusion defects as a cause for limited osteoclast function. We have re-written parts of the discussion, to take into account our new findings.

“Data showing normal osteoblasts in ninein-null mice was qualitative and requires further in-depth analysis and quantification of osteoblast …”

To address this point, quantification of osteoblast numbers in tibiae at E16.5 and E18.5 was performed in control and ninein-deleted mouse embryos. The data are presented in the new Figures 3G and J.

“In ninein knock-out mice, reduced TRAP+ve multinuclear cells were observed (Figure 6A and 6B). However, the magnitude of difference (about 5% decrease in multinucleated cells) is not consistent with the skeletal deformities reported in Figures 2-4, potentially suggesting the contribution of additional mechanisms.”

We agree that the difference appears to be small at first glance, but nevertheless it remains statistically significant (a more than three-fold difference). We would like to recall that these observations (Fig. 6A) were performed at E14.5, i.e. at a stage when no ossification has occurred yet. We are looking at the first fusion events of myeloid precursors, likely derived from the fetal liver, that colonize the area of the first bone to form, and small differences in the number of functional osteoclasts may account for different timing of ossification. We think that differences in osteoclast fusion also account for the premature appearance of ossification centers for other skeletal elements, at later time points during development.

“The fusion assay in Figure 6C needs further clarification. How was the syncytia perimeter defined to measure cell surface? The x-axis suggests that there are syncytia that contain up to 160 nuclei at day 3. How were the nuclei differentially stained and quantified?”

We provide now additional information on the experimental approach in the revised manuscript, on pages 16-17 (Materials and Methods). For information: high numbers of syncytial nuclei in cultures were also observed by other groups in the past (Tiedemann et al., 2017, Front Cell Dev Biol. 5:54). In addition, we performed new experiments and quantified the fusion of osteoclast precursors by staining for actin and nuclei (new Figure 7C). This allowed us to quantify several additional parameters related to cell fusion (as initially performed in Raynaud-Messina et al., 2018, PNAS, 115:E2556-E2565).

“Some text needs clarification. … What is the definition of "large syncytia"? Is the fusion index increase by day 5 diminished in later days? A graph of the syncytia size/ nuclei number or fusion index in the above-mentioned days will be helpful.”

Information on the definition of “large syncytia” is now provided on page 10 (1st paragraph). We added further experimental details on osteoclast size for days 3, 4, and 5 in the supplemental Figures 7A and B. Most importantly, we performed additional assays of the fusion index by quantifying syncytial versus non-syncytial nuclei in a semi-automated manner. The new data are presented in Figure 7C, and the methods are explained on page 17. Together with our new analysis of cell proliferation, cell viability, and cell adhesion (Figure 7C, D, suppl. Fig. 7C-G), we provide now solid evidence for a fusion defect at the origin of impaired formation of ninein del/del osteoclasts.

“Assessment of resorption was qualitative in Figure 6E and since the fusion deficiencies are transient, quantification of a corresponding resorption activity is needed. This should be described in the Materials and Methods section.”

Quantifications of the bone resorption activities are now provided in the new Figure 7E, and a reference for the methods is provided on page 16.

“Further experiments are needed to show connections between reduced centrosome clustering and reduced osteoclast formation as there is no evidence to date that suggest centrosome clustering is required for cell fusion. Multi-color live imaging and dynamic analysis can be used to determine if the ninein deficient cells show defective movement/migration/ fusion dynamics.”

We agree that it is an important question, and studying potential links between centrosomal microtubule organization and osteoclast fusion is an ongoing project of the team. However, we estimate that in order to obtain conclusive results this will require 1-2 additional years of research activity, and we intend to present this as a separate project in the future. At the current point of our investigation, we think that providing a solid link between ninein, osteoclast fusion, and controlled timing of ossification, as shown in this manuscript, represents valuable progress to understand previously published bone abnormalities in patients with ninein mutations.

“Quantification of the % of multinucleated osteoclasts that contain clustered and dispersed centrosomes is needed.”

New quantification experiments on centrosome clustering are now provided in Figure 8H. These quantifications demonstrate that the potential of centrosome clustering is almost completely lost in osteoclasts without ninein.

Reviewer #2 (Public Review):

“Based on the decrease in the number of osteoclasts (Fig 5E, G, and also per coverslip after 2 days in culture), the authors suggest that the loss of ninein impacts osteoclast proliferation. First, proliferation can be directly quantified using Ki67 staining or EdU incorporation. Second, other interpretations are also plausible and can also be experimentally tested. These include less adhesion and attachment of the mutants to the coverslips, but perhaps more relevant in vivo is cell death of the ninein mutant osteoclasts. It has been established that the loss of centrosome function activates p53- dependent cell death and osteoclasts might be a vulnerable cell population. Quantifying p53 immunoreactivity and/or cell death in osteoclasts might help clarify the phenotype of osteoclast reduction.”

In response to the reviewers, we have performed a series of new experiments that include

1) A careful analysis of the fusion index, using a semi-automated approach, indicating significant differences in the fusion of precursor cells into osteoclasts (Fig. 7C).

2) We have repeated the quantification of cell numbers prior to fusion and find variations between samples from different mice (also among mice of the same genotype), but we see on average comparable cell adhesion between samples from control mice and ninein-del/del mice. The data are provided in the supplemental Figure 7F. Moreover, we have quantified the expression of three main beta-integrins at the surface of control and ninein del/del osteoclast precursors (suppl. Fig. 7G), without detecting significant differences. Altogether, these data suggest the cell adhesion is comparable for the two genotypes.

3) We have addressed the question of altered cell proliferation, by performing flow cytometry experiments and by quantifying the different cell cycle stages (Fig. 7D), and by quantifying Ki67 expression (suppl. Fig. 7C). We see no significant differences between samples from control and ninein-del/del mice.

4) We have addressed the question of cell death, by performing Annexin V staining and flow cytometry (suppl. Fig. 7D), and by immunoblotting for cleaved caspase 3 and PARP (suppl. Fig. 7E). These experiments reveal no significant differences between the control and ninein del/del samples. Our data permit us to exclude cell death as a likely cause for the reduction of fused osteoclasts in the absence of ninein.

Overall, the new experiments show that the defects in osteoclast formation from ninein-deleted samples are due to defects in cell fusion, but not in cell proliferation, cell adhesion or viability.

Reviewer #3 (Public Review):

“The authors put much emphasis on the centrosome in the Introduction session. However, it was not until Figure 7 did they show abnormal centriole clustering in osteoclasts. The introduction should include more background on osteoclast and osteoblast balance during skeletal development.”

To address this, we included more background on the role of osteoclasts and osteoblasts in the revised introduction (page 4).

Reviewer #3 (Public Review):

Summary:

The authors consider several known aspects of PV and SOM interneurons and tie them together into a coherent single-cell model that demonstrates how the aspects interact. These aspects are:<br /> (1) While SOM interneurons target distal parts of pyramidal cell dendrites, PV interneurons target perisomatic regions.<br /> (2) SOM interneurons are associated with beta rhythms, PV interneurons with gamma rhythms.<br /> (3) Clustered excitation on dendrites can trigger various forms of dendritic spikes independent of somatic spikes. The main finding is that SOM and PV interneurons are not simply associated with beta and gamma frequencies respectively, but that their ability to modulate the activity of a pyramidal cell "works best" at their assigned frequencies. For example, distally targeting SOM interneurons are ideally placed to precisely modulate dendritic Ca-spikes when their firing is modulated at beta frequencies or timed relative to excitatory inputs. Outside those activity regimes, not only is modulation weakened, but overall firing reduced.

Strengths:

I think the greatest strength is the model itself. While the various individual findings were largely known or strongly expected, the model provides a coherent and quantitative picture of how they come together and interact.

The paper also powerfully demonstrates that an established view of "subtractive" vs. "divisive" inhibition may be too soma-focused and provide an incomplete picture in cells with dendritic nonlinearities giving rise to a separate, non-somatic all-or-nothing mechanism (Ca-spike).

Weaknesses:

While the authors overall did an admirable job of simulating the neuron in an in-vivo-like activity regime, I think it still provides an idealized picture that it optimized for the generation of the types of events the authors were interested in. That is not a problem per se - studying a mechanism under idealized conditions is a great advantage of simulation techniques - but this should be more clearly characterized. Specifics on this are very detailed and will follow in the comments to authors.

What disappointed me a bit was the lack of a concise summary of what we learned beyond the fact that beta and gamma act differently on dendritic integration. The individual paragraphs of the discussion often are 80% summary of existing theories and only a single vague statement about how the results in this study relate. I think a summarizing schematic or similar would help immensely.

Orthogonal to that, there were some points where the authors could have offered more depth on specific features. For example, the authors summarized that their "results suggest that the timescales of these rhythms align with the specialized impacts of SOM and PV interneurons on neuronal integration". Here they could go deeper and try to explain why SOM impact is specialized at slower time scales. (I think their results provide enough for a speculative outlook.)

Beyond that, the authors invite the community to reappraise the role of gamma and beta in coding. This idea seems to be hindered by the fact that I cannot find a mention of a release of the model used in this work. The base pyramidal cell model is of course available from the original study, but it would be helpful for follow-up work to release the complete setup including excitatory and inhibitory synapses and their activation in the different simulation paradigms used. As well as code related to that.

Impact:

Individually, most results were at least qualitatively known or at least expected. However, demonstrating that beta-modulation of dendritic events and gamma-modulation of soma spiking can work together, at the same time and in the same model can lead to highly valuable follow-up work. For example, by studying how top-down excitation onto apical compartments and bottom-up excitation on basal compartments interacts with the various rhythms; or what the impact of silencing of SOM neurons by VIP interneuron activation entails. But this requires - again - public release of the model and the code controlling the simulation setups.

Beyond that, the authors clearly demonstrated that a single compartment, i.e., only a soma-focused view is too simple, at least when beta is considered. Conversely, the authors were able to describe the impact of most things related to the apical dendrite on somatic spiking as "going through" the Ca-spike mechanism. Therefore, the setup may serve as the basis of constraining simplified two-compartment models in the future.

Reviewer #1 (Public Review):

This manuscript describes the pattern of relaxed selection observed at spermatogenesis genes in gorillas, presumably due to the low sperm competition associated with single-male polygyny. The analyses to detect patterns of selection are very thorough, as are the follow up analyses to characterize the function of these genes. Furthermore, the authors take the extra steps of in vivo determination of function with a Drosophila model.

This is an excellent paper. It addresses the interesting phenomenon of relaxation of selection as a genomic signal of reproductive strategies using multiple computational approaches and follow-up analyses by pulling in data from GO, mouse knockouts, human infertility database, and even Drosophila RNAi experiments. I really appreciate the comprehensive and creative approach to analyze and explore the data. As far as I can tell, the analyses were performed soundly and statistics are appropriate. The Introduction and Discussion sections are thoughtful and well-written. I have no major criticisms of the manuscript.

The main area that I would suggest for improvement is in the "Caveats and Limitations" section of the Discussion. Currently, the first paragraph of this section states the obvious that genetic manipulation of gorillas is not feasible. Beyond a reminder to the reader that this was a rationale for the Drosophila work, it isn't really adding much insight. The second paragraph is a brief discussion of the directionality of change. I think it comes across as overly simplistic, with a sort of "well, we can never know" feel. Obviously, there are plenty of researchers who do model change to infer direction and causation, and there are plenty of published papers attempting to do so with respect to mating systems in primates.

