I have never been so thoroughly opposed to everyone involved. He's right to scorn the rationalismists, but not for the fan fiction, Jesus.

So so true. I love a new update, not only because it removes the red notification from my screen, but because it brings a fresh design, a new efficient approach or just something more aesthetically pleasing to the tool. However, that shouldn't be the reason to use tools in learning. They should have an academic purpose, level up the learning, provide opportunitiy for creativity, allow students to more fully demonstrate their mastery.

I don't think this is a good definition. The example, at least, is a bad one. That resource could still exist (the same way a .docx that lives in the Documents directory and has been uploaded but later had the file host go down would still exist)—it just wouldn't be resolvable by URL.

Author Response

Reviewer #1 (Public Review):

In this study, Guggenmos proposes a process model for predicting confidence reports following perceptual choices, via the evidence available from stimuli of various intensities. The mechanisms proposed are principled, but a number of choices are made that should be better motivated - I develop below a number of concerns by order of importance.

I’d like to thank the reviewer for their thorough and excellent review. It’s no set phrase that this review substantially improved the manuscript.

1) Lack of separability of the two metacognitive modules.

Can the author show that the proposed model can actually discriminate between the noisy readout module and the noisy report module? The two proposed modules have a different psychological meaning, but seem to similarly impact the confidence output. Are these two mutually exclusive (as Fig 1 suggests), or could both sources of noise co-exist? It will be important to show model recovery for introducing readout vs. report at the metacognitive level, e.g., show that a participant best-fitted by a nested model or a subpart of the full model, with a restricted number of modules (some of the parameters set to zero or one), is appropriately recovered? (focusing on these two modules) This raises the question of how the two types of sigma_m are recoverable/separable from each other (and should they both be called sigma_m, even if they both represent a standard deviation)? If they capture independent aspects of noise, one could imagine a model with both modules. More evidence is needed to show that these two capture separate aspects of noise.

Testing the separability of the two noise types (readout, report) is a great idea and I have now performed a corresponding recovery analysis. Specifically, I have simulated data with both noise types for different regimes of sensory and metacognitive noise. As shown in the new Figure 7—figure supplement 6, the noise type can be precisely recovered in the most typical regimes.

I now refer to this analysis in the subsection 2.4 Model recovery (Line 521ff):

“One strength of the present modeling framework is that it allows testing whether inefficiencies of metacognitive reports are better described by metacognitive noise at readout (noisy-readout model) or at report (noisy-report model). To validate this type of application, I performed an additional model recovery analysis which tested whether data simulated by either model are also best fitted by the respective model. Figure 7—figure supplement 6 shows that the recovery probability was close to 1 in most cases, thus demonstrating excellent model identifiability. With fewer trials per observer, recovery probabilities decrease expectedly, but are still at a very good level. The only edge case with poorer recovery was a scenario with low metacognitive noise and high sensory noise. Model identification is particularly hard in this regime because low metacognitive noise reduces the relevance of the metacognitive noise source, while high sensory noise increases the general randomness of responses.”

In principle, both noise modules can co-exist and model inversion should be possible (though mathematically more complicated). On the other hand, I anticipate that parameter recovery would be extremely noisy in such a scenario. For this work, I decided to not test this possibility as it would add a lot of complexity, with a high probability of ultimately being unfeasible.

2) The trade-off between the flexibility of the model (modularity of the metacognitive part, choice of the link functions) and the generalisability of the process proposed seems in favor of the former. Does the current framework really allow to disambiguate between the different models? Or at least, the process modeled is so flexible that I am not sure it allows us to draw general conclusions? Fig 7 and section 3 of the results explain that all models are similar, regardless of module of functions specified; Fig 7 supp shows that half of participants are best fitted by noisy readout, while the other half is best fitted by noisy report; plus, idiosyncrasies across participants are all captured. Does this compromise the generalisability of the modeling of the group as a whole?

This is a fair point and I understand the question has two components: a) is the model too flexible, potentially preventing generalized conclusions? b) is the flexibility of the model recoverable?

Regarding a), I should emphasize that the manuscript (and toolbox) provides a modeling framework, rather than a single specific model. In other words, researchers applying the framework/toolbox must make a number of decisions: which noise type? which metacognitive biases should be considered? which link function? To ensure interpretability / generalizability, researchers have to sufficiently constrain the model. Due to this framework character, it makes sense that the manuscript is submitted under the Tools & Resources Article format rather than the Research Article format.

On the other hand, I agree that it is the duty of the manuscript introducing the framework to provide all necessary information to help the researcher make these decisions. This is where the reviewer’s point b) is critical and I hope that with the new parameter and model recovery analyses in the present revision (see other comments) I meet this requirement to a satisfactory degree.

To clarify the scope and aim of the paper, I now put a new subsection in front of the example application to the data from Shekhar and Rahnev, 2021 (Line 534ff):

“It is important to note that the present work does not propose a single specific model of metacognition, but rather provides a flexible framework of possible models and a toolbox to engage in a metacognitive modeling project. Applying the framework to an empirical dataset thus requires a number of user decisions: which metacognitive noise type is likely more dominant? which metacognitive biases should be considered? which link function should be used? These decisions may be guided either by a priori hypotheses of the researcher or can be informed by running a set of candidate models through a statistical model comparison. As an exemplary workflow, consider a researcher who is interested in quantifying overconfidence in a confidence dataset with a single parameter to perform a brain-behavior correlation analysis. The concept of under/overconfidence already entails the first modeling decision, as only a link function that quantifies probability correct (Equation 6) allows for a meaningful interpretation of metacognitive bias parameters. Moreover, the researcher must decide for a specific metacognitive bias parameter. The researcher may not be interested in biases at the level of the confidence report, but, due to a specific hypothesis, rather at metacognitive biases at the level of readout/evidence, thus leaving a decision between the multiplicative and the additive evidence bias parameter. Also, the researcher may have no idea whether the dominant source of metacognitive noise is at the level of the readout or report. To decide between these options, the researcher computes the evidence (e.g., AIC) for all four combinations and chooses the best-fitting model (ideally, this would be in a dataset independent from the main dataset).”

In addition, the website of the toolbox now provides a lot more information about typical use cases: https://github.com/m-guggenmos/remeta

3) More extensive parameter recovery needs to be done/shown. We would like to see a proper correlation matrix between parameters, and recovery across the parameter space, not only for certain regimes (i.e. more than fig 6 supp 3), that is, the full grid exploration irrespective of how other parameters were set.

The recovery of the three metacognitive bias parameters is displayed in Fig 4, but what about the other parameters? We need to see that they each have a specific role. The point in the Discussion "the calibration curves and the relationships between type 1 performance and confidence biases are quite distinct between the three proposed metacognitive bias parameters may indicate that these are to some degree dissociable" is only very indirect evidence that this may be the case.

A comprehensive parameter recovery analysis is indeed a key analysis that was missing in the first version of the manuscript. I now performed several analyses to address this, rewrote and extended section 2.3 on parameter recovery. The new parameter recovery analysis was performed as follows (Line 455ff):

“To ensure that the model fitting procedure works as expected and that model parameters are distinguishable, I performed a parameter recovery analysis. To this end, I systematically varied each parameter of a model with metacognitive evidence biases and generated data. Specifically, each of the six parameters (σs, ϑs, δs, σm, 𝜑m, δm) was varied in 500 equidistant steps between a sensible lower and upper bound. The model was then fit to each dataset. To assess the relationship between fitted and generative parameters, I computed linear slopes between each generative parameter (as the independent variable) and each fitted parameter (as the dependent variable), resulting in a 6 x 6 slope matrix. Note that I computed (robust) linear slopes instead of correlation coefficients, as correlation coefficients are sample-sizedependent and approach 1 with increasing sample size even for tiny linear dependencies. Thus, as opposed to correlation coefficients, slopes quantify the strength of a relationship. Comparability between the slopes of different parameters is given because i) slopes are – like correlation coefficients – expected to be 1 if the fitted values precisely recover the true parameter values (i.e., the diagonal of the matrix) and ii) all parameters have a similar value range which makes a comparison of off-diagonal slopes likewise meaningful. To test whether parameter recovery was robust against different settings of the respective other parameters, I performed this analysis for a coarse parameter grid consisting of three different values for each of the six parameters except σm, for which five different values were considered. This resulted in 35·51 = 1215 slope matrices for the entire parameter grid.”

In addition, I computed additional supplementary analyses assessing a case with fewer trials, a model with confidence biases, and models with mixed evidence and confidence biases. For details about these analyses, I kindly point the reviewer to section 2.3. Together, these new analyses demonstrate that parameter recovery works extremely well across different regimes and for all model parameters, including the metacognitive bias parameters mentioned in the reviewer’s comment.

1.8: It would be important to report under what regimes of other parameters these simulations were conducted. This is because, even if dependence of Mratio onto type 1 performance is reproduced, and that is not the case for sigma_m, it would be important to know whether that holds true across different combinations of the other parameter values.

I now repeated this analysis for various settings of other parameters and include the results as new Figure 6—figure supplement 2. While the settings of other parameters affect the type 1 performance dependency of Mratio (with some interesting effects such as Mratio > 1), parameter recovery of sigma_m is largely unaffected. The same basic point thus holds: Mratio shows a nonlinear dependency with type 1 performance, but sigma_m can be recovered largely without bias under most regimes (the main exception is a combination of low sensory noise and high metacognitive noise under the noisy-readout model, which is also mentioned in the manuscript).

Is lambda_m meaningfully part of the model, and if so, could it be introduced into the Fig 1 model, and be properly part of the parameter recovery?

I now reworked the part about metacognitive biases to make it more consistent and to introduce lambda_m on equal footing with the other metacognitive bias parameters. I now distinguish between metacognitive evidence biases (the two main bias parameters of the original model, phi_m and theta_m) and metacognitive confidence biases, i.e. lambda_m and a new additive confidence bias parameter kappa_m. The schematic presentation of the model framework in Figure 1 is updated in accordance:

This change also complies with reviewer 2, who rightfully pointed out that the original model framework put much stronger emphasis on bias parameters loading on evidence than on confidence. The metacognitive confidence bias parameters are now also part of the parameter recovery analyses (Figure 7—figure supplement 2).

While it is still feasible to combine the two evidence-related bias parameters and lambda_m – as queried by the reviewer – not all mixed combinations of evidence- and confidence-related bias parameters perform well in terms of model recovery (in particular, combining all four parameters; cf. Figure 7—figure supplement 3). Hence, a decision on the side of the modeler is required. I comment on this important aspect at the end of the section 1.4 about metacognitive biases (Line 276ff):

“Finally, note that the parameter recovery shown in Figure 4 was performed with four separate models, each of which was specified with a single metacognitive bias parameter (i.e., 𝜑m, δm, λm, or Km). Parameter recovery can become unreliable when more than two of these bias parameters are specified in parallel (see section 2.3; in particular, Figure 7—figure supplement 3). In practice, the researcher thus must make an informed decision about which bias parameters to include in a specific model (in most scenarios one or two metacognitive bias parameters are a good choice). While the evidence-related bias parameters 𝜑m and δm have a more principled interpretation (e.g., as an under/overestimation of sensory noise), it is not unlikely that metacognitive biases also emerge at the level of the confidence report (λm, km). The first step thus must always be a process of model specification or a statistical comparison of candidate models to determine the final specification (see also section 3.1).”

4) An important nuance in comparing the present sigma_m to Mratio is that the present model requires that multiple difficulty levels are tested, whereas instead, the Mratio model based on signal detection theory assumes a constant signal strength. How does this impact the (unfair?) comparison of these two metrics on empirical data that varied in difficulty level across trials? Relatedly, the Discussion paragraph that explained how the present model departs from type 2 AUROC analysis similarly omits to account for the fact that studies relying on the latter typically intend to not vary stimulus intensity at the level of the experimenter.

I thank the reviewer for this comment which made me realize that I incorrectly assumed that my model requires multiple stimulus difficulty levels. The only parameter that would require multiple stimulus intensities is the sensory threshold parameter, but for this parameter I already state that it requires additional stimulus difficulties close to threshold (Line 147ff). Otherwise I now made extensive tests that the model works just fine with constant stimuli. My reasoning mistake (iirc) was related to the fact that I fit a metacognitive link function, which I thought would require variance on the x-axis; but of course there is already plenty of variance introduced through noise at the sensory level, so multiple difficulty levels are not required to fit the metacognitive level. I now removed the relevant references to this requirement from the manuscript.

Nevertheless, I agree that it is interesting to perform the comparison between Mratio and sigma_m also for a scenario with constant stimuli. See both the new Figure 6–supplement 1 with constant stimuli, and the (updated) main Figure 6 with multiple stimulus levels for comparison.

The general point still holds also for constant stimuli: Mratio is not independent of type 1 performance. Thus, the observed dependence on type 1 performance is not due to the presence of varying stimulus levels. I now reference this new supplementary figure in Result section 1.8 (Line 389).

5) 'Parameter fitting minimizes the negative log-likelihood of type 1 choices (sensory level) or type 2 confidence ratings (metacognitive level)'. Why not fitting both choices and confidence at the same time instead of one after the other? If I understood correctly, it is an assumption that these are independent, why not allow confidence reports to stem from different sources of choice and metacognitive noise? Is it because sensory level is completely determined by a logistic (but still, it produces the decision values that are taken up to the metacognitive level)?

The decision to separate the two levels during parameter inference was deliberate. I now explain this choice in the beginning of Result section 2 (Line 416ff):

“The reason for the separation of both levels is that choice-based parameter fitting for psychometric curves at the type 1 / sensory level is much more established and robust compared to the metacognitive level for which there are more unknowns (e.g., the type of link function or metacognitive noise distribution). Hence, the current model deliberately precludes the possibility that the estimates of sensory parameters are influenced by confidence ratings.”

Indeed, I would regard it as highly problematic if the estimates of sensory parameters were influenced by confidence ratings, which are shaped by a manifold of interindividual quirks and biases and for which computational models are still in a developmental stage. Yet, from a pure simulation-based parameter recovery perspective, in which the true confidence model is known, using confidence ratings would indeed make sensory parameter estimation more precise (because of the rich information contained in continuous confidence ratings which is lost in the binarization of type 1 choices).

6) Fig 4 left panels: could you clarify the reasoning that due to sensory noise, overconfidence is expected, instead of having objective and subjective probability correct aligning on the diagonal? Shouldn't the effects of sensory noise average out? In other words, why would the presence of sensory noise systematically push towards overconfidence rather than canceling out on average?

As an intuitive explanation consider the case that no signal is present in a stimulus, e.g., a line grating in a clockwise/counterclockwise orientation discrimination task with an angle of 0 degrees. Since there is no true information in the stimulus, type 1 performance will be at chance level irrespective of sensory noise.

However, sensory noise matters for the metacognitive level. Assuming no sensory noise (i.e., sigma_s = 0), the observer’s stimulus/decision variable would be zero and thus confidence would be zero. Thus, confidence would exactly match type 1 performance. Yet, assuming the presence of sensory noise, the stimulus estimate (“decision value”) will be always different from point-zero, if ever so slightly. While the average estimate of the stimulus variable across trials will indeed cancel out to zero, each individual trial will be different from zero (in either direction) and hence also the confidence will be different from zero in each trial. Since confidence is unsigned, the average confidence will be greater than zero and thus give the impression of an overconfident observer.

Note that this explanation was implicitly included in the paragraph on the 0.75 signature of confidence (“When evidence discriminability is zero, an ideal Bayesian metacognitive observer will show an average confidence of 0.75 and thus an apparent (over)confidence bias of 0.25. Intuitively this can be understood from the fact that Bayesian confidence is defined as the area under a probability density in favor of the chosen option. Even in the case of zero evidence discriminability, this area will always be at least 0.5 − otherwise the other choice option would have been selected, but often higher.”, Line 257ff).

7) The same analysis as Fig 6 but for noisy readout instead of noisy reports do not show the same results: both sigma_m and m-ratio vary as a function of type 1 performance. Does this mean that the present model with readout module does not solve the issue of dependency upon type 1 performance?

I refer to this in the Result section: “The exception is a regime with very high metacognitive noise and low sensory noise under the noisy-readout model, in which recovery becomes biased” (Line 391ff). Indeed, the type 1 performance dependency of sigma_m recovery in this edge case is not as good as in the noisyreport model. However, note that recovery is stable across a large range of d’ including the range typical aimed for in metacognition experiments (i.e., medium performance levels to ensure sufficient variance in confidence ratings).

It is also important to point out that a failure to recover true parameters under certain conditions is not a failure of the model, but a reflection of the fact that information can be lost at the level of confidence reports. For example, if sensory noise is very high, the relationship between evidence and confidence becomes essentially flat (Figure 3), producing confidence ratings close to zero irrespective of the level of stimulus evidence. It becomes increasingly impossible to recover any parameters in such a scenario. Vice versa if sensory noise is extremely low, confidence ratings approach a value of 1 irrespective of stimulus evidence, and the same issue arises. In both cases there is no meaningful variance for an inference about latent parameters. This issue is more pronounced in the noisy-readout case because it requires an inversion of precisely the relationship between evidence and confidence.

8) In Eq8, could you explain why only the decision values consistent with the empirical choice are filtered. Is this an explicit modeling of the 'decision-congruence' phenomenon reported elsewhere (eg. Peters et al 2017)? What are the implications of not keeping only the congruent decision values?

I apologize, this was a mistake in the manuscript. The integration is over all decision values, not just those consistent with the choice. I corrected it accordingly.

Reviewer #2 (Public Review):

This paper presents a novel computational model of confidence that parameterises links between sensory evidence, metacognitive sensitivity and metacognitive bias. While there have been a number of models of confidence proposed in the literature, many of these are tailored to bespoke task designs and/or not easily fit to data. The dominant model that sees practical use in deriving metacognitive parameters is the meta-d' framework, which is tailored for inference on metacognitive sensitivity rather than metacognitive biases (over- and underconfidence). This leaves a substantial gap in the literature, especially as in recent years many interesting links between metacognitive bias and mental health have started to be uncovered. In this regard, the ReMeta model and toolbox is likely to have significant impact on the field, and is an excellent example of a linked publication of both paper and code. It's possible that this paper could do for metacognitive bias what the meta-d' model did for metacognitive sensitivity, which is to say have a considerable beneficial impact on the level of sophistication and robustness of empirical work in the field.

The rationale for many of the modelling choices is clearly laid out and justified (such as the careful handling of "flips" in decision evidence). My main concern is that the limits to what can be concluded from the model fits need much clearer delineation to be of use in future empirical work on metacognition. Answering this question may require additional parameter/model recovery analysis to be convincing.

I thank the reviewer for these encouraging and constructive comments!

Specific comments:

• The parameter recovery demonstrated in Figure 4 across range of d's is impressive. But I was left wondering what happens when more than one parameter needs to be inferred, as in real data. These plots don't show what the other parameters are doing when one is being recovered (nor do the plots in the supplement to Figure 6). The key question is whether each parameter is independently identifiable, or whether there are correlations in parameter estimates that might limit the assignment of eg metacognitive bias effects to one parameter rather than another. I can think of several examples where this might be the case, for instance the slope and metacognitive noise may trade off against each other, as might the slope and delta_m. This seems important to establish as a limit of what can be inferred from a ReMeta model fit.

This is an excellent point and was also raised by reviewer #1. See major comment 3 of reviewer #1 for a detailed response. In short, I now provide comprehensive analyses that demonstrate successful parameter recovery across different regimes and both noisy types (noisy-readout, noisy-report). See Figure 7.