I do not think the authors need to remove these paragraphs, but I do encourage them to turn the "Caveats and Limitations" section into something more meaningful by addressing limitations of the work that was actually done rather than limitations of hypothetical things that were not done. A few areas come to mind. First, the authors should discuss the effect of gene-tree vs species-tree inconsistencies in the analyses, which could affect the identification of gorilla-specific amino acid changes and/or the dN/dS estimates. Incomplete lineage sorting is very common in primates including the gorilla-chimp-human splits (Rivas-González et al. 2023). It would be nice to hear the authors' thoughts on how that might affect their analyses. Second, the dN/dS-based analyses assume the neutrality of synonymous substitutions. Of course, that assumption is not completely true; it might be true enough, and the authors should at least note it as a caveat. Third, and potentially related, is the consideration that these protein-coding genes may be functioning in other ways such as via antisense transcription. The genes under relaxed selection may be on their way to becoming pseudogenes and evolving as such at the sequence level, but many pseudogenes continue to be transcribed sense or anti-sense in a regulatory purpose. I don't think there is a way to incorporate this into the authors' analyses but it would be nice to see it acknowledged as a caveat or limitation.

I think intersectionality is essential because it helps us understand how different identity traits can intersect to affect an individual's experience. For example, an Asian pansexual male may face discrimination based on both race and sexual orientation, and the impact of such compounded discrimination may be much more complex than discrimination based on a single identity. This situation shows that we cannot consider only one factor when addressing discrimination and inequality; we must consider how multiple identity factors interact and may lead to more complex injustices. Such insights are critical to developing more effective equality policies and interventions.

When it comes to mental health issues in our generation, I can say with almost 100% confidence, that social media is the main culprit. With technology integrated in every aspect of our lives nowadays, we can blame social media for being so easily accessible and influential on our generation and generations to come. From my experiences, many social media sites encourage people to really only showcase the good aspects of our lives, which leaves a very one-sided angle of everyone. This is very harmful as many younger people (and probably older people too) tend to compare themselves to the influencers and accounts, leading to accelerating conditions like depression, anxiety, and jealousy. Additionally, with all these way to edit photos, these posts may not even be real but they sure seem real, leading to negative self body image thoughts and unhealthy diet and workout plans. I think in some ways social media can be good for your mental health, whether that is watching funny videos to lighten the mood, or looking into what other people with similar hobbies do to mimic and get inspiration. However, it is hard to filter out the harmful content with the content with want and enjoy.

The article addresses misconceptions about autism being a gradient rather than a true spectrum. This article also use vivid analogies like the visible light spectrum to illustrate its point. It highlights the diversity within autism by explaining how different individuals can have unique combinations of traits, emphasizing that autism is not just one condition but a complex collection of related neurological conditions. The author thinks we shouldn't compare different types of autism as "more" or "less" severe, and emphasizes the importance of understanding individual strengths and challenges rather than making assumptions based on outward behaviors.

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Reply to the reviewers

Reply to reviewer comments

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We extend our gratitude to the reviewers for their time and valuable feedback on our manuscript. We especially appreciate the insightful suggestions that have significantly contributed to refining our work and elucidating our findings. With the revisions made to the text and the inclusion of new experimental data, we believe our manuscript now effectively addresses all reviewer comments. We eagerly await your evaluation of our revised submission.

Small ARF-like GTPases play fundamental roles in dynamic signaling processes linked with vesicular trafficking in eukaryotes. Despite of their evolutionary conservation, there is little known about the ARF-like GTPase functions in plants. Our manuscript reports the biochemical and cell biological characterization of the small ARF-like GTPase TTN5 from the model plant Arabidopsis thaliana*. Fundamental investigations like ours are mostly lacking for ARF and ARL GTPases in Arabidopsis. *

We employed fluorescence-based enzymatic assays suited to uncover different types of the very rapid GTPase activities for TTN5. The experimental findings are now illustrated in a more comprehensive modified Figure 2 and in the form of a summary of the GTPase activities for TTN5 and its mutant variants in the NEW Figure 7A in the Discussion part. Taken together, we found that TTN5 is a non-classical GTPase based on its enzymatic kinetics. The reviewers appreciated these findings and highlighted them as being „impressive in vitro biochemical characterization" and "major conceptual advance". Since such experiments are "uncommon" for being conducted with plant GTPases, reviewers regarded this analysis as "useful addition to the plant community in general". The significance of these findings is given by the circumstance that „the ARF-like proteins are poorly addressed in Arabidopsis while they could reveal completely different function than the canonical known ARF proteins". Reviewers saw here clearly a "strength" of the manuscript.

With regard to the cell biological investigation and initial assessment of cell physiological roles of TTN5, we now provide requested additional evidence. First of all, we provide NEW data on the localization of TTN5 by immunolocalization using a complementing HA3-TTN5 construct, supporting our initial suggestions that TTN5 may be associated with vesicles and processes of the endomembrane system. The previous preprint version had left the reviewers „less convinced" of cell biological data due to the lack of complementation of our YFP-TTN5 construct, lack of Western blot data and the low resolution of microscopic images. We fully agree that these points were of concern and needed to be addressed. We have therefore intensively worked on these „weaknesses" and present now a more detailed whole-mount immunostaining series with the complementing HA3-TTN5 transgenic line (NEW Figure 4, NEW Figure 3P), Western blot data (NEW Supplementary Figures S7C and D), and we will provide all original images upon publication of our manuscript at BioImage Archives which will provide the high quality for re-analysis. BioImage Archives is an online storage for biological image data associated with a peer-reviewed publication. This way, readers will be able to inspect each image in detail. The immunolocalization data are of particular importance as they indicate that HA3-TTN5 can be associated with punctate vesicle structures and BFA bodies as seen with YFP studies of YFP-TTN5 seedlings. We have re-phrased very carefully and emphasized those localization patterns which are backed up by immunostaining and YFP fluorescence detection of YFP-TTN5 signals. To improve the comprehension, the findings are summarized in a schematic overview in NEW Figure 7B of the Discussion. We have also addressed all other comments related to the cell biological experiments to "provide the substantial improvement" that had been requested. We emphasize that we found two cell physiological phenotypes for the TTN5T30N mutant. YFP-TTN5T30N confers phenotypes, which are differing mobility of the fluorescent vesicles in the epidermis of hypocotyls (see Video material and NEW Supplementary Video Material S1M-O), and a root growth phenotype of transgenic HA3-TTN5T30N seedlings (NEW Figure 3O). We explain the cell physiological phenotypes in relation to enzymatic GTPase data. These findings convince us of the validity of the YFP-TTN5 analysis indicative of TTN5 localization.

*We are deeply thankful to the reviewers for judging our manuscript as "generally well written", "important" and "of interest to a wide range of plant scientists" and "for scientists working in the trafficking field" as it "holds significance" and will form the basis for future functional studies of TTN5. *

We prepared very carefully our revised manuscript in which we address all reviewer comments one by one. Please find our revision and our detailed rebuttal to all reviewer comments below. Changes in the revised version are highlighted by yellow and green color. In the "revised version with highlighted changes".

With these adjustments, we hope that our peer-reviewed study will receive a positive response.

We are looking forward to your evaluation of our revised manuscript and thank you in advance,

Sincerely

Petra Bauer and Inga Mohr on behalf of all authors

*

• *

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

The manuscript from Mohr and collaborators reports the characterization of an ARF-like GTPase of Arabidopsis. Small GTPases of the ARF family play crucial role in intracellular trafficking and plant physiology. The ARF-like proteins are poorly addressed in Arabidopsis while they could reveal completely different function than the canonical known ARF proteins. Thus, the aim of the study is important and could be of interest to a wide range of plant scientists. I am impressed by the biochemical characterization of the TTN5 protein and its mutated versions, this is clearly a very nice point of the paper and allows for proper interpretations of the other results. However, I was much less convinced on the cell biology part of this manuscript and aside from the subcellular localization of the TTN5 I think the paper would benefit from a more functional angle. Below are my comments to improve the manuscript:

1- In the different pictures and movies, TTN5 is quite clearly appearing as a typical ER-like pattern. The pattern of localization further extends to dotty-like structures and structures labeled only at the periphery of the structure, with a depletion of fluorescence inside the structure. These observations raise several points. First, the ER pattern is never mentioned in the manuscript while I think it can be clearly observed. Given that the YFP-TTN5 construct is not functional (the mutant phenotype is not rescued) the ER-localization could be due to the retention at the ER due to quality control. The HA-TTN5 construct is functional but to me its localization shows a quite different pattern from the YFP version, I do not see the ER for example or the periphery-labeled structures. In this case, it will be a crucial point to perform co-localization experiments between HA-TTN5 and organelles markers to confirm that the functional TTN5 construct is labeling the Golgi and MVBs, as does the non-functional one. I am also quite sure that a co-localization between YFP-TTN5 and HA-TTN5 will not completely match... The ER is contacting so many organelles that the localization of YFP-TTN5 might not reflects the real location of the protein.

__Our response: __

At first, we like to state that specific detection of intracellular localization of plant proteins in plant cells is generally technically very difficult, when the protein abundance is not overly high. In this revised version, we extended immunostaining analysis to different membrane compartments, including now immunostaining of complementing HA3-TTN5 in the absence and presence of BFA, along with immunodetection of ARF1 and FM4-64 labeling in roots (NEW Figure 3P, NEW Figure 4A, B). In the revised version, we focus the analysis and conclusions on the fluorescence patterns that overlap between YFP-TTN5 detection and HA3-TTN5 immunodetection. With this, we can be most confident about subcellular TTN5 localization. Please find this NEW text in the Result section (starting Line 323):

„For a more detailed investigation of HA3-TTN5 subcellular localization, we then performed co-immunofluorescence staining with an Alexa 488-labeled antibody recognizing the Golgi and TGN marker ARF1, while detecting HA3-TTN5 with an Alexa 555-labeled antibody (Robinson et al. 2011, Singh et al. 2018) (Figure 4A). ARF1-Alexa 488 staining was clearly visible in punctate structures representing presumably Golgi stacks (Figure 4A, Alexa 488), as previously reported (Singh et al. 2018). Similar structures were obtained for HA3-TTN5-Alexa 555 staining (Figure 4A, Alexa 555). But surprisingly, colocalization analysis demonstrated that the HA3-TTN5-labeled structures were mostly not colocalizing and thus distinct from the ARF1-labeled ones (Figure 4A). Yet the HA3-TTN5- and ARF1-labeled structures were in close proximity to each other (Figure 4A). We hypothesized that the HA3-TTN5 structures can be connected to intracellular trafficking steps. To test this, we performed brefeldin A (BFA) treatment, a commonly used tool in cell biology for preventing dynamic membrane trafficking events and vesicle transport involving the Golgi. BFA is a fungal macrocyclic lactone that leads to a loss of cis-cisternae and accumulation of Golgi stacks, known as BFA-induced compartments, up to the fusion of the Golgi with the ER (Ritzenthaler et al. 2002, Wang et al. 2016). For a better identification of BFA bodies, we additionally used the dye FM4-64, which can emit fluorescence in a lipophilic membrane environment. FM4-64 marks the plasma membrane in the first minutes following application to the cell, then may be endocytosed and in the presence of BFA become accumulated in BFA bodies (Bolte et al. 2004). We observed BFA bodies positive for both, HA3-TTN5-Alexa 488 and FM4-64 signals (Figure 4B). Similar patterns were observed for YFP-TTN5-derived signals in YFP-TTN5-expressing roots (Figure 4C). Hence, HA3-TTN5 and YFP-TTN5 can be present in similar subcellular membrane compartments."