Regarding the anticipated trade-offs between the confidence slope (now referred to as multiplicative evidence bias) and metacognitive noise / delta_m (now additive evidence bias), there is a single scenario in which this becomes an issue. I describe this in the Results section as follows (Line 480ff):

“Here, the only marked trade-off emerges between metacognitive noise σm and the metacognitive evidence biases (𝜑m, δm) in the noisy-readout model, under conditions of low sensory noise. In this regime, the multiplicative evidence bias 𝜑m becomes increasingly underestimated and the additive evidence bias δm overestimated with increasing metacognitive noise. Closer inspection shows that this dependency emerges only when metacognitive noise is high – up to σm  0.3 no such dependency exists. It is thus a scenario in which there is little true variance in confidence ratings (due to low sensory noise many confidence ratings would be close to 1 in the absence of metacognitive noise), but a lot of measured variance due to high metacognitive noise. It is likely for this reason that parameter inference is problematic. Overall, except for this arguably rare scenario, all parameters of the model are highly identifiable and separable.” In my experience, certain trade-offs in specific edge cases are almost inescapable for more complex models. Overall, I think it is fair to say that parameter recovery works extremely well, including the ‘trinity’ of metacognitive noise / multiplicative evidence bias / additive evidence bias.

• Along similar lines, can the noisy readout and noisy report models really be distinguished? I appreciate they might return differential AICs. But qualitatively, it seems like the only thing distinguishing them is that the noise is either applied before or after the link function, and it wasn't clear whether this was sufficient to distinguish one from the other. In other words, if you created a 2x2 model confusion matrix from simulated data (see Wilson & Collins, 2019 eLife) would the correct model pathway from Figure 1 be recovered?

Great point. I introduced a new subsection 2.4 “Model recovery”, in which I demonstrate successful recovery of noisy-readout versus noisy-report models. See also my response to the first comment of Reviewer #1, which includes the new model recovery figure and the associated paragraph in the manuscript. The key new figure is Figure 7—figure supplement 6.

• Again on a similar theme: isn't the slope parameter rho_m better considered a parameter governing metacognitive sensitivity, given that it maps the decision values onto confidence? If this parameter approaches zero, the function flattens out which seems equivalent to introducing additional metacognitive noise. Are these parameters distinguishable?

Indeed, the parameter recovery analysis shows a slight negative correlation between the slope parameter (now termed multiplicative evidence bias) and metacognitive noise (Figure 7). As the reviewer mentions, this is likely caused by the fact that both parameters lead to a flattening /steepening of the evidenceconfidence relationship. For reference, in the empirical dataset by Shekhar & Rahnev, the correlation between AUROC2 and the multiplicative evidence bias is almost absent at r = −0.017. Critically, however, while an increase of the metacognitive noise parameter σm will ultimately lead to a truly flat/indifferent relationship between evidence and confidence, the multiplicative evidence parameter 𝜑m only affects the slope (i.e., asymptotically confidence will still reach 1). This is one reason why parameter recovery for both σm and 𝜑m works overall very well. The differential effects of σm and 𝜑m are now better illustrated in the updated Figure 3:

Also conceptually, the multiplicative evidence parameter 𝜑m plausibly represents a metacognitive bias, with either interpretation that I suggest in the manuscript: as a an under/overestimation of the evidence or as a an over/underestimation of one’s own sensory noise, leading to under/overconfidence, respectively. In sum, I think there are strong arguments for the present formalization and interpretation.

• The final paragraph of the discussion was interesting but potentially concerning for a model of metacognition. It explains that data on empirical trial-by-trial accuracy is not used in the model fits. I hadn't appreciated this until this point in the paper. I can see how in a process model that simulates decision and confidence data from stimulus features, accuracy should not be an input into such a model. But in terms of a model fit, it seems odd not to use trial by trial accuracy to constrain the fits at the metacognitive level, given that the hallmark of metacognitive sensitivity is a confidence-accuracy correlation. Is it not possible to create accuracy-conditional likelihood functions when fitting the confidence rating data (similar to how the meta-d' model fit is handled)? Psychologically, this also makes sense given that the observer typically knows their own response when giving a confidence rating.

While I agree of course that metacognitive sensitivity quantifies the relationship confidence-accuracy relationship, a process model is a distinct approach and requires distinct methodology. Briefly, the current model fit cannot be improved upon, as it is based on a precise inversion of the forward model. Computing accuracy-conditional likelihoods would lead to a biased parameter estimates, because it would incorrectly imply that the observer has access to the accuracy of their choice. While the observer knows their choice, as the reviewer correctly notes, they do not know the true stimulus category and hence not their accuracy.

I argue in the manuscript that both approaches (descriptive meta-d’, explanatory process model) have their advantages and disadvantages. The concept of meta-d’ / metacognitive sensitivity does not care why a particular confidence rating is the way it is, or whether an incorrect response is caused by sensory noise or by an attentional lapse. On the one hand, this implies that one cannot draw any conclusions about the causes and mechanisms of metacognitive inefficiency, which could be perceived as a major drawback. In this respect, it is a purely descriptive measure (cf. last comment of Reviewer #1). On the other hand, because it is descriptive, it can simply compare the confidence between correct and incorrect choices and thus, in a sense, capture a more thorough picture of metacognitive sensitivity; that is, being metacognitively aware not only of the consequences one’s own sensory noise (as in typical process models), but also of all other sources of error (attentional lapses, finger errors, etc.). I now added an additional paragraph in which I summarize the comparison of type 2 ROC / meta-d’ and process models along these lines (Line 800ff):

“In sum, while a type 2 ROC analysis, as a descriptive approach, does not allow any conclusions about the causes of metacognitive inefficiency, it is able to capture a more thorough picture of metacognitive sensitivity: that is, it quantifies metacognitive awareness not only about one’s own sensory noise, but also about other potential sources of error (attentional lapses, finger errors, etc.). While it cannot distinguish between these sources, it captures them all. On the other hand, only a process model approach will allow to draw specific conclusions about mechanisms – and pin down sources – of metacognitive inefficiency, which arguably is of major importance in many applications.”

• I found it concerning that all the variability in scale usage were being assumed to load onto evidencerelated parameters (eg delta_m) rather than being something about how subjects report or use an arbitrary confidence scale (eg the "implicit biases" assumed to govern the upper and lower bounds of the link function). It strikes me that you could have a similar notion of offset at the level of report - eg an equivalent parameter to delta_m but now applied to c and not z. Would these be distinguishable? They seem to have quite different interpretations psychologically: one is at the level of a bias in confidence formation, and the other at the level of a public report.

I substantially reworked the section about metacognitive biases, including an additive metacognitive bias (κm) also at the level of confidence. The previous version of the manuscript already included a multiplicative bias parameter loading onto confidence (previously referred to as ‘confidence scaling’ parameter, now multiplicative confidence bias λm), but it was considered optional and e.g. not part of the parameter recovery analyses.

My previous emphasis on biases that load onto evidence-related variables was due to a more principled interpretation (e.g. ‘underestimation of sensory noise’), but I agree that metacognitive biases must not necessarily be principled and may be driven e.g. by the idiosyncratic usage of a particular confidence scale. Updated Figure 1 sketches the new, more complete model.

Is a mix of evidence- and confidence-related metacognitive bias parameters distinguishable? I tested this in Figure 7—figure supplement 3.

The slope matrices show that e.g., the model suggested by the reviewer (two evidence-related bias parameters 𝜑m and δm + an additive confidence-based bias parameter κm) is to some degree dissociable, although slight tradeoffs start to emerge with such a complex model. By contrast, a mix of only one evidence-related and one confidence-related bias parameter is much more robust. In general, I thus recommend using at most two metacognitive bias parameters, which are selected either based on a priori hypotheses or on a model comparison. I comment on the necessity of choosing one’s bias parameters in a new paragraph in section 1.4 about metacognitive biases (Line 276ff):

“Finally, note that the parameter recovery shown in Figure 4 was performed with four separate models, each of which was specified with a single metacognitive bias parameter (i.e., 𝜑m, δm, λm, or m). Parameter recovery is more unreliable when more than two of these bias parameters are specified in parallel (see section 2.3; in particular, Figure 7—figure supplement 3). In practice, the researcher thus must make an informed decision about which bias parameters to include in a specific model (in most scenarios 1 or 2 metacognitive bias parameters is a good choice). While the evidence-related bias parameters 𝜑m and δm have a more principled interpretation (e.g., as an under/overestimation of sensory noise), it is not unlikely that metacognitive biases also emerge at the level of the confidence report (λm, km). The first step thus must always be a process of model specification or a statistical comparison of candidate models to determine the final specification (see also section 3.1).”

Reviewer #1 (Public Review):

This article considers the common scenario wherein it is desired to construct a dose-response curve. Response is a continuously valued number between 0 and 1, e.g. % cell viability. A typical approach is to transform response using a logistic function, log(x/[1-x]), so that the transformed response spans the real number line and then applying linear regression models. However, when some responses are very close to 0 (or very close to 1), this typical approach will be susceptible to these highly influential values, and the fitted model will not estimate well parameters of usual interest, such as the slope or intercept of the regression or the IC50 or the back-transformed dose response curve.

The proposed approach is an adaptation of Beta regression. The underlying systematic component of the generalized linear model is the same as in the standard case described above but the random component is based upon the beta distribution. Further, rather than using maximum likelihood to fit the model, the authors propose to use 'robust minimum density power divergence estimators'. This method is called REAP, for Robust and Efficient Assessment of Potency. The authors present an online, interactive application where users can upload data and fit the authors' method.

Overall, the REAP methodology seems useful and achieves exactly what they are intended to achieve: robustly fitting dose-response curves subject to extreme measurement error. The mathematical arguments are sound and further supported by simulation. A potentially straightforward alternative solution to this problem would be to directly model the measurement error process by using a heavy-tailed Cauchy distribution to model the measurement error process, thereby downweighting the influence of extreme responses but not removing them altogether. This would seem to have a 'familiarity advantage' over the authors' method.