We did not find evidence that HA3-TTN5 can localize at the ER using whole-mount immunostaining (NEW Figure 3P; NEW Figure 4A, B). Hence, we are careful with describing that fluorescence at the ER, as seen in the YFP-TTN5 line (Figure 3M, N) reflects TTN5 localization. We therefore do not focus the text on the ER pattern in the Result section (starting Line 295):

„Additionally, YFP signals were also detected in a net-like pattern typical for ER localization (Figure 3M, N). (...) We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

*And we discuss in the Discussion section (starting Line 552): *

„We based the TTN5 localization data on tagging approaches with two different detection methods to enhance reliability of specific protein detection. Even though YFP-TTN5 did not complement the embryo-lethality of a ttn5 loss of function mutant, we made several observations that suggest YFP-TTN5 signals to be meaningful at various membrane sites. We do not know why YFP-TTN5 does not complement. There could be differences in TTN5 levels and interactions in some cell types, which were hindering specifically YFP-TTN5 but not HA3-TTN5. (...) Though constitutively driven, the YFP-TTN5 expression may be delayed or insufficient at the early embryonic stages resulting in the lack of embryo-lethal complementation. On the other hand, the very fast nucleotide exchange activity may be hindered by the presence of a large YFP-tag in comparison with the small HA3-tag which is able to rescue the embryo-lethality. The lack of complementation represents a challenge for the localization of small GTPases with rapid nucleotide exchange in plants. Despite of these limitations, we made relevant observations in our data that made us believe that YFP signals in YFP-TTN5-expressing cells at membrane sites can be meaningful."

2- What are the structures with TTN5 fluorescence depleted at the center that appear in control conditions? They look different from the Golgi labeled by Man1 but similar to MVBs upon wortmannin treatment, except that in control conditions MVBs never appear like this. Are they related to any kind of vacuolar structures that would be involved in quality control-induced degradation of non-functional proteins?

Our response:

The reviewer certainly refers to fluorescence images from N. benthamiana leaf epidermal cells where different circularly shaped structures are visible. In these respective structures, the fluorescent circles are depleted from fluorescence in the center, e.g. in Figure 5C, YFP- fluorescent signals in TTN5T30N transformed leaf discs. We suspect that these structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al., 2020 for ANNI-GFP (reference in manuscript). The reviewer certainly does not refer to swollen MVBs that are seen following wortmannin treatment, as in Figure 5N-P, which look similar in their shape but are larger in size. Please note that we always included the control conditions, namely the images recorded before the wortmannin treatment, so that we were able to investigate the changes induced by wortmannin. Hence, we can clearly say that the structures with depleted fluorescence in the center as in Figure 5C are not wortmannin-induced swollen MVBs.To make these points clear to the reader, we added an explanation into the text (Line 385-388):

„We also observed YFP fluorescence signals in the form of circularly shaped ring structures with a fluorescence-depleted center. These structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al. (2020) for ANNI-GFP."

3- The fluorescence at nucleus could be due to a proportion of YFP-TTN5 that is degraded and released free-GFP, a western-blot of the membrane fraction vs the cytosolic fraction could help solving this issue.

Our response:

In an α-GFP Western blot using YFP-TTN5 Arabidopsis seedlings, we detected besides the expected and strong 48 kDa YFP-TTN5 band, three additional weak bands ranging between 26 to 35 kDa (NEW Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins expressed from aberrant transcripts. α-HA Western blot controls performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size (Supplementary Figure S7D). We must therefore be cautious about nuclear TTN5 localization and we rephrased the text carefully (starting Line 300):

„We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

4- It is not so easy to conclude from the co-localization experiments. The confocal pictures are not always of high quality, some of them appear blurry. The Golgi localization looks convincing, but the BFA experiments are not that clear. The MVB localization is pretty convincing but the images are blurry. An issue is the quantification of the co-localizations. Several methods were employed but they do not provide consistent results. As for the object-based co-localization method, the authors employ in the text co-localization result either base on the % of YFP-labeled structures or the % of mCherry/mRFP-labeled structures, but the results are not going always in the same direction. For example, the proportion of YFP-TTN5 that co-localize with MVBs is not so different between WT and mutated version but the proportion of MVBs that co-localize with TTN5 is largely increased in the Q70L mutant. Thus it is quite difficult to interpret homogenously and in an unbiased way these results. Moreover, the results coming from the centroid-based method were presented in a table rather than a graph, I think here the authors wanted to hide the huge standard deviation of these results, what is the statistical meaning of these results?

Our response:

First of all, we like to point out that, as explained above, the BFA experiments are now more clear. We performed additional BFA treatment coupled with immunostaining using HA3-TTN5-expressing Arabidopsis seedlings and coupled with fluorescence analysis using YFP-TTN5-expressing Arabidopsis plants. In both experiments, we observed the typical BFA bodies very clearly (NEW Figure 4B, C).

Second, we like to insist that we performed colocalization very carefully and quantified the data in three different manners. We like to state that there is no general standardized procedure that best suits the idea of a colocalization pattern. Results of colocalization are represented in stem diagrams and table format, including statistical analysis. Colocalization was carried out with the ImageJ plugin JACoP for Pearson's and Overlap coefficients and based on the centroid method. The plotted Pearson's and Overlap coefficients are presented in bar diagrams in Supplementary Figure S8A and C, including statistics. The obtained values by the centroid method are represented in table format in Supplementary Figure S8B and D, which *can be considered a standard method (see Ivanov et al., 2014). *

Colocalization of two different fluorescence signals was performed for the two channels in a specific chosen region of interest (indicating in % the overlapping signal versus the sum of signal for each channel). The differences between the YFP/mRFP and mRFP/YFP ratios indicate that a higher percentage of ARA7-RFP signal is colocalizing with YFP-TTN5Q70L signal than with the TTN5WT or the TTN5T30N mutant form signals, while the YFP signals have a similar overlap with ARA7-positive structures. This is not a contradiction. Presumably this answers well the questions on colocalization.

Please note that upon acceptance for publication, we will upload all original colocalization data to BioImage Archive. Hence, the high-quality data can be reanalyzed by readers.

5- The use of FM4-64 to address the vacuolar trafficking is a hazardous, FM4-64 allows the tracking of endocytosis but does not say anything on vacuolar degradation targeting and even less on the potential function of TTN5 in endosomal vacuolar targeting. Similarly, TTN5, even if localized at the Golgi, is not necessarily function in Golgi-trafficking. __Our response: __

*Perhaps our previous description was misleading. Thank you for pointing this out. We reformulated the text and modified the schematic representation of FM4-64 in NEW Figure 6A: *

"(A), Schematic representation of progressive stages of FM4-64 localization and internalization in a cell. FM4-64 is a lipophilic substance. After infiltration, it first localizes in the plasma membrane, at later stages it localizes to intracellular vesicles and membrane compartments. This localization pattern reflects the endocytosis process (Bolte et al. 2004)."

6- The manuscript lacks in its present shape of functional evidences for a role of TTN5 in any trafficking steps. I understand that the KO mutant is lethal but what are the phenotypes of the Q70L and T30N mutant plants? What is the seedling phenotype, how are the Golgi and MVBs looking like in these mutants? Do the Q70L or T30N mutants perturbed the trafficking of any cargos?

__Our response: __

*We agree fully that functional evidences are interesting to assign roles for TTN5 in trafficking steps. A phenotype associated with TTN5T30N and TTN5Q70L is clearly meaningful. *

First of all, we like to emphasize that it is incorrect that the manuscript lacks functional evidences for a role of TTN5 and the two mutants. In fact, the manuscript even highlights several functional activities that are meaningful in a cellular context. These include different types of kinetic GTPase enzyme activities, subcellular localization in planta and association with different endomembrane compartments and subcellular processes such as endocytosis. We surely agree that future research can focus even more on cell physiological aspects and the physiological functions in plants to examine the proposed roles of TTN5 in intracellular trafficking steps. For such studies, our findings are the fundamental basis.

Concerning the aspect of colocalization of the mutants with the markers we show in Figure 5C, D and G, H that YFP-TTN5T30N- and YFP-TTN5Q70L-related signals colocalize with the Golgi marker GmMan1-mCherry. Figure 5K, L and O, P show that YFP-TTN5T30N and YFP-TTN5Q70L-related signals can colocalize with the MVB marker, and this may affect relevant vesicle trafficking processes and plasma membrane protein regulation involved in root cell elongation.

*At present, we have not yet investigated perturbed cargo trafficking. These aspects are certainly interesting but require extensive work and testing of appropriate physiological conditions and appropriate cargo targets. We discuss future perspectives in the Discussion. We agree that such functional information is of great importance, but needs to be clarified in future studies. *

__Reviewer #1 (Significance (Required)): __

In conclusion, I think this manuscript is a good biochemical description of an ARF-like protein but it would need to be strengthen on the cell biology and functional sides. Nonetheless, provided these limitations fixed, this manuscript would advance our knowledge of small GTPases in plants. The major conceptual advance of that study is to provide a non-canonical behavior of the active/inactive cycle dynamics for a small-GTPase. Of course this dynamic probably has an impact on TTN5 function and involvement in trafficking, although this remains to be fully demonstrated. Provided a substantial amount of additional experiments to support the claims of that study, this study could be of general interest for scientist working in the trafficking field.

__Our response: __

We thank reviewer 1 for the very fruitful comments. We hope that with the additional experiments, NEW Figures and NEW Supplementary Figures as well as our changes in the text, all comments by the reviewer have been addressed.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

The manuscript by Mohr and colleagues characterizes the Arabidopsis predicted small GTPase TITAN5 in both biochemical and cell biology contexts using in vitro and in planta techniques. In the first half of the manuscript, the authors use in vitro nucleotide exchange assays to characterise the GTPase activity and nucleotide binding properties of TITAN5 and two mutant variants of it. The in vitro data they produce indicates that TITAN5 does indeed have general GTPase and nucleotide binding capability that would be expected for a protein predicted to be a small GTPase. Interestingly, the authors show that TITAN5 favors a GTP-bound form, which is different to many other characterized GTPases that favor GDP-binding. The authors follow their biochemical characterisation of TITAN with in planta experiments characterizing TITAN5 and its mutant variants association with the plant endomembrane system, both by stable expression in Arabidopsis and transient expression in N.benthamiana.

The strength of this manuscript is in its in vitro biochemical characterisation of TITAN5 and variants. I am not an expert on in vitro GTPase characterisation and so cannot comment specifically on the assays they have used, but generally speaking this appears to have been well done, and the authors are to be commended for it. In vitro characterisation of plant small GTPases is uncommon, and much of our knowledge is inferred for work on animal or yeast GTPases, so this will be a useful addition to the plant community in general, especially as TITAN5 is an essential gene. The in planta data that follows is sadly not as compelling as the biochemical data, and suffers from several weaknesses. I would encourage the authors to consider trying to improve the quality of the in planta data in general. If improved and then combined with the biochemical aspects of the paper, this has the potential to make a nice addition to plant small GTPase and endomembrane literature.

The manuscript is generally well written and includes the relevant literature.

Major issues:

1. The authors make use of a p35s: YFP-TTN5 construct (and its mutant variants) both stably in Arabidopsis and transiently in N.benthamiana. I know from personal experience that expressing small GTPases from non-endogenous promoters and in transient expression systems can give very different results to when working from endogenous promoters/using immunolocalization in stable expression systems. Strong over-expression could for example explain why the authors see high 'cytosolic' levels of YFP-TTN5. It is therefore questionable how much of the in planta localisation data presented using p35S and expression in tobacco is of true relevance to the biological function of TITAN5. The authors do present some immunolocalization data of HA3-TTN5 in Arabidopsis, but this is fairly limited and it is very difficult in its current form to use this to identify whether the data from YFP-TTN5 in Arabidopsis and tobacco can be corroborated. I would encourage the authors to consider expanding the immunolocalization data they present to validate their findings in tobacco. __Our response: __

We are aware that endogenous promoters may be preferred over 35S promoter. However, the two types of lines we generated with endogenous promoter did both not show fluorescent signals so that we could unfortunately not use them (not shown). Besides 35S promoter-mediated expression we were also investigating inducible expression vectors for fluorescence imaging in N. benthamiana (not shown). Both inducible and constitutive expression showed very similar expression patterns so that we chose characterizing in detail the 35S::YFP-TTN5 fluorescence in both N. bethamiana*and Arabidopsis. *

We have expanded immunolocalization using the HA3-TTN5 line and compare it now along with YFP fluorescence signal in YFP-TTN5 seedlings (NEW Figure 3P; NEW Figure 4).