The current version of the interactive application, although relatively easy to use, seems a little buggy. For example, the 'width of CI' argument accepts any positive number, including those greater than 1, and it's not clear how to interpret the different choices that one can plug in for this parameter. Also, even if there is just one 'Agent' provided in the data, the numerical comparisons below the plot are still presented in triplicate.

Passive voice: who sees it this way? Is this even the mainstream view?

I wonder if, given that boards are supposed to expire anyway, the key rotation shouldn't involve publishing old secrets such that it's not possible to use an old key to pin someone down as having published something (which would be long after its intended expiry anyway)? With the caveat that I don't know nothing about cryptography, I'm just a horrible little goblin typing in a Hypothes.is pane.

Has the deep dream aesthetic gotten this at all?

It's uncommon that marijuana may be found to contain fentanyl. The first possible report of this in the US was just late last year in Connecticut, and though popular literature talks about its being possible, it's way, way more likely to find fentanyl deaths related to opioids. Rather than talk about something nurses are unlikely to ever see, let's hit hard on fentanyl analogs. https://www.ama-assn.org/system/files/issue-brief-increases-in-opioid-related-overdose.pdf and https://portal.ct.gov/DPH/Press-Room/Press-Releases---2021/Officials-From-The-Connecticut-Overdose-Response-Strategy-And-The-DPH-Issue-Warning

The minor variations in titles that the author considered have sparked a flow of thoughts in me. I re-read and re-evaluated the lines a number of times to figure out why I was also captivated by such minor adjustments -- whether or not to include the article the. I feel that Digital Humanities without the would be another general subject covering several items within it. But like, nothing is specific about it. But with the article the, the distinction occurs. Now, the Digital Humanities feels very specific. It sounds like it is a department itself, just not as inclusive as Digital Humanities in the first case, but it is something that can be considered a particular segment, a particular academic concentration. There are no wrong cases, and I'm very pumped about the author's choice in the end. It's meaningful to think about this naming process and have follow-up questions about this decision's influence on the contents discussed in the lunchtime talk. It doesn't seem like a big deal for many, but the title helps guide the direction that the author guides her audience in understanding the field that she's been working hard on.

If there is an argument that lets women know that they are created equal, seeing them as men with "penises located inside" -- a similar yet reverse characteristic and functionality -- is not a perspective that should be encouraged.

I read a [Psychology Today article] (https://www.psychologytoday.com/us/blog/the-red-light-district/201105/female-misrepresentations) written ten years ago by Naveed Saleh. The report lists seven misperceptions about the vagina in a 2001 UK study titled "Socio-cultural representations of the vagina" by Braun and Wilkinson. It's disillusioning to know such points were made and widespread about the vagina. Why should the vagina be inferior to the penis on the basis that the penis penetrates it? Indeed, something must hold still like a bowl for the other to figure out which ingredients to put in the bowl. All for the best cake they can bake. Such natural and sacred collaboration was seen as a process of a vagina submitting to a penis. It was a statement by men controlling women and manipulating the gender hierarchy for themselves.

Coming back to the statement, this first appeared to me like a chill unawareness that women and men were not categorized and that women were seen as "different kinds of men." Nevertheless, it started to feel unsuitable as to why I should feel delighted and validateed because women were compared to men?

This is fantastic. Internet persona lore. Who is brave enough to make themselves lore? Kicks has legendary-tier lore. (I still get mad thinking about hacker news dweebs failing to have even a single bone in their body capable of recognizing non-literal text, I am not cool, I have no chill, etc.) I like that this isn't quite a statement about bluelander themself, but about the context they inhabit.

I'm somehow reminded of

Now, when getting into book discussions with a certain kind of man, I often say “I can’t read” as soon as possible. This is a pretty transparent defense mechanism, but it works for me, sort of.

from that essay on David Foster Wallace that I treasure in my heart more than I can explain. I love the idea of instrumentalizing one's angle on reality. Sorry, having this conversation with you in the frame you want to use is just not going to work for me. I can't read.

Raccoons traditionally put punctuation in their names because they don't like to be tracked.

Phenomenal.

Perhaps it may be helpful to dramatically reframe the question of how to keep a zettelkasten? One page blog posts from people who've only recently seen the idea and are synopsizing it without a year or more practice themselves are highly confusing at best. Can I write something we don't see enough of in spaces relating to zettelkasten? Perhaps we should briefly consider the intellectual predecessor of the slip box?

Start out by forgetting zettelkasten exist. Instead read about what a commonplace book is and how that (simpler) form of note taking works. This short article outlined as a class assignment is a fascinating way to start and has some illustrative examples: https://www.academia.edu/35101285/Creating_a_Commonplace_Book_CPB_. If you're a writer, researcher, or journalist, perhaps Steven Johnson's perspective may be interesting to you instead: https://stevenberlinjohnson.com/the-glass-box-and-the-commonplace-book-639b16c4f3bb

Collect interesting passages, quotes, and ideas as you read. Keep them in a notebook and call it your commonplace book. If you like call these your "fleeting notes" as some do.

As you do this, start building an index of subject headings for your ideas, perhaps using John Locke's method (see: https://publicdomainreview.org/collection/john-lockes-method-for-common-place-books-1685).

Once you've got this, you've really mastered the majority of what a zettelkasten is and have a powerful tool at your disposal. If you feel it's useful, you can add a few more tools and variations to your set up.

Next instead of keeping the ideas in a notebook, put them on index cards so that they're easier to sort through, move around, and re-arrange. This particularly useful if you want to use them to create an outline of your ideas for writing something with them.

Next, maybe keep some index cards that have the references and bibliographies from which your excerpting and note taking comes from. Link these bibliographical cards to the cards with your content.

As you go through your notes, ideas, and excerpts, maybe you want to further refine them? Write them out in your own words. Improve their clarity, so that when you go to re-use them, you can simply "excerpt" material you've already written for yourself and you're not plagiarizing others. You can call these improved notes, as some do either "permanent notes" or "evergreen notes".

Perhaps you're looking for more creativity, serendipity, and organic surprise in your system? Next you can link individual notes together. In a paper system you can do this by following one note with another or writing addresses on each card and using that addressing system to link them, but in a digital environment you can link one note to many multiple others that are related. If you're not sure where to start here, look back to your subject headings and pull out cards related to broad categories. Some things will obviously fit more closely than others, so be more selective and only link ideas that are more intimately connected than just the subject heading you've used.

Now when you want to write or create something new on a particular topic, ask your slip box a question and attempt to answer it by consulting your index. Find cards related to the topic, pull out those and place them in a useful order to create an outline perhaps using the cross links that already exist. (You've done that linking work as you went, so why not use it to make things easier now?) Copy the contents into a document and begin editing.

Beyond the first few steps, you're really just creating additional complexity to a system to increase the combinatorial complexity of juxtaposed ideas that you could potentially pull back out of your system for writing more interesting text and generating new ideas. Some people may neither want nor need this sort of complexity in their working lives. If you don't need it, then just keep a simple commonplace book (or commonplace card file) to remind you of the interesting ideas and inspirations you've seen and could potentially reuse throughout your life.

The benefit of this method is that beyond creating your index, you'll always have something useful even if you abandon things later on and quit refining it. If you do go all the way, concentrate on writing out just two short solid ideas every day (Luhmann averaged about 6 per day and Roland Barthes averaged 1 and change). Do it until you have between 500 and 1000 cards (based on some surveys and anecdotal evidence), and you should begin seeing some serendipitous and intriguing results as you use your system for your writing.

We should acknowledge that that (visual) artists and musicians might also keep commonplaces and zettelkasten. As an example, Eminem keeps a zettelkasten, but it is so minimal that it is literally just a box and slips of paper with no apparent organization beyond this. If this fits your style and you don't get any value out of having cards with locators like 3a4b/65m1, then don't do that useless work. Make sure your system is working for you and you're not working for your system.

Sadly, it's generally difficult to find a single blog post that can accurately define what a zettelkasten is, how it's structured, how it works, and why one would want one much less what one should expect from it. Sonke Ahrens does a reasonably good job, but his explanation is an entire book. Hopefully this distillation will get you moving in a positive direction for having a useful daily practice, but without an excessive amount of work. Once you've been at it a while, then start looking at Ahrens and others to refine things for your personal preferences and creative needs.

Actually this is like a lot of content creator stuff, because it is hard to believe that you can do the unconventional lifestyle

Virtuous justification for cheating.

Defines Deontology as an ethical theory based on duties, rights, or principles rather than consequences.

to me, success is not achieving a desired outcome. it is designing a process that you follow, and tweaking that process over time to make it better and better, so that you succeed more often. it's not individual successful outcomes; it's designing a process or system that makes doing the thing that leads to success just-challenging enough, and just-easy enough, that you'll keep on doing it, get better at it over time, and learn from it over time. success is working on the process, it's not meeting a goal.

Reviewer #3 (Public Review): 

I think this is an interesting study on an important topic. I agree that there is not enough research to understand how the dopaminergic system interfaces with goal-directed planning, and I like the focus on specific types of dopamine receptors. It is interesting that they seem to find a specific effect on just the dopamine antagonist. I also appreciate the clarity with which the authors describe this field of research and their results. However, I also feel that there are several concerns with this paper, both in terms of framing and in terms of the experimental design and analysis. For completeness, I must note that I am not a dopamine expert. 

I felt that the introduction of the paper did not sufficiently motivate the focus on the comparison between neurotransmitters systems, and (for the dopaminergic system) the distinction between D1/D2 receptors. Why is the mapping between stability/flexibility and D1/D2 receptors important? How does this relate to model-based control? Why do the authors predict that model-based control would increase when D2 receptors are blocked? If the hypothesis is about contrasting the contribution of D1 and D2 receptors to goal-directed control, why did the authors not use antagonists directly targeting these two systems? 

In addition, the predictions that are more explicit, for example, that blocking D2 receptors increases MB control by stabilizing goal-relevant information, are fairly specific. However, the current version of the two-step task is not amenable to testing such a specific hypothesis, because it doesn't allow us to measure the specific components of planning (e.g., maintaining goals, the representation of the structure, prospective reasoning). Moreover, MB control in this version of the two-step task is marked by flexibility, because it requires the agent to be sensitive to switching starting states. 

The predictions for the opioid system are also lacking. Why are the authors targeting this system? Why are they comparing the effects of the D2 antagonist with the opioid agonist? Why do the authors predict that amisulpride should have a stronger effect than naltrexone? In my opinion, these predictions were not sufficiently laid out, which made it difficult to appreciate the authors' motivation to run the study. 

Related to this, I felt that the introduction was a bit too quiet on the genetic markers. Their discussion in the results was a bit surprising, and it wasn't quite clear why the authors decided to investigate these interaction effects. 

I found some of the core results confusing. Most importantly, why does amisulpride make people less like to stay after a reward when the first-stage state is the same? When first-stage states repeat, both an MB agent and an MF agent will be more likely to stay after a reward. To me, this kind of behavior doesn't seem particularly model-based. Why does this behavior occur under amisulpride? I was surprised that the authors did not really address it. 

With regards to the design, it is unfortunate that the order of drug administration is not counterbalanced. As far as I understand, model-based control is always measured without a drug in the first session, and then with the drug (or placebo) in the second. The change between sessions is then tested for all three conditions. Of course, it is possible that the increase in model-based control in the amisulpride condition is only driven by the drug. However, given the lack of counterbalancing, it's also possible that amisulpride increases model-based control only after the experience with the task. That is, if the authors had counterbalanced the drug effect, they may have found that amisulpride had a different effect if it was administered in the first session. That would have changed their interpretation quite a bit! As it stands, they are unable to verify their (admittedly simpler) hypothesis that there is only a main effect.

Exponential change can happen if social tipping points are triggered by a few influencers have a change of heart because they have become educated on how the collective illusion operations, and how congruency of these few influencers can cause exponential change.

One of the key triggers for social tipping points is shifting the values of influential people under the spell of collective illusion from incongruency to congruency.

If people who are incongruent can imagine how our societies would flourish were we as individuals more congruent, this could be a powerful leverage point for system change that dispells the collective illusion.

Politicians are exquisitely sensitive to collective illusions, and that means educating politicians is a leverage point!

In summary, the collective illusions principle applies at the level of two people all the way up to nations.

What we know about a person thinks and feels and what the other person actually thinks and feels are two entirely different things.

This harkens back to Uexkull and Husserl and Umwelt and Lebenswelt respectively.

In summary, women don't do well in politics (and probably business) due to a collective illusion effect of the gatekeepers who believe everyone else thinks that they are rocking the boat.

Groups can influence a cult like power on individual group members.

Deep education of the concept of collective illusion by referring to their reference systems is critical...meet them where they are and scaffold on ideas they are familiar with.

In summary, the collective illusion hides the enormous common ground we share. By buying into the collective illusion, it becomes a self-fulfilling prophecy creating the very antagonism which it holds to exist.

From a Deep Humanity perspective, this research validates the approach of collectively finding the Common Human Denominators (CHD).

Introducing the concept of collective illusion within highly polarized context tends to reduce its conceptual understanding. The political impact of collective illusions is that it is a self-reinforcing feedback loop that drives further entrenchment, misunderstanding of one's own ingroup as well as the outgroup.

Collective illusions do great damage to society. Think about political polarization, climate change, the pandemic to name a few crisis marred by polarization.

Applying the conjoin technique, Rose et al discovered this surprising result: that while publicly, fame was perceived as the idea to conform to, privately, it was the lowest priority of all.

This is the key problem that makes current social media dangerous, it can be easily gamed due to this evolutionary shortcut of the brain, the fast system of biases aka Daniel Kahneman's research.

Collective illusion is having major harmful social impacts today, bring about polarization and conflict. It has also been called pluralistic ignorance and ignorance of universality.

Reviewer #3 (Public Review):

The authors present a systematic assessment of low complexity sequences (LCRs) apply the dotplot matrix method for sequence comparison to identify low-complexity regions based on per-residue similarity. By taking the resulting self-comparison matrices and leveraging tools from image processing, the authors define LCRs based on similarity or non-similarity to one another. Taking the composition of these LCRs, the authors then compare how distinct regions of LCR sequence space compare across different proteomes.

The paper is well-written and easy to follow, and the results are consistent with prior work. The figures and data are presented in an extremely accessible way and the conclusions seem logical and sound.

My big picture concern stems from one that is perhaps challenging to evaluate, but it is not really clear to me exactly what we learn here. The authors do a fine job of cataloging LCRs, offer a number of anecdotal inferences and observations are made - perhaps this is sufficient in terms of novelty and interest, but if anyone takes a proteome and identifies sequences based on some set of features that sit in the tails of the feature distribution, they can similarly construct intriguing but somewhat speculative hypotheses regarding the possible origins or meaning of those features.

The authors use the lysine-repeats as specific examples where they test a hypothesis, which is good, but the importance of lysine repeats in driving nucleolar localization is well established at this point - i.e. to me at least the bioinformatics analysis that precedes those results is unnecessary to have made the resulting prediction. Similarly, the authors find compositional biases in LCR proteins that are found in certain organelles, but those biases are also already established. These are not strictly criticisms, in that it's good that established patterns are found with this method, but I suppose my concern is that this is a lot of work that perhaps does not really push the needle particularly far.

As an important caveat to this somewhat muted reception, I recognize that having worked on problems in this area for 10+ years I may also be displaying my own biases, and perhaps things that are "already established" warrant repeating with a new approach and a new light. As such, this particular criticism may well be one that can and should be ignored.

That overall concern notwithstanding, I had several other questions that sprung to mind.

Dotplot matrix approach<br /> The authors do a fantastic job of explaining this, but I'm left wondering, if one used an algorithm like (say) SEG, defined LCRs, and then compared between LCRs based on composition, would we expect the results to be so different? i.e. the authors make a big deal about the dotplot matrix approach enabling comparison of LCR type, but, it's not clear to me that this is just because it combines a two-step operation into a one-step operation. It would be useful I think to perform a similar analysis as is done later on using SEG and ask if the same UMAP structure appears (and discuss if yes/no).

LCRs from repeat expansions<br /> I did not see any discussion on the role that repeat expansions can play in defining LCRs. This seems like an important area that should be considered, especially if we expect certain LCRs to appear more frequently due to a combination of slippy codons and minimal impact due to the biochemical properties of the resulting LCR. The authors pursue a (very reasonable) model in which LCRs are functional and important, but it seems the alternative (that LCRs are simply an unavoidable product of large proteomes and emerge through genetic events that are insufficiently deleterious to be selected against). Some discussion on this would be helpful. it also makes me wonder if the authors' null proteome model is the "right" model, although I would also say developing an accurate and reasonable null model that accounts for repeat expansions is beyond what I would consider the scope of this paper.

Minor points<br /> Early on the authors discuss the roles of LCRs in higher-order assemblies. They then make reference to the lysine tracts as having a valence of 2 or 3. It is possibly useful to mention that valence reflects the number of simultaneous partners that a protein can interact with - while it is certainly possible that a single lysine tracts interacts with a single partner simultaneously (meaning the tract contributes a valence of 1) I don't think the authors can know that, so it may be wise to avoid specifying the specific valence.

The authors make reference to Q/H LCRs. Recent work from Gutiérrez et al. eLife (2022) has argued that histidine-richness in some glutamine-rich LCRs is above the number expected based on codon bias, and may reflect a mode of pH sensing. This may be worth discussing.

Eric Ross has a number of very nice papers on this topic, but sadly I don't think any of them are cited here. On the question of LCR composition and condensate recruitment, I would recommend Boncella et al. PNAS (2020). On the question of proteome-wide LCR analysis, see Cascarina et al PLoS CompBio (2018) and Cascarina et al PLoS CompBio 2020.

Author Response

Reviewer #1 (Public Review):

This work offers a simple explanation to a fundamental question in cell biology: what dictates the volume of a cell and of its nucleus, focusing on yeast cells. The central message is that all this can be explained by an osmotic equilibrium, using the classical Van't Hoff's Law. The novelty resides in an effort to provide actual numbers experimentally.

In this work, Lemière and colleagues combine physical modeling and quantitative measures to establish the basic principles that dictate the volume of a cell and of its nucleus. By doing so, they also explain an observation reported many times and in many different types of cells, of a proportionality between the volume of the cell and of its nucleus. The central message is that all this can be explained by an osmotic equilibrium, using the classical Van't Hoff's Law. This is because, in yeast cells, while the cell has a wall that can contribute to the equilibrium, the nucleus does not have a lamina and there is thus no elastic contribution in the force balance for the nucleus, as the authors show very nicely experimentally, using both cells and protoplasts and measuring the cell and nucleus volume for various external osmotic pressures (the Boyle Van't Hoff Law for a perfect gas, also sometimes called the Ponder relation) ¬- this was performed before for mammalian cells (Finan et al.), as cited and commented in the discussion by the authors, showing that mammalian cells have no significant elastic wall (linear relation) while the nucleus has one (non linear relation). This is well explained by the authors in the discussion. It is one of the clearer experimental results of the article. Together, the data and model presented in this article offer a simple explanation to a fundamental question in cell biology. In this matter, the principles are indeed seemingly simple, but what really counts are the actual numbers. While this article sheds some light on this aspect, it does not totally solve the question. The experiments are very well done and quantified, but some approximations made in the modeling are questionable and should at least be discussed in more length. Overall, this article is extremely valuable in the context of the recent effort of the cell biology and biophysics communities to understand the fundamental question of what dictates the size of cells and organelles. I have a few concerns detailed below. Importantly, there are many very interesting points of the article that I am not discussing below, simply because I completely agree with them.

1) The main concern is about the assumption made by the authors that the small osmolytes do not count to establish the volume of the nucleus. It was shown that small osmolytes such as ions are a vast majority of the osmolytes in a cell (more than ten times more abundant than proteins for example, which represent about 10 mM, for a total of 500 mM of osmolytes). This means that just a small imbalance in the amount of these between the nucleus and cytoplasm might have a much larger effect than the number of proteins, which is the osmolyte that authors choose to consider for the nuclear volume.