„For a more detailed investigation of HA3-TTN5 subcellular localization, we then performed co-immunofluorescence staining with an Alexa 488-labeled antibody recognizing the Golgi and TGN marker ARF1, while detecting HA3-TTN5 with an Alexa 555-labeled antibody (Robinson et al. 2011, Singh et al. 2018) (Figure 4A). ARF1-Alexa 488 staining was clearly visible in punctate structures representing presumably Golgi stacks (Figure 4A, Alexa 488), as previously reported (Singh et al. 2018). Similar structures were obtained for HA3-TTN5-Alexa 555 staining (Figure 4A, Alexa 555). But surprisingly, colocalization analysis demonstrated that the HA3-TTN5-labeled structures were mostly not colocalizing and thus distinct from the ARF1-labeled ones (Figure 4A). Yet the HA3-TTN5- and ARF1-labeled structures were in close proximity to each other (Figure 4A). We hypothesized that the HA3-TTN5 structures can be connected to intracellular trafficking steps. To test this, we performed brefeldin A (BFA) treatment, a commonly used tool in cell biology for preventing dynamic membrane trafficking events and vesicle transport involving the Golgi. BFA is a fungal macrocyclic lactone that leads to a loss of cis-cisternae and accumulation of Golgi stacks, known as BFA-induced compartments, up to the fusion of the Golgi with the ER (Ritzenthaler et al. 2002, Wang et al. 2016). For a better identification of BFA bodies, we additionally used the dye FM4-64, which can emit fluorescence in a lipophilic membrane environment. FM4-64 marks the plasma membrane in the first minutes following application to the cell, then may be endocytosed and in the presence of BFA become accumulated in BFA bodies (Bolte et al. 2004). We observed BFA bodies positive for both, HA3-TTN5-Alexa 488 and FM4-64 signals (Figure 4B). Similar patterns were observed for YFP-TTN5-derived signals in YFP-TTN5-expressing roots (Figure 4C). Hence, HA3-TTN5 and YFP-TTN5 can be present in similar subcellular membrane compartments."

• *

Many of the confocal images presented are of poor quality, particularly those from N.benthamiana.

Our response:

All confocal images are of high quality in their original format. To make them accessible, we will upload all raw data to BioImage Archive upon acceptance of the manuscript.

The authors in some places see YFP-TTN5 in cell nuclei. This could be a result of YFP-cleavage rather than genuine nuclear localisation of YFP-TTN5, but the authors do not present western blots to check for this.

__Our response: __

As described in our response to reviewer 1, comment 3, Fluorescence signals were detected within the nuclei of root cells of YFP-TTN5 plants, while immunostaining signals of HA3-TTN5 were not detected in the nucleus. In an α-GFP Western blot using YFP-TTN5 Arabidopsis seedlings, we detected besides the expected and strong 48 kDa YFP-TTN5 band, three additional weak bands ranging between 26 to 35 kDa (NEW Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins expressed from aberrant transcripts. α-HA Western blot controls performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size (Supplementary Figure S7D). We must therefore be cautious about nuclear TTN5 localization and we rephrased the text carefully (starting Line 300):

• *

„We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

That YFP-TTN5 fails to rescue the ttn5 mutant indicates that YFP-tagged TTN5 may not be functional. If the authors cannot corroborate the YFP-TTN5 localisation pattern with that of HA3-TTN5 via immunolocalization, then the fact that YFP-TTN5 may not be functional calls into question the biological relevance of YFP-TTN5's localisation pattern.

__Our response: __

This refers to your comment 1, please check this comment for a detailed response. Please also see our answer to reviewer 1, comment 1.

At first, we like to state that specific detection of intracellular localization of plant proteins in plant cells is generally technically very difficult, when the protein abundance is not overly high. In this revised version, we extended immunostaining analysis to different membrane compartments, including now immunostaining of complementing HA3-TTN5 in the absence and presence of BFA, along with immunodetection of ARF1 and FM4-64 labeling in roots (NEW Figure 3P, NEW Figure 4A, B). In the revised version, we focus the analysis and conclusions on the fluorescence patterns that overlap between YFP-TTN5 detection and HA3-TTN5 immunodetection. With this, we can be most confident about subcellular TTN5 localization. Please find this NEW text in the Result section (starting Line 323):

„For a more detailed investigation of HA3-TTN5 subcellular localization, we then performed co-immunofluorescence staining with an Alexa 488-labeled antibody recognizing the Golgi and TGN marker ARF1, while detecting HA3-TTN5 with an Alexa 555-labeled antibody (Robinson et al. 2011, Singh et al. 2018) (Figure 4A). ARF1-Alexa 488 staining was clearly visible in punctate structures representing presumably Golgi stacks (Figure 4A, Alexa 488), as previously reported (Singh et al. 2018). Similar structures were obtained for HA3-TTN5-Alexa 555 staining (Figure 4A, Alexa 555). But surprisingly, colocalization analysis demonstrated that the HA3-TTN5-labeled structures were mostly not colocalizing and thus distinct from the ARF1-labeled ones (Figure 4A). Yet the HA3-TTN5- and ARF1-labeled structures were in close proximity to each other (Figure 4A). We hypothesized that the HA3-TTN5 structures can be connected to intracellular trafficking steps. To test this, we performed brefeldin A (BFA) treatment, a commonly used tool in cell biology for preventing dynamic membrane trafficking events and vesicle transport involving the Golgi. BFA is a fungal macrocyclic lactone that leads to a loss of cis-cisternae and accumulation of Golgi stacks, known as BFA-induced compartments, up to the fusion of the Golgi with the ER (Ritzenthaler et al. 2002, Wang et al. 2016). For a better identification of BFA bodies, we additionally used the dye FM4-64, which can emit fluorescence in a lipophilic membrane environment. FM4-64 marks the plasma membrane in the first minutes following application to the cell, then may be endocytosed and in the presence of BFA become accumulated in BFA bodies (Bolte et al. 2004). We observed BFA bodies positive for both, HA3-TTN5-Alexa 488 and FM4-64 signals (Figure 4B). Similar patterns were observed for YFP-TTN5-derived signals in YFP-TTN5-expressing roots (Figure 4C). Hence, HA3-TTN5 and YFP-TTN5 can be present in similar subcellular membrane compartments."

We did not find evidence that HA3-TTN5 can localize at the ER using whole-mount immunostaining (NEW Figure 3P; NEW Figure 4A, B). Hence, we are careful with describing that fluorescence at the ER, as seen in the YFP-TTN5 line (Figure 3M, N) reflects TTN5 localization. We therefore do not focus the text on the ER pattern in the Result section (starting Line 295):

„Additionally, YFP signals were also detected in a net-like pattern typical for ER localization (Figure 3M, N). (...) We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

*And we discuss in the Discussion section (starting Line 552): *

„We based the TTN5 localization data on tagging approaches with two different detection methods to enhance reliability of specific protein detection. Even though YFP-TTN5 did not complement the embryo-lethality of a ttn5 loss of function mutant, we made several observations that suggest YFP-TTN5 signals to be meaningful at various membrane sites. We do not know why YFP-TTN5 does not complement. There could be differences in TTN5 levels and interactions in some cell types, which were hindering specifically YFP-TTN5 but not HA3-TTN5. (...) Though constitutively driven, the YFP-TTN5 expression may be delayed or insufficient at the early embryonic stages resulting in the lack of embryo-lethal complementation. On the other hand, the very fast nucleotide exchange activity may be hindered by the presence of a large YFP-tag in comparison with the small HA3-tag which is able to rescue the embryo-lethality. The lack of complementation represents a challenge for the localization of small GTPases with rapid nucleotide exchange in plants. Despite of these limitations, we made relevant observations in our data that made us believe that YFP signals in YFP-TTN5-expressing cells at membrane sites can be meaningful."

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Without a cell wall label/dye, the plasmolysis data presented in Figure 5 is hard to visualize.

__Our response: __

Figure 6E-G (previously Fig. 5) show the results of plasmolysis experiments with YFP-TTN5 and the two mutant variant constructs. It is clearly possible to observe plasmolysis when focusing on the Hechtian strands. Hechtian strands are formed due to the retraction of the protoplast as a result of the osmotic pressure by the added mannitol solution. Hechtian strands consist of PM which remained in contact with the cell wall, visible as thin filamental structures. We stained the PM and the Hechtian strands by the PM dye FM4-64. This is similary done in Yoneda et al., 2020. We could detect in the YFP-TTN5-transformed cells, colocalization with the YFP channels and the PM dye in filamental structures between two neighbouring FM4-64-labelled PMs. Although an additional labeling of the cell wall may further indicate plasmolysis, it is not needed here.

Please consider that we will upload all original image data to BioImage Archive so that a detailed re-investigation of the images can be done.

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__Minor issues: __

In some of the presented N.benthamiana images, it looks like YFP-TTN5 may be partially ER-localised. However, co-localisation with an ER marker is not presented.

Our response:

*Referring to our response to comments 1 and 3 of reviewer 2 and to comment 1 of reviewer 1: *

We did not find evidence that HA3-TTN5 can localize at the ER using whole-mount immunostaining (NEW Figure 3P; NEW Figure 4A, B). Hence, we are careful with describing that fluorescence at the ER, as seen in the YFP-TTN5 line (Figure 3M, N) reflects TTN5 localization. We therefore do not focus the text on the ER pattern in the Result section (starting Line 295):

„Additionally, YFP signals were also detected in a net-like pattern typical for ER localization (Figure 3M, N). (...) We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

*And we discuss in the Discussion section (starting Line 552): *

„We based the TTN5 localization data on tagging approaches with two different detection methods to enhance reliability of specific protein detection. Even though YFP-TTN5 did not complement the embryo-lethality of a ttn5 loss of function mutant, we made several observations that suggest YFP-TTN5 signals to be meaningful at various membrane sites. We do not know why YFP-TTN5 does not complement. There could be differences in TTN5 levels and interactions in some cell types, which were hindering specifically YFP-TTN5 but not HA3-TTN5. (...) Though constitutively driven, the YFP-TTN5 expression may be delayed or insufficient at the early embryonic stages resulting in the lack of embryo-lethal complementation. On the other hand, the very fast nucleotide exchange activity may be hindered by the presence of a large YFP-tag in comparison with the small HA3-tag which is able to rescue the embryo-lethality. The lack of complementation represents a challenge for the localization of small GTPases with rapid nucleotide exchange in plants. Despite of these limitations, we made relevant observations in our data that made us believe that YFP signals in YFP-TTN5-expressing cells at membrane sites can be meaningful."

• *

There is some inconsistency within the N.benthamiana images. For example, compare Figure 4C of YFP-TTN5T30N to Figure 4O of YFP-TTN5T30N. Figure 4O is presented as being significant because wortmannin-induced swollen ARA7 compartments are labelled by YFP-TTN5T30N. However, structures very similar to these can already been seen in Figure 4C, which is apparently an unrelated experiment. This, to my mind, is likely a result of the very different expression levels between different cells that can be produced by transient expression in N.benthamiana.