The point of the authors to disregard small osmolytes is that they can freely diffuse between the cytoplasm and the nucleus through the nuclear pores. They thus consider that the nuclear volume is established thanks to the barrier function of the nuclear envelope, which would retain larger osmolytes inside the nucleus and that the rest is balanced. This reasoning is not correct: for example, the volume of charged polymers depends on the concentration of ions in the polymer while there is no membrane at all to retain them. This is because of an important principle that the authors do not include in their reasoning, which is electro-neutrality.

Because most large molecules in the cell are charged (proteins and also DNA for the nucleus), the number of counterions is large, and is probably much larger than the number of proteins. So it is hard to argue that this could be ignored in the number of osmotically active molecules in the nucleus. This is known as the Donnan equilibrium and the question is thus whether this is actually the principle which dictates the nuclear volume.

The question then becomes whether the number of counterions differs between the cytoplasm and the nucleus, and more precisely whether the difference is larger than the difference considered by the authors in the number of proteins.

How is it possible to estimate this number? One of the numbers found in the literature is the electric potential across the nuclear envelope (Mazanti Physiological Reviews 2001). The number is between 1 and 10 mV, with more cations in the nucleus than in the cytoplasm. This number could correspond to much more cations than the number of proteins, although the precise number is not so simple to compute and the precision of the measure matters a lot, since there is an exponential relation between the concentrations and the potential.

This point above is simply made to explain that the authors cannot rule out the contribution of small osmolytes to the nuclear volume and should at least leave this possibility open in the discussion of their article.

As a conclusion, I totally agree with equation 3 which defines the N/C ratio, but I think that the Ns considered might not be the number of large macromolecules which cannot pass the nuclear envelope, but rather the small ones. Whether it is the case or not and what is actually the important species to consider depends on the actual numbers and these numbers are not established in this article. It is likely out of the scope of the article to establish them, but the point should at least be discussed and left open for future studies.

We appreciate these excellent points made by the reviewer and their numerous consultants. We amend the discussion of colloid osmotic pressure in the text to reflect these points.

2) The authors refer to the notion of colloidal pressure, discussed in the review by Mitchison et al. This term could be confusing and the authors should either explain it better or just not use it and call it perfect gas pressure or Van't Hoff pressure. Indeed, what is meant by colloidal pressure is simply the notion that all molecules could be considered as individual objects, independently of their size, and that it is then possible to apply the Van't Hoff Law just as it was a perfect gas, hence the notion of 'colloidal' pressure, which would be the osmotic pressure of all the individual molecules. The authors might want to discuss, or at least mention, that it is a bit surprising that all these crowded large macromolecules would behave like a perfect osmometer and that the Van't Hoff law applies to them. Alternatively, it could be simpler to consider that what actually counts for the volume is mostly small freely diffusing osmolytes, to which this law applies well, and which are much more numerous.

3) Very small point: on page 7 the authors refer to BVH's Law (Nobel, 1969). It is not clear what they mean. If they refer to the Nobel prize of Van't Hoff, it dates from 1901 (he died in 1911) and not 1969. I am not sure if there is something in one of the Nobel prizes delivered in 1969 which relates to this law. I checked but it does not seem to be the case, so it is probably a mistake in the date.

The citation is correct. It's a JTB paper by Park S. Nobel describing the BHV relation in biology.

4) On page 11, bottom, the result of the maintenance of the N/C ratio in protoplast is presented as an additional result, while it is a simple consequence of the previous results: both the cell and nuclear volume change linearly with the external osmotic pressure, so it is obvious that their ratio does not change when the external pressure is changed.

This result was not trivial. Although both cells and nuclei volume change linearly with the inverse of the external osmotic concentration in protoplasts, it was not obvious whether the two volumes change with the same proportion (ie same slope on the BVH graph).

Another result, not commented by the authors, is that this should be true only in protoplasts, since in whole cells, the cell wall is affecting the response of the cell volume, but not the nucleus, so the ratio should change.

In whole cells, the maintenance of the N/C ratio is in fact also maintained, consistent with the model. This result is now clarified in the manuscript (Figure 1C and D plus Figures 3D and S1C).

5) The results in Figure 5, with the inhibition of export from the nucleus, are presented as supporting the model. It is not really clear that they do. First the effect is very small, even if very clear. Again, the numbers matter here, so the interpretation of this result is not really direct and more calculation should be made to understand whether it can really be explained by a change of number of proteins. The result in panel F is even more problematic. The authors try to argue that the nucleus transiently gets denser, based on the diffusion of the GEMs and then adapts its density. It rather seems that it is overall quite constant in density, while it is the cell which has a decreasing density ¬- maybe, as suggested by the authors, because there are less ribosomes in the cytoplasm, so protein production is reduced. This could have an indirect effect on the number of amino acids (which would then be less consumed). A recent article by Neurohr et al (Trends in cell biology, 2020) suggests that such an effect can lead to cell dilution, in yeast, because the number of amino acids increases. In this particular case, this increase would affect the nuclear volume rather than the cell volume because of the presence of the cell wall and the rather small change.

We agree that there are different possible interpretations for these results. We have carefully reconsidered the interpretation and have rewritten the entire text for Figure 5

6) Page 16: it seems to me that the experiments presented in the chapter lines 360 to 376, on the ribosomal subunits, simply confirm that export is impaired, and they do not really contribute to confirm the hypothesis of the authors that it is the number of proteins in the nucleus which counts.

We agree. We highlight the ribosomal subunit proteins as they are very abundant nuclear shuttling proteins that provide a good example for the dynamics of nuclear protein accumulation.

The next paragraph with the estimation of the number of proteins in the nucleus and cytoplasm and how they change relatively upon export inhibition also appears to mostly demonstrate that export has been inhibited.

The authors propose to use the number they find, 8%, to compare it to the change in the N/C ratio, which is of the same order. Given how small these numbers are, and the precision of such measures, it is very hard to believe that these 8% are really precise at a level which could allow such a comparison. The authors should really estimate the precision of their measures if they want to claim that. It is more likely that what they observe is a small but significant change in both cases; a small change means it is small compared to the total, so it is a fraction of it, and it is measurable, which means it is more than just a few percent, which is usually not possible to measure. So it means that it is in the order of 10%. This is the typical value of any small but measurable change given a method for the measure which can detect changes around 10%. In conclusion, these numbers might not prove anything.

It could also be that the numbers match not just by chance, but that the osmolyte which matters is, for this type of experiment, changing in proportion to the amount of proteins (which would be possible for counter ions for example). But determining all that requires precise calculations and additional measures. It is thus more a matter of discussion and should be left more open by the authors.

We agree that these measurements are not so precise. We have carefully reworded this section and removed these specific comparisons.

Reviewer #2 (Public Review):

The goal of the paper is to test the idea that colloidal osmotic pressure controls nuclear growth as suggested by Tim Mitchison in a recent review.

In fleshing out the idea, Lemiere and colleagues develop a simple mathematical model that focuses on the forces generated by the movement of macromolecules across the nuclear-cytoplasmic boundary, ignoring any contribution of ions or small molecules which they assume equilibrate across the nuclear envelope. In testing this model, they focus their quantitative analysis on the response of cells that lack a wall (protoplasts) to osmotic shocks and to perturbations of nuclear export, protein synthesis and symmetric cell division. They also analyse the motion of small 40nm particles to test how diffusion is affected by these perturbations in both compartments.

Their analysis leads them to make some important observations that suggest that the system is even simpler than they might have hoped, since under the conditions tested nuclei (which lack lamins) behave as ideal osmometers. That is, the nuclei and cytoplasm grow and shrink in concert following sudden osmotic shocks. This suggests that the tension in the nuclear envelope, which gives nuclei their spherical shape, plays no role in constraining nuclear size.

While most of the paper's claims are well supported by their data under the assumptions of the model, there are a few claims that are less convincing.

For example, while their data are consistent with the idea that cells regulate their nuclear/cytoplasmic size ration using an adder type mechanism, in which a fix ratio of nuclear and cytoplasmic proteins are synthesised per unit time as cells grow, this has not been rigorously put to the test. In addition, while the diffusion analysis is very interesting, it does not fully support the authors' simple model linking diffusion, molecular crowding and colloidal osmotic pressure, something that could be more thoroughly discussed in the manuscript.

We added new data showing that slowing growth rate leads to a proportionate decrease in N/C ratio correction. This strengthens this portion of the paper.

We have added an improved discussion of the GEMs data and its limitations.

Reviewer #3 (Public Review):

This manuscript by Lemière and colleagues presents a view on how nuclear size is set by simple physical principles. The first part of the work describes a theoretical framework with the nucleus and the cell as two nested osmometers. Using fission yeast as a model, the authors then show that protoplasts and nuclei behave as ideal osmometers, i.e. show linear changes in volume upon change in external osmotic pressure. Consequently, the nuclear to cell volume ratio remains constant upon osmotic changes, but increases upon block of nuclear export, which leads to higher nuclear protein contents. Measurements of diffusion in the cytoplasm and nucleoplasm back these data. Finally, in the last part of the manuscript, the authors show that nuclear growth through a passive osmotic model can explain the previously described homeostasis of nuclear volume.

The manuscript is clearly written, and the data are clean and overall solid. I very much liked the simple view on the phenomenon of constant nuclear to cytosol ratio and the mix of modelling and experiments supporting the model that nuclear size is set passively by osmotic principles.

There are however a few points that are slightly at odds with the model and/or require further explanation to make the model compelling and discuss it in view of previous findings.

1) Isn't the finding that diffusion rates are faster in the nucleus (line 298, Fig S4C), indicating lower crowding in the nucleus, at odds with the finding that the non-osmotic volumes are similar in the two compartments? If the nucleus is less crowded, does this not suggest a lower pressure than the cytosol? I would also like to see this finding appear in Figure 4, which only reports on the normalized diffusion rates in both nuclei and cytosol.