__Our response: __

Former Figure 4 is now Figure 5. As detailed in our response to comment 2 of reviewer 1:

The reviewer certainly refers to fluorescence images from N. benthamiana leaf epidermal cells where different circularly shaped structures are visible. In these respective structures, the fluorescent circles are depleted from fluorescence in the center, e.g. in Figure 5C, YFP- fluorescent signals in TTN5T30N transformed leaf discs. We suspect that these structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al., 2020 for ANNI-GFP (reference in manuscript). The reviewer certainly does not refer to swollen MVBs that are seen following wortmannin treatment, as in Figure 5N-P, which look similar in their shape but are larger in size. Please note that we always included the control conditions, namely the images recorded before the wortmannin treatment, so that we were able to investigate the changes induced by wortmannin. Hence, we can clearly say that the structures with depleted fluorescence in the center as in Figure 5C are not wortmannin-induced swollen MVBs.To make these points clear to the reader, we added an explanation into the text (Line 385-388):

„We also observed YFP fluorescence signals in the form of circularly shaped ring structures with a fluorescence-depleted center. These structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al. (2020) for ANNI-GFP."

**Referees cross-commenting**

It sems that all of the reviewers have converged on the conclusion that the in planta characterisation of TTN5 is insufficient to be of substantial interest to the field, highlighting the fact that major improvements are required to strengthen this part of the manuscript and increase its relevance.

__Reviewer #2 (Significance (Required)): __

General assessment: the strengths of this work are in its in vitro characterisation of TITAN5, however, the in planta characterisation lacks depth.

Significance: the in vitro characterisation of TITAN5 is commendable as such work is lacking for plant GTPases. However, the significance of the work would be boosted substantially by better in planta characterisation, which is where most the most broad interest will lie.

My expertise: my expertise is in in planta characterisation of small GTPases and their interactors.

__Our response: __

We thank the reviewer for the kind evaluation of our manuscript. We are confident that the changes in the text and NEW Figures and NEW Supplementary Figures will be convincing to consider our work.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

Summary: Cellular traffic is an important and well-studied biological process in animal and plant systems. While components involved in transport are known the mechanism by which these components control activity or destination remains to be studied. A critical step in regulating traffic is proper budding and tethering of vesicles. A critical component in determining this step is a family proteins with GTPase activity, which act as switches facilitating vesicle interaction between proteins, or cytoskeleton. The current manuscript by Mohr and colleagues have characterized a small GTPase TITAN5 (TTN5) and identified two residues Gln70 and Thr30 in the protein which they propose to have functional roles. The authors catalogue the localization, GTP hydrolytic activity, and discuss putative functions of TTN5 and the mutants.

__Major comments: __

The core of the manuscript, which is descriptive characterization of TTN5, lies in reliably demonstrating putative roles. While the GTP hydrolysis rates are well-quantified (though the claims need to be toned down), the microscopy data especially the association of TTN5 with different endomembrane compartments is not convincing due to the quality (low resolution) of the figures submitted. The manuscript text is difficult to navigate due to repetition and inconsistency in the order that the mutants are referred. I am requesting additional experiments which should be feasible considering the authors have all the materials required to perform the experiments and obtain high-quality images which support their claims.

In general the figure quality needs to be improved for all microscopy images. I would suggest that the authors highlight 1-2 individual cells to make their point and use the current images as supplementary to establish a broader spread. __Our response: __

*We have worked substantially on the text and figures to make the content well comprehensive. The mutants are referred to in a consistent manner in the text and figures. We have addressed requested experiments. *

As we pointed out in the cover letter and our responses to reviewers 1 and 2, we will upload all raw image data to BioImage Archive upon acceptance of the manuscript so that they can be re-examined without any reduction of resolution. Furthermore, we have conducted new experiments on immunolocalization of HA3-TTN5 (NEW Figure 3P, NEW Figure 4A, B). The text has been improved in several places (see highlighted changes in the manuscript and as detailed in the responses to reviewer 1. We think, this addresses well the reviewers' concerns.

Fig. S1 lacks clarity. __Our response: __

Supplementary Figure S1 shows TTN5 gene expression in different organs and growing stages as revealed by transcriptomic data, made available through the AtGenExpress eFB tool of the Bio-Analytic Resource for Plant Biology (BAR). The figure visualizes that TTN5 is ubiquitously expressed in different plant organs and tissues, e.g. the epidermis layers that we investigated here, and throughout development including embryo development. In accordance with the embryo-lethal phenotype, this highlights well that TTN5* is needed throughout for plant growth and it emphasizes that our investigation of TTN5 localization in epidermis cells is valid. *

We have added a better description to the figure legend. We now also mention the respective publications from which the transcriptome data-sets are derived. The modified figure legend is:

"Supplementary Figure S1. Visualization of TTN5 gene expression levels during plant development based on transcriptome data. Expression levels in (A), different types of aerial organs at different developmental stages; from left to right and bottom to top are represented different seed and plant growth stages, flower development stages, different leaves, vegetative to inflorescence shoot apex, embryo and silique development stages; (B), seedling root tissues based on single cell analysis represented in form of a uniform manifold approximation and projection plot; (C), successive stages of embryo development. As shown in (A) to (C), TTN5 is ubiquitously expressed in these different plant organs and tissues. In particular, it should be noted that TTN5 transcripts were detectable in the epidermis cell layer of roots that we used for localization of tagged TTN5 protein in this study. In accordance with the embryo-lethal phenotype, the ubiquitous expression of TTN5 highlights its importance for plant growth. Original data were derived from (Nakabayashi et al. 2005, Schmid et al. 2005) (A); (Ryu et al. 2019) (B); (Waese et al. 2017) (C). Gene expression levels are indicated by local maximum color code, ranging from the minimum (no expression) in yellow to the maximum (highest expression) in red."

For the supplementary videos, it is difficult to determine if punctate structures are moving or is it cytoplasmic streaming? Could this be done with a co-localized marker? Considering that such markers have been used later in Fig. 4? __Our response: __

We had detected movement of YFP fluorescent structures in all analyzed YFP-TTN5 plant parts except the root tip. Movement of fluorescence signals in YFP-TTN5T30N seedlings was slowed in hypocotyl epidermis cells. To answer the reviewer comment, we added three NEW supplemental videos (NEW Supplementary Video Material S1M-O) generated with all the three YFP-TTN5 constructs imaged over time in N. benthamiana leaf epidermal cells upon colocalization with the cis-Golgi marker GmMan1-mCherry as requested by the reviewer. In these NEW videos, some of *the YFP fluorescent spots seem to move together with the Golgi stacks. GmMan1 is described with a stop-and-go directed movement mediated by the actino-myosin system (Nebenführ 1999) and similarly it might be the case for YFP-TTN5 signals based on the colocalization. *

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It would be good if the speed of movement is quantified, if the authors want to retain the current claims in results and the discussion. __Our response: __

*We describe a difference in the movement of YFP fluorescent signal for the YFP-TTN5T30N variant in the hypocotyl compared to YFP-TTN5 and YFP-TTN5Q70L. In hypocotyl cells, we could observe a slowed down or arrested movement specifically of YFP-TTN5T30N fluorescent structures, and we describe this in the Results section (Line 278-291). *

"Interestingly, the mobility of these punctate structures differed within the cells when the mutant YFP-TTN5T30N was observed in hypocotyl epidermis cells, but not in the leaf epidermis cells (Supplementary Video Material S1E, compare with S1B) nor was it the case for the YFP-TTN5Q70L mutant (Supplementary Video Material S1F, compare with S1E)."

*The slowed movement in the YFP-TTN5T30N mutant is well visible even without quantification. We checked that the manuscript text does not contain overstatements in this regard. *

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Fig.2 I am not sure what the unit / scale is in Fig. 2D/E if each parameter (Kon, Koff, and Kd) are individually plotted? Could the authors please clarify/simplify this panel?

__Our response: __

We presented kinetics for nucleotide association (kon) and dissociation (koff) and the dissociation constant (Kd) in a bar diagram for each nucleotide, mdGDP (Figure 2D) and mGppNHp (Figure 2E). We modified and relabeled the bar diagram representation. It should be now very clear which are the parameters and units. Please see also the other modified figures (NEW modified Figure 2A-H). We also modified the legend of Figure 2D and E:

"(D-E), Kinetics of association and dissociation of fluorescent nucleotides mdGDP (D) or mGppNHp (E) with TTN5 proteins (WT, TTN5T30N, TTN5Q70L) are illustrated as bar charts. The association of mdGDP (0.1 µM) or mGppNHp (0.1 µM) with increasing concentration of TTN5WT, TTN5T30N and TTN5Q70L was measured using a stopped-flow device (see A, B; data see Supplementary Figure S3A-F, S4A-E). Association rate constants (kon in µM-1s-1) were determined from the plot of increasing observed rate constants (kobs in s-1) against the corresponding concentrations of the TTN5 proteins. Intrinsic dissociation rates (koff in s-1) were determined by rapidly mixing 0.1 µM mdGDP-bound or mGppNHp-bound TTN5 proteins with the excess amount of unlabeled GDP (see A, C, data see Supplementary Figure S3G-I, S4F-H). The nucleotide affinity (dissociation constant or Kd in µM) of the corresponding TTN5 proteins was calculated by dividing koff by kon. When mixing mGppNHp with nucleotide-free TTN5T30N, no binding was observed (n.b.o.) under these experimental conditions."

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Are panels D and E representing values for mdGDP and GppNHP? This is not very clear from the figure legend.

__Our response: __

Yes, Figure 2D and E represent the kon, koff and Kd values for mdGDP (Figure 2D) and mGppNHP (Figure 2E). As detailed in our previous response to comment 2a, we modified figure and figure legend to make the representation more clear.

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Fig. 3 Same comments as in para above - improve resolution fo images, concentrate on a few selected cells, if required use an inset figure to zoom-in to specific compartments. Our response:

As detailed in our responses to reviewers 1 and 2, we will upload all original image data to BioImage Archive upon acceptance of the manuscript, so that a detailed investigation of all our images is possible without any reduction of resolution.

Please provide the non-fluorescent channel images to understand cell topography __Our response: __

*We presented our microscopic images with the respective fluorescent channel and for colocalization with an additional merge. We did not present brightfield images as the cell topography was already well visible by fluorescent signal close to the PM. Therefore, brightfield images would not provide any benefit. Since we will upload all original data to BioImage Archive for a detailed investigation of all our images, the data can be obtained if needed. *

Is the nuclear localization seen in transient expression (panel L-N) an artefact? If so, this needs to be mentioned in the text. Our response:

As explained in our responses to reviewers 1 and 2, fluorescence signals were detected within the nuclei of root cells of YFP-TTN5 plants, while immunostaining signals of HA3-TTN5 were not detected in the nucleus.

In an α-GFP Western blot using YFP-TTN5 Arabidopsis seedlings, we detected besides the expected and strong 48 kDa YFP-TTN5 band, three additional weak bands ranging between 26 to 35 kDa (NEW Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins expressed from aberrant transcripts. α-HA Western blot controls performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size (Supplementary Figure S7D). We must therefore be cautious about nuclear TTN5 localization and we rephrased the text carefully (starting Line 300):

„We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

Fig. 4 - In addition to the points made for Fig. 3 The authors should consider reducing gain/exposure to improve image clarity. Especially for the punctate structures, which are difficult to observe in TTN5, likely because of the cytoplasmic localization as well.

__Our response: __

Thank you for this comment. We record image z-stacks and represent in single z-planes. Reducing the gain to decrease the cytoplasmic signal does not increase the clarity of the punctate structures as the signal strength will become weak.. As mentioned above, we will upload all original image data to BioImage Archive for a detailed investigation of all our images without any reduction of resolution.

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Reducing Agrobacterial load could be considered. OD of 0.4 is a bit much, 0.1 or even 0.05 could be tried. If available try expression in N. tabaccum, which is more amenable to microscopy. However, this is OPTIONAL, benthamiana should suffice. __Our response: __

Thank you for the suggestion. We are routinely using N. benthamiana leaf infiltration. When setting up this method at first, we did not observe different localization results by using different ODs of bacterial cultures. Hence, an OD600 of 0.4 is routinely used in our institute. This value is comparable with the literature although some literature reports even higher OD values for infiltration (Norkunas et al., 2018; Drapal et al., 2021; Zhang et al., 2020, Davis et al., 2020; Stephenson et al., 2018).

A standard norm now is to establish the level of colocalization is by quantifying a pearson's or Mander's correlation. Which I believe has been done in the text, I didn't find a plot representing the same? Could the data (which the authors already have) be plotted alongwith "n" as a table or graph? __Our response: __

*Please check our response to reviewer 1, comment 4. *

We like to insist that we performed colocalization very carefully and quantified the data in three different manners. We like to state that there is no general standardized procedure that best suits the idea of a colocalization pattern. Results of colocalization are represented in stem diagrams and table format, including statistical analysis. Colocalization was carried out with the ImageJ plugin JACoP for Pearson's and Overlap coefficients and based on the centroid method. The plotted Pearson's and Overlap coefficients are presented in bar diagrams in Supplementary Figure S8A and C, including statistics. The obtained values by the centroid method are represented in table format in Supplementary Figure S8B and D, which *can be considered a standard method (see Ivanov et al., 2014). *

Colocalization of two different fluorescence signals was performed for the two channels in a specific chosen region of interest (indicating in % the overlapping signal versus the sum of signal for each channel). The differences between the YFP/mRFP and mRFP/YFP ratios indicate that a higher percentage of ARA7-RFP signal is colocalizing with YFP-TTN5Q70L signal than with the TTN5WT or the TTN5T30N mutant form signals, while the YFP signals have a similar overlap with ARA7-positive structures. This is not a contradiction. Presumably this answers well the questions on colocalization.

Please note that upon acceptance for publication, we will upload all original colocalization data to BioImage Archive. Hence, the high-quality data can be reanalyzed by readers.

The cartoons for the action of chemicals are useful, but need a bit more clarity. Our response:

The schematic explanations of pharmacological treatments and expected outcomes are useful to readers. For a better understanding, we added additional explaining sentences to the figure legends (Figure 5E, M; Figure 6A). We also modified Figure 6A and the corresponding legend.

"(E), Schematic representation of GmMan1 localization at the ER upon brefeldin A (BFA) treatment. BFA blocks ARF-GEF proteins which leads to a loss of Golgi cis-cisternae and the formation of BFA-induced compartments due to an accumulation of Golgi stacks up to a redistribution of the Golgi to the ER by fusion of the Golgi with the ER (Renna and Brandizzi 2020)."

"(M), Schematic representation of ARA7 localization in swollen MVBs upon wortmannin treatment. Wortmannin inhibits phosphatidylinositol-3-kinase (PI3K) function leading to the fusion of TGN/EE to swollen MVBs (Renna and Brandizzi 2020)."

"(A), Schematic representation of progressive stages of FM4-64 localization and internalization in a cell. FM4-64 is a lipophilic substance. After infiltration, it first localizes in the plasma membrane, at later stages it localizes to intracellular vesicles and membrane compartments. This localization pattern reflects the endocytosis process (Bolte et al. 2004)."

• *

Fig. 5 does the Q70L mutant show reduced endocytosis ?

__Our response: __

We have not investigated this question. As detailed in our response to reviewer 1, *we like to emphasize that we agree fully that functional evidences are interesting to assign role for TTN5 in trafficking steps. A phenotype associated with TTN5T30N and TTN5Q70L would be clearly meaningful. *

Concerning the aspect of colocalization of the mutants with the markers we show in Figure 5C, D and G, H that YFP-TTN5T30N- and YFP-TTN5Q70L-related signals colocalize with the Golgi marker GmMan1-mCherry. Figure 5K, L and O, P show that YFP-TTN5T30N and YFP-TTN5Q70L-related signals can colocalize with the MVB marker, and this may affect relevant vesicle trafficking processes and plasma membrane protein regulation involved in root cell elongation.

*At present, we have not yet investigated perturbed cargo trafficking. These aspects are certainly interesting but require extensive work and testing of appropriate physiological conditions and appropriate cargo targets. We discuss future perspectives in the Discussion. We agree that such functional information is of great importance, but needs to be clarified in future studies. *

• *

The main text needs to be organized in a way that a reader can separate what is the hypothesis/assumption from actual results and conclusions (see lines #143-149).

Our response:

*Thank you for this comment. We reformulated text throughout the manuscript. *

The text is repeated in multiple places, while I understand that this is not plagiarism, the repetitiveness makes it difficult to read and understand the text. I highlight a couple of examples here, but please check the whole text thoroughly and edit/delete as necessary. a. Lines #124-125 with Lines #149-151 Lines #140-143

__Our response: __

*We checked the text and removed unnecessary repetitions. *

• *

• Could the authors elaborate on whether there are plan homologs of TTN5? Also, have other ARF/ARLs been compared to TTN5 beyond HsARF1? *

Our response:

Phylogenetic trees of the ARF family in Arabidopsis in comparison to human ARF family were already published by Vernoud et al. (2003). In this phylogenetic tree ARF, ARL and SAR proteins of Arabidopsis are compared with the members in humans and S. cervisiae. It is difficult to deduce whether the proteins are homologs or orthologs. In this setting, an ortholog of TTN5 may be HsARL2 followed by HsARL3. In Figure 1A we represented some human GTPases as closely related in sequence to TTN5, these are HsARL2, HsARF1 and AtARF1 since they are the best studied ARF GTPases. HRAS is a well-known member of the RAS superfamily which we used for kinetic comparison in Figure 2. We additionally compared published kinetics of RAC1, HsARF3, *CDC42, RHOA, ARF6, RAD, GEM, and RAS GTPases. *

• *

On a related note, a major problem I have with these kinetic values is the assumption of significance or not. For eg. Line#180 the values represent and 2 and 6-fold increase, if these numbers do not matter can a significance threshold be applied so as to understand how much fold-change is appreciable?

Our response:

The kinetics of TTN5 and its two mutant variants can be compared with those of other studied GTPases. To provide a basis for the statements about differences in GTPase activities, we modified the text and added respective references in the text for comparisons of fold changes.

The new text is now as follows Line 175-231):

„ We next measured the dissociation (koff) of mdGDP and mGppNHp from the TTN5 proteins in the presence of excess amounts of GDP and GppNHp, respectively (Figure 2C) and found interesting differences (Figure 2D, E; Supplementary Figures S3G-I, S4F-H). First, TTN5WT showed a koff value (0.012 s-1 for mGDP) (Figure 2D; Supplementary Figure S3G), which was 100-fold faster than those obtained for classical small GTPases, including RAC1 (Haeusler et al. 2006)and HRAS (Gremer et al. 2011), but very similar to the koff value of HsARF3 (Fasano et al. 2022). Second, the koffvalues for mGDP and mGppNHp, respectively, were in a similar range between TTN5WT (0.012 s-1 mGDP and 0.001 s-1mGppNHp) and TTN5Q70L (0.025 s-1 mGDP and 0.006 s-1 mGppNHp), respectively, but the koff values differed 10-fold between the two nucleotides mGDP and mGppNHp in TTN5WT (koff = 0.012 s-1 versus koff = 0.001 s-1; Figure 2D, E; Supplementary Figure S3G, I, S4F, H). Thus, mGDP dissociated from proteins 10-fold faster than mGppNHp. Third, the mGDP dissociation from TTN5T30N (koff = 0.149 s-1) was 12.5-fold faster than that of TTN5WT and 37-fold faster than the mGppNHp dissociation of TTN5T30N (koff = 0.004 s-1) (Figure 2D, E; Supplementary Figure S3H, S4G). Mutants of CDC42, RAC1, RHOA, ARF6, RAD, GEM and RAS GTPases, equivalent to TTN5T30N, display decreased nucleotide binding affinity and therefore tend to remain in a nucleotide-free state in a complex with their cognate GEFs (Erickson et al. 1997, Ghosh et al. 1999, Radhakrishna et al. 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al. 2003, Nassar et al. 2010, Huang et al. 2013, Chang and Colecraft 2015, Fisher et al. 2020, Shirazi et al. 2020). Since TTN5T30N exhibits fast guanine nucleotide dissociation, these results suggest that TTN5T30N may also act in either a dominant-negative or fast-cycling manner as reported for other GTPase mutants (Fiegen et al. 2004, Wang et al. 2005, Fidyk et al. 2006, Klein et al. 2006, Soh and Low 2008, Sugawara et al. 2019, Aspenström 2020).

The dissociation constant (Kd) is calculated from the ratio koff/kon, which inversely indicates the affinity of the interaction between proteins and nucleotides (the higher Kd, the lower affinity). Interestingly, TTN5WT binds mGppNHp (Kd = 0.029 µM) 10-fold tighter than mGDP (Kd = 0.267 µM), a difference, which was not observed for TTN5Q70L (Kd for mGppNHp = 0.026 µM, Kd for mGDP = 0.061 µM) (Figure 2D, E). The lower affinity of TTN5WT for mdGDP compared to mGppNHp brings us one step closer to the hypothesis that classifies TTN5 as a non-classical GTPase with a tendency to accumulate in the active (GTP-bound) state (Jaiswal et al. 2013). The Kd value for the mGDP interaction with TTN5T30N was 11.5-fold higher (3.091 µM) than for TTN5WT, suggesting that this mutant exhibited faster nucleotide exchange and lower affinity for nucleotides than TTN5WT. Similar as other GTPases with a T30N exchange, TTN5T30Nmay behave in a dominant-negative manner in signal transduction (Vanoni et al. 1999).

To get hints on the functionalities of TTN5 during the complete GTPase cycle, it was crucial to determine its ability to hydrolyze GTP. Accordingly, the catalytic rate of the intrinsic GTP hydrolysis reaction, defined as kcat, was determined by incubating 100 µM GTP-bound TTN5 proteins at 25{degree sign}C and analyzing the samples at various time points using a reversed-phase HPLC column (Figure 2F; Supplementary Figure S5). The determined kcat values were quite remarkable in two respects (Figure 2G). First, all three TTN5 proteins, TTN5WT, TTN5T30N and TTN5Q70L, showed quite similar kcatvalues (0.0015 s-1, 0.0012 s-1, 0.0007 s-1; Figure 2G; Supplementary Figure S5). The GTP hydrolysis activity of TTN5Q70L was quite high (0.0007 s-1). This was unexpected because, as with most other GTPases, the glutamine mutations at the corresponding position drastic impair hydrolysis, resulting in a constitutively active GTPase in cells (Hodge et al. 2020, Matsumoto et al. 2021). Second, the kcat value of TTN5WT (0.0015 s-1) although quite low as compared to other GTPases (Jian et al. 2012, Esposito et al. 2019), was 8-fold lower than the determined koff value for mGDP dissociation (0.012 s-1) (Figure 2E). This means that a fast intrinsic GDP/GTP exchange versus a slow GTP hydrolysis can have drastic effects on TTN5 activity in resting cells, since TTN5 can accumulate in its GTP-bound form, unlike the classical GTPase (Jaiswal et al. 2013). To investigate this scenario, we pulled down GST-TTN5 protein from bacterial lysates in the presence of an excess amount of GppNHp in the buffer using glutathione beads and measured the nucleotide-bound form of GST-TTN5 using HPLC. As shown in Figure 2H, isolated GST-TTN5 increasingly bonds GppNHp, indicating that the bound nucleotide is rapidly exchanged for free nucleotide (in this case GppNHp). This is not the case for classical GTPases, which remain in their inactive GDP-bound forms under the same experimental conditions (Walsh et al. 2019, Hodge et al. 2020)."

Another issue with the kinetic measurements is the significance levels. Line #198-201. The three proteins are claimed to have similar values and in the nnext line, the Q70L mutant is claimed to be high.