We have added this figure to the main Figure 4, as requested. We agree that this raises some interesting questions. Our current interpretation is that composition of the nucleoplasm and cytoplasm are different and therefore affect GEMs diffusion and colloid osmotic pressure slightly differently.

2) Similarly, I don't understand the observed change in diffusion rates of GEMs upon LMB treatment (Fig 5F). If the nucleus behaves as an ideal osmometer, then any change in protein density between the nucleus and the cytosol, leading to change in osmotic pressure, will lead to a change in nuclear size that should re-equilibrate the osmotic pressures between the two compartments. The prediction would thus be that, if LMB treatment does not change overall protein concentration, at equilibrium there is no change in either osmotic pressure or density as measured by GEM diffusion rates. This is indeed illustrated by the constant normalized non-osmotic volume of the nucleus after LMB treatment. Is the change in diffusion rates perhaps only transient until a new steady state is reached? Or is there a change upon total protein content in the cell after LMB treatment?

3) In the experiments labelling proteins with FITC, are the reported values really those of protein concentrations or rather protein amounts? Isn't the enlargement of the nucleus upon LMB treatment compensating for this increase in amounts, returning the nucleus to a similar concentration as before treatment? A change in concentration is not in agreement with the reported constant non-osmotic volume of the nucleus.

These measurements of intensity are of concentrations. We add in the text this prediction that changes in concentration will be compensated for by swelling in nuclear volume and now interpret the data in light of this prediction. We add new data that total FITC staining for protein and RNA shows no change in concentration in compartments, consistent with this model.

4) The authors state that "a previous paper proposed a model for N/C ratio homeostasis based upon an active feedback mechanism (Cantwell and Nurse, 2019)" (lines 471-472). My understanding of this previous study is that nuclear size was proposed to be set by a limiting component, itself proportional to cell volume. No feedback was postulated. This previous model is in fact not too different from what the authors propose here, with the previously proposed limiting component now corresponding to the nuclear macromolecules that produce colloid osmotic pressure and thus set nuclear size. Though the present study goes significantly further in presenting the passive role of osmosis in setting nuclear size, it is a misrepresentation to portray this previous model as fundamentally different. Furthermore, it is not clear whether the new osmotic pressure-based model produces a better fit than the previous 'limiting component model'. Figure 7E here is very similar to Fig 4I in Cantwell and Nurse 2019, but it is difficult to judge the similarity of the fits.

The Cantwell and Nurse paper tested two models. The first was based upon nuclear growth being a fraction of cell growth. This model is qualitatively similar to ours. However, they discarded this initial model because it fitted poorly with their data. They then went to propose a second model, which contains a critical equation in which nuclear growth rate is a function of the N/C ratio, i.e. the system is sensing the N/C ratio and adjusting nuclear growth rate as a function of the N/C ratio. In other words, this is a feedback mechanism. The Cantwell paper does not describe this "feedback" term explicitly in the text, but it is clearly present in the equations. Therefore, our model which lacks any feedback term is fundamentally different from the Cantwell limiting component model.

We show that our model fits our data much better than the Cantwell model. We believe that the different views in these studies arise from differences in the experimental data. These differences may arise from two technical differences: 1) Their use of binning could be responsible for flattening the nuclear growth rate as a function of the nuclear volume at start. 2) Their estimates of cell and nuclear volumes using a 2D image and geometric assumptions may be less accurate than our automated 3D volume method.

5) If nuclear size is set purely by osmotic regulation, how do you explain that mutants in membrane regulation (such as nem1 and spo7, see Kume et al 2017; or lem2, see Kume et al 2019) previously shown to have an enlarged nucleus, display increased nuclear size?

This is an interesting question that we are currently pursuing. It is likely that these mutants affect multiple processes besides nuclear envelope expansion. For example, at least some of these mutants have altered chromatin organization could cause increase in colloid pressure. There may also be significant defects in chromosome segregation, which leads to production of different-sized nuclei with abnormal number of chromosomes. Some of the N/C ratio defects reported in these papers may arise from their 2D measurement methods, which are not accurate for misshapen nuclei. In our preliminary results, lem2 mutants do not have N/C ratio defects.

As an aside, I've always wrestled with the term 'universal' in UDL - simply because it's not universal (as in applicable to all). Instead, it's applicable to as many as possible. I'm conscious that this might be an impossibility - it just seems like the wrong word.

Is this arguing that she knows that what she's doing is not just, she just doesn't know what actually would be just, or she just doesn't care that it's not just?

The things that these men carry are not just weapons, ammo, or rations. It's not just photographs, lucky rocks, or rabbit feet. These men carry emotions like love, grief, regret, and fear. Every burden these men carry is added weight, even if it's not physically. Their memories and their imagination are almost the only way for them to escape the torture they endure every day.

It's strange how the same people who imagine a disarmed populace as a good thing are playing catch-up to arm Ukranian civilians against a military. I've lost count of the children massacred by militaries that are the only groups of people magically trustworthy enough to be armed apparently.

If you left a murderer alone with a room full of kids and a knife for 77 minutes, you'd have the same result - and if you're a student of recent history, you'd know that's exactly the kind of attack that has happened time and again in gun-free victim zones around the world.

To address this issue properly, citizens must understand the #JustPowers Clause of The Declaration of Independence, the foundation that the US Constitution is laid upon; and a universal document that recognizes the rights of ALL humans.

Put simply, it states that neither you, nor I, nor anyone else may justly grant powers to others that we do not have.

If you or I stole our neighbors' firearms, even if we claimed it was for "safety" or "the common good", we'd face criminal charges. We all know this, and the evidence is in our conduct.

Instead, why not focus just powers such has holding adults responsible for the safety of others accountable for negligence?

He is saying that people try not to suffer but sometimes there is no way out of it.

He is saying here that heroin makes him feel in control just like the woman's voice which he is saying he has to have sometimes.

This is another mention about darkness which the theme across the text has portrayed.

Sonny's friend and the narrator really go back and forth here. The friend thinks if they let him out then it's just going to go back to where he started.

Here is another mention of the motifs of light and darkness. I saw that someone earlier mentioned a theme that every person will experience suffering and I feel this passage describes that idea.

He has children growing up the same way he did. He knows that his children are going to struggle the way he and Sonny did, and he feels guilty and disappointed he couldn't provide them with better. He also feels guilty for bringing Sonny back into the suffering.

lots of imagery

Sonny's friend and the narrator really go back and forth here. The friend thinks if they let him out then it's just going to go back to where he started.

true! But it's also important to know that without American military terror neoliberalism would not have maintained its reign. like colonialism, violence, not just epistemology alone, is the driving force. many chinese challenges to colonialism, epistemological & philosophical challenges failed, not because of lack of imagination & sophistication but because ... 秀才遇見兵有理說不清

Niantic 首席执行官 John Hanke：

“大多数人使用来自大公司之一的身份。它是我们所做的一切、应用程序和在线活动的主要通行证。从某种意义上说，当我们这样做时，我们正在出卖我们的数字灵魂。这是我们养成的习惯，也是目前的工作方式。但是 web3 将使我们能够拥有自主权的身份。因此，您可以使用不会泄露您的个人信息的 web3 系统，而不是使用其中一个按钮，并且不会在您和您正在使用的服务之间放置一个中间人，该中间人可能会以某种方式拦截或存储信息你可能不想发生。所以对我来说，这真的是它应该工作的方式。

[…]

正是这样的用途让我想押注它是未来互联网不可或缺的一部分。区块链在这种情况下实际上很有用，因为没有中央权威。”

Author Response

Reviewer #1 (Public Review):

Using Tet-off system, Kir2.1 was expressed (or not) during the key time of callosal development from E15 to P15. Restoring activity either by adding Dox during a critical period from P6 to P15 or using DREADDs from P10-14 could rescue the callosal projection to the cortex, whereas later restoration of activity (with Dox) was not successful. Did this successful rescue lead to normal activity? Calcium imaging in animals with Kir2.1 had low levels of any kind of activity, both highly correlated and low correlation, but P6-13 dox treatment partially restored only low-correlation activity and not high correlation activity at P13. The effects of DREADDs on activity was not similarly measured though it was effective for at least partially restoring the callosal projection.

Overall this study builds on earlier findings regarding the importance of neuronal activity in the formation of a normal callosal projection, using in utero electroporation which is particularly well suited for this subject. It makes the case very compellingly that near-normal callosal connectivity can be produced if activity is permitted during a critical period window from P6 or P10 to P15, though the exact timing of this window is imprecise because the elimination of Kir expression was not systematically quantified. For transmembrane proteins like channels it can often take many days for protein expression to completely abate.

We thank the reviewer for their positive evaluation and the constructive comments. Based on the comment on Kir expression, we conducted new experiments using pTRE-Tight2Kir2.1EGFP, with which EGFP signals reflect localization of over-expressed Kir2.1, and examined when the expression of Kir2.1EGFP went down after Dox treatment at P6. At P6 (before Dox treatment), the signals of Kir2.1EGFP (stained with anti-GFP antibody) were observed in the periphery of the soma and along dendrites, implying that Kir2.1EGFP was transported to the cellular membrane. At P10 and P15 (4 days and 9 days after Dox treatment), Kir2.1EGFP signals were not observed in the periphery of the soma and along dendrites. We noted that low-level green signals were observed in the central part of the cell body. These may stem from low-level expression of Kir2.1EGFP in nuclei or cytosol even after Dox treatment. Alternatively, and more likely, these may reflect bleed-through of RFP signals into GFP channel. Overall, we confirmed that Kir2.1 proteins that were localized to the cellular membrane were largely down-regulated. We described these observations in detail in the figure legend of Figure 1-figure supplement 3, and added the result as Figure 1-figure supplement 3.

I found the quantification of the callosal projection to be rather minimal and the normalization approach not entirely transparent. For example does activity from P10-15 restore the full normal PATTERN of callosal connectivity or merely the density of input overall?