Our response:

Please see our response and changes in the text according in our response to the previous comment 9. We have provided extra explanations and references to clarify why the kinetic behavior of TTN5 is unusual in several respects (Line 215-220).

„First, all three TTN5 proteins, TTN5WT, TTN5T30N and TTN5Q70L, showed quite similar kcat values (0.0015 s-1, 0.0012 s-1, 0.0007 s-1; Figure 2G; Supplementary Figure S5). The GTP hydrolysis activity of TTN5Q70L was quite high (0.0007 s-1). This was unexpected because, as with most other GTPases, the glutamine mutations at the corresponding position drastic impair hydrolysis, resulting in a constitutively active GTPase in cells (Hodge et al. 2020, Matsumoto et al. 2021)."

Provide data for conclusion in line#214-215

Our response:

We agree that a reference should be added after this sentence to make this sentence clearer (Line 228-231).

"As shown in Figure 2H, isolated GST-TTN5 increasingly bonds GppNHp, indicating that the bound nucleotide is rapidly exchanged for free nucleotide (in this case GppNHp). This is not the case for classical GTPases, which remain in their inactive GDP-bound forms under the same experimental conditions (Walsh et al. 2019, Hodge et al. 2020)."

• *

How were the mutants studied here identified? random mutation or was it directed based on qualified assumptions?

__Our response: __

We used the T30N and the Q70L point mutations as such types of mutants had been reported to confer specific phenotypes in these well-conserved amino acid positions in multiple other small GTPases (Erickson et al. 1997, Ghosh et al. 1999, Radhakrishna et al. 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al. 2003, Nassar et al. 2010, Huang et al. 2013, Chang and Colecraft 2015, Fisher et al. 2020, Shirazi et al. 2020). In particular, these positions affect the interaction between small GTPases and their respective guanine nucleotide exchange factor (GEF; T30N) or on GTP hydrolysis (Q70L). We introduced the mutants and described their potential effect on the GTPase cycle in the introduction and cited exemplary literature. Please see also our response to comment 6 and the proposed text changes (Line 142-151).

Could more simplification be provided for deifitinition of Kon/Koff values. And can these values be compared between mutants directly?

__Our response: __

*We introduce kon and koff in the modified Figure 2D, E, and they are described in the figure legends. Moreover, we present the data for calculations in Supplementary Figures S3, 4, where again we define the values in the respective figure legends. *

• *

Data provided are not convincing to claim that both the mutant forms have lower association with the Golgi.

__Our response: __

*Our conclusion is that both YFP-TTN5 and YFP-TTN5Q70L fluorescence signals tend to colocalize more with the Golgi-marker signals compared to YFP-TTN5T30N signals as deduced from the centroid-based colocalization method (Line 404-405). *

"Hence, the GTPase-active TTN5 forms are likely more present at cis-Golgi stacks compared to TTN5T30N."

The Pearson coefficients of all three YFP-TTN5 constructs were nearly identical, but we could identify differences in overlapping centers between the YFP and mCherry channel. 48 % of the GmMan1-mCherry fluorescent cis-Golgi stacks were overlapping with signal of YFP-TTN5Q70L, while for YFP-TTN5T30N an overlap of only 31 % was detected. This means that less cis*-Golgi stacks colocalized with signals in the YFP-TTN5T30N mutant than in YFP-TTN5Q70L, which is the statement in our manuscript. *

• *

IN general the Authors should strongly consider the claims made in the manuscript. For eg. "This study lays the foundation for studying the functional relationships of this small GTPase" (line 125) is unqualified as this is true for every protein ever studied and published. Considering that TTN was not isolated/identified in this study for the first time this claim doesn't stand.

__Our response: __

*We reformulated the sentence (Line 123-124). *

"This study paves the way towards future investigation of the cellular and physiological contexts in which this small GTPase is functional."

• *

Line #185 - "characterestics of a dominant-negative...." What is this based on? From the text it is not clear what are the paremeters. Considering that no complementation phenotypes have been presented, this is a far-fetched claim Our response:

Small GTPases in general are a well studied protein family and the here used mutations T30N and Q70L are conserved amino acids and commonly used for the characterization of the Ras superfamily members. We added explaining sentences with references to the text. The characteristics referred to in the above paragraph is based on the kinetic study.

We modified the text as follows (Line 186-197 ):

„Third, the mGDP dissociation from TTN5T30N (koff = 0.149 s-1) was 12.5-fold faster than that of TTN5WT and 37-fold faster than the mGppNHp dissociation of TTN5T30N (koff = 0.004 s-1) (Figure 2D, E; Supplementary Figure S3H, S4G). Mutants of CDC42, RAC1, RHOA, ARF6, RAD, GEM and RAS GTPases, equivalent to TTN5T30N, display decreased nucleotide binding affinity and therefore tend to remain in a nucleotide-free state in a complex with their cognate GEFs (Erickson et al. 1997, Ghosh et al. 1999, Radhakrishna et al. 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al. 2003, Nassar et al. 2010, Huang et al. 2013, Chang and Colecraft 2015, Fisher et al. 2020, Shirazi et al. 2020). Since TTN5T30N exhibits fast guanine nucleotide dissociation, these results suggest that TTN5T30N may also act in either a dominant-negative or fast-cycling manner as reported for other GTPase mutants (Fiegen et al. 2004, Wang et al. 2005, Fidyk et al. 2006, Klein et al. 2006, Soh and Low 2008, Sugawara et al. 2019, Aspenström 2020)."

The claims in Line #224-227 are exaggerated. Please tone down or delete __Our response: __

*We rephrased the sentence (Line 240-243). *

"Therefore, we propose that TTN5 exhibits the typical functions of a small GTPase based on in vitro biochemical activity studies, including guanine nucleotide association and dissociation, but emphasizes its divergence among the ARF GTPases by its kinetics."

Line#488-489 - This conclusion is not really supported. At best Authors can claim that TTN5 is associated with trafficking components, but the functional relevance of this association is not determined. Our response:

*We toned down our statement (Line 604-608). *

„The colocalization of FM4-64-labeled endocytosed vesicles with fluorescence in YFP-TTN5-expressing cells may indicate that TTN5 is involved in endocytosis and the possible degradation pathway into the vacuole. Our data on colocalization with the different markers support the hypothesis that TTN5 may have functions in vesicle trafficking."

__Minor comments: __

Line #95 - " This rolein vesicle....." - please clarify which role? Our response:

We rephrased the sentence (Line 96-99).

„These roles of ARF1 and SAR1 in COPI and II vesicle formation within the endomembrane system are well conserved in eukaryotes which raises the question of whether other plant ARF members are also involved in functioning of the endomembrane system."

Line #168 - "we did not observed" please change to "not able to measure/quantify" __Our response: __

*We changed the text accordingly (Line 169-171). *

„A remarkable observation was that we were not able to monitor the kinetics of mGppNHp association with TTN5T30N but observed its dissociation (koff = 0.026 s-1; Figure 2E)."

Line#179 - ARF# is human for Arabidopsis?

Our response:

*The study of Fasano et al., 2022 is based on human ARF3 and we added the information to the text (Line 180-181) *

"(...) very similar to the koff value of HsARF3 (Fasano et al. 2022)."

• *

Line #181 - compared to what is the 10-fold difference?

__Our response: __

The 10-fold difference is between the nucleotides mGDP and mGppNHp, for both TTN5WT and TTN5Q70L. We added the information on specific nucleotides to this sentence for a better understanding (Line 181-185).

„Second, the koff values for mGDP and mGppNHp, respectively, were in a similar range between TTN5WT (0.012 s-1mGDP and 0.001 s-1 mGppNHp) and TTN5Q70L (0.025 s-1 mGDP and 0.006 s-1 mGppNHp), respectively, but the koffvalues differed 10-fold between the two nucleotides mGDP and mGppNHp in TTN5WT (koff = 0.012 s-1 versus koff = 0.001 s-1; Figure 2D, E; Supplementary Figure S3G, I, S4F, H)."

Lines #314-323 - are diffciult to understand, consider reframing. Same goes for the conclusion following these lines.

__Our response: __

We added an explanation to these sentences for a better understanding (Line 392-405).

„We performed an additional object-based analysis to compare overlapping YFP fluorescence signals in YFP-TTN5-expressing leaves with GmMan1-mCherry signals (YFP/mCherry ratio) and vice versa (mCherry/YFP ratio). We detected 24 % overlapping YFP- fluorescence signals for TTN5 with Golgi stacks, while in YFP-TTN5T30N and YFP-TTN5Q70L-expressing leaves, signals only shared 16 and 15 % overlap with GmMan1-mCherry-positive Golgi stacks (Supplementary Figure S8B). Some YFP-signals did not colocalize with the GmMan1 marker. This effect appeared more prominent in leaves expressing YFP-TTN5T30N and less for YFP-TTN5Q70L, compared to YFP-TTN5 (Figure 5B-D). Indeed, we identified 48 % GmMan1-mCherry signal overlapping with YFP-positive structures in YFP-TTN5Q70L leaves, whereas 43 and only 31 % were present with YFP fluorescence signals in YFP-TTN5 and YFP-TTN5T30N-expressing leaves, respectively (Supplementary Figure S8B), indicating a smaller amount of GmMan1-positive Golgi stacks colocalizing with YFP signals for YFP-TTN5T30N. Hence, the GTPase-active TTN5 forms are likely more present at cis-Golgi stacks compared to TTN5T30N."

Authors might consider a longer BFA treatment (3-4h) to see more clearer ER-Golgi fusion (BFA bodies)

__Our response: __

We perforned addtional BFA treatments for HA3-TTN5-expressing Arabidopsis seedlings followed by whole-mount immunostaining and for YFP-TTN5-expressing Arabidopsis lines. In both experiments we could obtain the typical BFA bodies. We included the NEW data in NEW Figure 4B, C

**Referees cross-commenting**

I agree with both my co-reviewers that the manuscript needs substantial improvement in its cell biology based experiments and conclusions thereof. I think the concensus of all reviewers points to weakness in the in-planta experiments which needs to be addressed to understand and characterize TTN5, which is the main goal of the manuscript.

Reviewer #3 (Significance (Required)):

Significance: The manuscript has general significance in understanding the role of small GTPases which are understudied. Although the manuscript does not advance the field of either intracellular trafficking or organization it holds significance in attempting to characterize proteins involved, which is a prerequisite for further functional studies.

__Our response: __

Thank you for your detailed analysis of our manuscript and positive assessment. Our study is an advance in the plant vesicle trafficking field.

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

Compared to our initial submission to Review Commons, we have addressed all the reviewers' comments. We have extensively re-written the manuscript to make it clearer to a larger audience. In particular, we have transferred Figure EV1 to Figure 1 with more complete panels and included a scheme (Figure EV3) on the steps of D2R internalization which we measure with live cell imaging. We have added a new paragraph to the start of the Discussion to summarize our main conclusions and reordered the discussion on the possible mechanisms of membrane PUFA enrichment on D2R endocytosis. All the changes in the text are in red for easier comparison with the previous version.

As suggested by reviewer 1, we have performed additional experiments to test the specificity of the effects of PUFA treatments on D2R endocytosis, reinforcing the results shown in Figure 4 using feeding assays. We show with live cell TIRF imaging and the ppH assay that TfR-SEP endocytosis is not affected (Figure EV5) and that SEP-β2AR endocytosis and βarr2-mCherry recruitment to the plasma membrane are not affected (Figure EV6).

Reviewer #1

Evidence, reproducibility and clarity

*The manuscript, using different live and fixed cell trafficking assays, demonstrates that incorporation of poly-unsaturated, but not saturated, free fatty acids in the membrane phospholipids reduce agonist induced internalization of the D2 dopamine receptor but not the adrenergic beta2 receptors or the transferrin receptor. Pulsed pH (ppH) live microscopy further demonstrated that the reduced internalization by incorporation of free fatty acid was accompanied by a blunted recruitment of Beta-arrestin for the D2R.

I believe said claims put forward in the manuscript are overall well supported by the data and as such I do not believe that further experiments are necessarily needed to uphold these key claims. Also, the methodology is satisfactorily reported, and statistics are robust, although two-way Anova like used in Fig 1 seems appropriate for Fig 2 and 3*

We thank the reviewer for his/her positive assessment of our work. We have checked the statistical tests used for all our measures. For Figure 2 and 3 (now 3 and 4) we test for only one factor (PUFA treatment or not) so we ran ordinary one-way ANOVA using Graphpad Prism.

That said, I suggest that the fixed cell internalization experiments (Fig 2 and 3), which relate the effect on the D2R to B2AR and transferrin are revised. This is important since this is relevant to judge whether the effect is a general or a selective molecular mechanism since this is the one of the three assay which this comparison relies on. Alternatively, I suggest omitting this data and include the B2AR in the Live DERET assay and both B2AR and TfR in the ppH assay. Specifically, my concerns with the fixed cell internalization are: • The analysis is based on counting the number of endosomes, which is not necessarily equivalent to the number of receptors internalized

The number of puncta, as well as their fluorescence, is reported by the analysis program (written in Matlab2021 and available upon request). We chose to show number of puncta because they reflect more directly the number of labelled endosomes (in Figures 3 and 4). As shown in the figure below, we found slight but significant differences between groups for FLAG-D2R (88.6 % and 87.6 % of average fluorescence in DHA and DPA treated cells compared to control cells), (panel A), and no differences for FLAG-β2AR (panel B). We find a significant decrease in puncta fluorescence for transferrin uptake in cells incubated with DHA (but not DPA) relative to control cells (panel C). However, because we did not detect differences in the number of puncta or in the frequency and amplitude of endocytic vesicle creation events (see below), we still conclude that enrichment with exogenous PUFAs does not affect clathrin mediated endocytosis.

In conclusion, the most robust measure of endocytosis for this assay is the number of detected puncta per cell rather than their fluorescence.

• The analysis relies on fully effective stripping of the surface pool of receptors - i.e clustered surface receptors not stripped by the protocol will be assessed as internalized. It is often very difficult to obtain full efficiency of the Flag-tag stripping and this is somewhat expression dependent. • The protocol for the constitutive and agonist induced internalization is different and yet shown on the same absolute graph. Although I take it the microscope gain setting are unaltered between the constitutive and agonist induced internalization I don't believe the quantification can be directly related. This is confusing at the very least. More critically however, the membrane signal from the non-stripped condition of constitutive internalization will likely fully shield internalized receptors in the Rab4 membrane proximal recycling pathway leading to under-estimation of the in the constitutive endocytosis. I believe this methodological limitation underlies the massive relative difference in the constitutive endocytosis between panel 2A,B and 2C,D. For comparison, by a quantitative dual color FACS endocytosis assay, we have previously demonstrated the ligand endocytosis a ~4 fold increased over constitutive (in concert with Fig 2A,B here) (Schmidt et al 20XX). Importantly, high relative variability by this methodology could well shield an actual effect of incorporation of FFAs on the constitutive endocytosis. We thank the reviewer for pointing this difference in the protocol. As a matter of fact, we have not used acid stripping in all the conditions used for the uptake assays (Figures 3 and 4). We apologize for the confusion and we have clarified this point in the Methods section. In early experiments we compared conditions with or without stripping but we concluded from these experiments that indeed, the stripping was not complete. Moreover, we noticed early on that many cells treated with DHA or DPA did not have any detectable cluster (13 cells out of 58 quantified cells treated with DHA after addition of QPL, 12/56 cells treated with DPA, 0/68 for cells treated with vehicle). Stripping the antibody would have made these cells undetectable, biasing the analysis. Therefore, to make our results more consistent we decided to use non-stripping conditions. To detect endosomes specifically, we used a segmentation tool developed earlier (see Rosendale et al.* 2019). This tool is based on wavelet transforms which recognizes dot-like structures. In addition, we excluded from the cell mask the labelled plasma membrane by a mask erosion.

We agree the design of experiments was not aimed at comparing the effect of PUFA treatment on low levels of constitutive D2R endocytosis. This would require more sensitive assays and be addressed in subsequent studies.

'Optional' Also, it would be informative to see the ppH Beta-arrestin experiments with the B2AR to assess, whether the putative discrepancy between D2R and B2AR is upstream or downstream of the blunted Beta-arrestin recruitment. To the same point, it would be very informative to assess how the incorporation of the free fatty acids affect receptor signalling, which would also help relate the effect of incorporation of the FFA's in the phospholipids to previous experiment using short term incubation with FFA's

We have now performed live imaging experiments in HEK293 cells expressing SEP-β2AR, GRK2 and βarr2-mCherry and stimulated with isoproterenol (Figure EV6). We show that the clustering of SEP-β2AR, of βarr2-mCherry, as well as endocytosis, are not affected by treatments with DHA or DPA. In this study, we focused on the early trafficking steps of D2R internalization. It will be interesting in a future study to address its consequences on G protein dependent and independent signaling. Moreover, and for good measure, we performed experiments to assess TfR-SEP endocytosis with the ppH assay. Again, we found no difference between cells treated or not with PUFAs (Figure EV5)

*References overall seem appropriate although Schmidt et al would be relevant for reference of the constitutive vs agonist induced endocytosis of D2R and B2AR. *

We have now cited Schmidt et al. 2020 doi 10.1111/bcpt.13274 in the discussion with the following sentences: "D2R also shows constitutive endocytosis (Schmidt et al, 2020) which may be modulated by PUFAs although we did not detect any significant difference in our measures (see Figure 3) which were aimed at detecting high levels of internalization induced by agonists. Further work will be required to specifically examine the effect of PUFAs on constitutive GPCR internalization."

Overall, the figures are well composed and convey the messages fairly well. Specific point that would strengthen the rigor include: • Chosing actual representative pictures of the quantitative data in Fig 2 and 3 (e.g. hard to see 25 endocytic events in Fig 2A constitutive endo, EtOH)

We apologize for the confusion. We employ a normalization procedure to account for cell size. In addition, all numbers have been normalized to the condition stimulated with agonist with no PUFA treatment). In fact, we detect in unstimulated cells very few puncta (on average 0.6, range 0-5) compared to 27.3 clusters (range 2-87) in cells stimulated with QPL.

• Showing actual p values for the statistical comparisons* For easier reading, we have kept the stars convention for the figures but added two tables with all statistical tests and the p values for both main figures and EV figures.

Moreover, for ease of reading the figures (without consulting the legend repeatedly) it would be very helpful to headline individual panel with what the experiments assesses. Figure 1a and 1b for example can't be distinguished at all before reading the figure legend. Also, y-axis could be more informative on what I measured rather than just giving the unit.

We have added titles to panels (in particular for Figure 2A,B which correspond to former Figure 1A,B) and we have given new titles to Y axes to make them clearer. We hope that the reading of our figures will now be easier.

Finally, the figure presentation and description of S1 is very hard to follow. I cannot really make out what is assessed in the different panels.

We have changed substantially Figure EV1 (now Figure 1) with new presentation of data: all 4 conditions (control, treated with DHA, DPA or BA) systematically presented in the same graph, and clearer titles for the parameter displayed on the Y axes. We hope that this figure is now easier to follow.

Significance

*The strength of the manuscript is the use and validation of incorporation of FFA's in the plasma membrane, which more closely mimics the physiological situation than brief application of FFAs as often done. Is addition, the blunted recruitment of beta-arrestin as assessed by the ppH protocol is quite intriguing mechanistically. The limitation are the relative narrow focus on the D2 receptor (and not multiple GPCRs) that does not really speak to as or assess the physiological, pathophysiological or therapeutic role of the observations (except from referring the relation between FFAs and disease). Also, despite the putative role of Beta-arrestin recruitment in the process, the actual causation in the process is not clear. This shortcoming is underscored by the putative effect on the constitutive internalization described above.

My specific expertise for assessing the paper is within general trafficking processes (including the trafficking methodology applied), trafficking of GPCRs and function of the dopamine system including the role of D2 receptors.*

• *

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

• *

The only conclusion that I was able to understand from the study was that enrichment of cell membranes with polyunsaturated fatty acids specifically inhibited agonist-induced internalization of D2 receptors. However, I think that the experiments used to conclude that PUFAs do not alter D2R clustering but reduce the recruitment of β-arrestin2 and D2R endocytosis need some clarification (i.e. data depicted in Fig. 2-5). This lack of clarity might be due to the fact I am not familiar enough with the employed technologies or to the unclear writing style of the paper. There was an overuse of acronyms, initialisms and abbreviations, which are difficult to understand for researchers outside of the specific lipid field. I think that the manuscript should be written in a way to be legible also for researchers not working in the immediate filed.

The paper was not written in a manner that a general audience of cell biologists or those interested in GPCR biology could understand and judge. It is indeed interesting that polyunsaturated fatty acids specifically inhibit D2R internalization in HEK293 cells, and it could be significant. But, it is difficult to judge the significance of the observation without more in vivo data.

I would suggest the following. Remove all acronyms and abbreviations. Significantly, expand the Materials and Methods section, either in the manuscript or in the Supplemental section. I suggest clearly explaining each construct used, and the function of each module in the construct, with diagrams. In addition, provide a comprehensive step by step description of each experimental protocol, providing the reader with the rationale for each step in the protocol with explanatory diagrams. The authors should also more clearly explain the rationale and logic that was utilized to make the conclusions that they did from the depicted observations. Only then can a broader audience determine if the authors' conclusions are justified.

We thank the reviewer for his/her comments. Indeed, our main message was that two types of PUFAs (DHA and DPA) specifically alter D2R endocytosis by reducing the recruitment of β-arrestin2 without changing D2R clustering at the plasma membrane. We are sorry that our writing was not clear enough. We also found out that in the last steps of the submission to Review Commons, the first paragraph of the Discussion was inadvertently erased. This made our main conclusions, summarized in this first paragraph, less clear. We have now put back this important paragraph. Moreover, we have extensively rewritten the manuscript thriving to make it as clear as possible to a large audience. We have reduced the use of acronyms to keep only the most used ones [e.g. PUFA (used 99 times), DHA (37 times), GPCR (34 times), D2R (126 times), GRK (17 times)] and made them consistent throughout the manuscript. Following the reviewer's suggestion, we have also added a scheme of the steps following D2R activation by agonist leading to its internalization (Figure EV3).

We understand that the reviewer implies by "in vivo data" results obtained in the brain of animals. As written in the Introduction and in the Discussion, the current work follows up on a recently published manuscripts by a subset of the authors, namely (i) Ducrocq et al. 2020 (doi 10.1016/j.cmet.2020.02.012) in which we show that deficits in motivation in animals deprived in ω3-PUFAs can be restored specifically by conditional expression of a fatty acid desaturase from c. elegans (FAT1) that allows restoring PUFA levels specifically in D2R-expressing striatal projection neurons (which mediate the so-called indirect pathway), and (ii) Jobin et al. 2023 (doi: 10.1038/s41380-022-01928-6) which combines in cellulo (HEK 293 cells) and in vivo data to show that PUFAs affects the ligand binding of the dopamine D2 receptor and its signaling in a lipid context that reflects patient lipid profiles regarding poly-unsaturation levels.

Reviewer #2 (Significance (Required)):

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In summary, I will reiterate that the reported experiments need to be much better explained to make the study understandable to a broader audience and for that audience to determine whether the conclusions are justified.

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