We thank the reviewer for this comment. Based on the comment, we added analyses of the pattern of callosal projections; the width of callosal axon innervation zone in layers 2/3 and 5, and densitometric line scans across all cortical layers. Our original quantification showed that the density of callosal axons reaching their target layer (i.e. cortical layer 2/3) is almost recovered in P6-P15 DOX condition (Fig1B-D), but new analyses suggest some aspects of callosal axon projections (the width of the innervation zone in layer 2/3 and 5 (Figure 1-figure supplement 4A,B), and lamina specific innervation pattern (Figure 1-figure supplement 4C)) might be only partially recovered. We have added these new results as Figure 1-figure supplement 4. In future study, we would like to assess the effect of the manipulations at finer resolution by 3D morphological reconstruction of axons of individual neurons.

Also in the discussion it would be nice to more clearly establish whether activity is thought to be maintaining a projection already formed by P10 or permitting the emergence of such a pattern.

Thank you for the suggestion. We have added thorough discussions about this point as follows. Page 7, lines 198-208:

“In the previous study, we showed that callosal axons could reach the innervation area almost normally under activity-reduction, and that the effects of activity-reduction became apparent afterwards (Mizuno et al., 2007). Callosal axons elaborate their branches extensively in P10P15 (Mizuno et al., 2010), and axon branching is regulated by neuronal activity (Matsumoto and Yamamoto, 2016). It is likely that activity is required for the processes of formation, rather than the maintenance of the connections already formed by P10, but the current study employed massive labeling of callosal axons which is not suited to clarify this. In addition, the restoration of activity in the Tet-off (Figure 1) or DREADD (Figure 2) experiment may not completely rescue the ramification pattern of individual axons. Single axon tracing experiments (Mizuno et al., 2010; Dhande et al., 2011) would be required to clarify this. Nonetheless, our findings suggest that callosal axons retain the ability, or are permitted, to grow and make region- and lamina-specific projections in the cortex during a limited period of postnatal cortical development under an activity-dependent mechanism.”

The calcium imaging is a valuable validation of the Kir expression approach, but it the study here appears to overinterpret what may simply be an intermediate level of activity restoration rather than a specific restoration of L events, as it seems that L events would be the most likely to occur under conditions of reduced overall activity. One possibility is that the absence of H events at P13 in the calcium is due to residual Kir expression creating a drag on high level network activation rather than any more complicated change in patterned spontaneous activity/connectivity. The conclusions from this study regarding the permissive role of activity during a critical window and the lack of a requirement for highly correlated activity are valuable, even if somewhat imprecise on both counts. The authors should probably refrain from use of the term patterned activity given that this was measured but not systematically compared to unpatterned spontaneous activity.

We thank the reviewer for this constructive comment. Based on this comment, we removed the term “patterned activity” throughout the manuscript and revised the title, abstract, introduction, results, and discussion extensively. For example, in the Discussion, we revised as follows.

“We have shown that the projections could be established even without fully restoring highly synchronous activity (Figure 4). L events, but not H events, were present in P13 cortex after Dox treatment at P6. L events may be sufficient for the formation of callosal projections. Alternatively, any form of activity with certain level(s) (i.e., “sufficiently” high activity with no specific pattern) could be permissive for the formation of callosal connections.”

Reviewer #2 (Public Review):

Tezuka et al. use in vivo manipulations of spontaneous activity to identify the activitydependent mechanisms of callosal projection development. Previous research of the authors' and other labs had shown that overexpressing the potassium channel Kir2.1, which reduces activity levels in the developing cortical network, blocks the formation of callosal connections almost entirely.

The current manuscript corroborates and extends these previous discoveries by:

1) Demonstrating that the effect of Kir overexpression can be rescued by pharmacogenetic network activation using DREADDs.<br /> 2) Revealing the requirement of network activity for the development of callosal projections during a particular developmental time window and by<br /> 3) Directly relating perturbed callosal development to the actual changes in activity patterns caused by the experimental manipulations.

Thus, this paper is important for our understanding of the role of neuronal activity in the development of long-range connections in the brain. In addition it provides strong evidence for a role of specific activity patterns in this process.

In general, the approach is very straightforward and the results clearly interpreted. Nevertheless, there are a few points to consider.

We thank the reviewer for these positive and supportive comments.

1) It is not clear in which cortical area(s) the in vivo 2-photon recordings were performed and in how far cortical areas that actually receive/send callosal projections were included or not in the analysis.

In response to this comment, we revised the text in the method section as follows.

“We aimed to record spontaneous neuronal activity in putative binocular zones in V1 (2.5 mm lateral of midline and 1 mm anterior of the posterior suture). Since the boundaries between V1 and higher visual areas, AL/LM are not as obvious as those in adult, our recordings likely contained juxtaposed lateral monocular V1 and AL/LM as well.”

Based on our colleaguesʼ unpublished observations, V1 and AL/LM can be distinguished solely by spontaneous activity patterns even before eye-opening. They also found frequencies of spontaneous activity are similar across mono/binocular regions of V1 and AL/LM (Murakami, Ohki, et al. unpublished). Thus, our results should hold even with the variability in recording sites.

2) It is not discussed what the duration of the CNO effect is. Do daily injections rescue activity patterns for 24 hours or a significant proportion of this period?

In response to this critical comment, we revised the text in the method section as follows.

“A previous study showed that an intraperitoneally injected CNO was effective (in terms of increasing activity) for about 9hrs (Alexander et al., 2009). The “partial rescue” effect we observed (Figure 2) may suggest that activity was not fully restored during 24hrs by our daily CNO injections.”

Reviewer #3 (Public Review):

The manuscript by Tezuka adds to an emerging story about the role of activity in the formation of callosal connections across the brain. Here, the authors show that they can use a TET system to switch off the activity of an exogenous potassium channel, in order to probe when activity might be necessary or sufficient for the formation of callosal connections. The authors find that artificial restoration of activity with DREADS is sufficient to rescue the formation of callosal connections, and that there is a critical period (somewhere between P5-P15) where activity must occur in order for the connections to form within the cortex. Finally, the authors show that when the potassium channel is removed during the critical period, the cortex exhibits activity, but few highly synchronous events. These results indicate that it is activity in general and not specifically highly synchronous activity that is necessary for the final innervation of the callosal cortex.

In general, the study is well done, and the writeup is polished, well summarized. The figures are solid. There are only a few criticisms/suggestions.

We thank the reviewer for the positive evaluation.

Major issue: Have the authors demonstrated a requirement for "patterned spontaneous activity"?

The authors claim variously in the abstract ("a distinct pattern of spontaneous activity") and in the results (pg 6, "our observations indicate that patterned spontaneous activity") and discussion (pg 6, "we demonstrated that patterned spontaneous activity") that it is "patterned" spontaneous activity that is key for the formation of callosal connections. However, when I was reading the paper, I came to the opposite conclusion: that any sufficiently high spontaneous activity is sufficient for the formation of these connections.

The authors showed that relieving the KIR expression from P5-15 allows the connections to form; however, in Figure 4, the authors show that the nature of the activity produced in the cortex (in terms of mixtures of H and L events) is very different. Nevertheless, the connections can form. Further, the authors showed that increasing activity when KIR is expressed using DREADS restores the connections. The pattern of activity produced by this DREADS + KIR expression is likely to be very different from the pattern of activity of a typically-developing animal. In total, I thought that the authors demonstrated, quite nicely, that it is just the presence of sufficient activity that is key to the innervation of the contralateral cortex. (It's not cell autonomous, as the authors showed before; there seems to be a "sufficient activity" requirement).

Therefore, I think the authors should remove references to the requirement of patterned activity and instead say something about sufficiently high activity (or some characterization that the authors choose). I think they've shown quite nicely that a specific pattern of the spontaneous activity is not important.

We thank the reviewer for this very important insight and interpretation. After considering all the currently presented data again, we have come to agree with the interpretation stated by the reviewer. We removed the term “patterned activity” throughout the manuscript and revised the title, abstract, introduction, results, and discussion extensively. Nevertheless, we would not completely discard the possibility that specific patterns of spontaneous activity, such as L-events, could potentially have some active contribution to the development of projection circuits, and would like to further address this in future study.

For example, in the Discussion, we revised the text as follows.

“We have shown that the projections could be established even without fully restoring highly synchronous activity (Figure 4). L events, but not H events, were present in P13 cortex after Dox treatment at P6. L events may be sufficient for the formation of callosal projections. Alternatively, any form of activity with certain level(s) (i.e., “sufficiently” high activity with no specific pattern) could be permissive for the formation of callosal connections.”

erste grobe Beschreibung woraus ein PLN besteht

I feel like the time isn't empty so much as it's hollow. Maybe they're the same thing. But there's something there pretending to social substance that is just a pale imitation of the real thing.

Q: So, it's better to be at strategy rather than military action? Is that the point?

Whatever your end goal is try to get better at that. The less proxies the better.

People need to feel like they are in control but also some stressors or goals are required in order to feel alive (challenged and progression)

Earlier in the story it said, "I used to run into him from time to time and he'd often work around to asking me for a quarter or fifty cents." In this sentence it's providing evidence to that statement.

Another absurd page that suggests Alexa has feelings. In the strictest sense Alexa doesn't even qualify as a partial AI. It's just a glorified (although extremely helpful) lookup table. There is no reason to believe that even a true AI, such as a self-teaching, building and growing neural network (which Alexa is not), has feelings. Of what we know of feelings, the hard question of consciousness is only a prerequisite ... doesn't even guarantee having feelings, and even whether machines can be conscious is doubtful by many if not most experts of AI within existentialism. Even all the theories of consciousness are rooted in correlations that have little to do with scientific tenets, so to make the leap to an AI having feelings, let alone Alexa which isn't even a theoretical AI, is just a sad to see. At best we should not be "rude" to machine because it might be hard for some to distinguish between machine and a feeling thing. but in that case the problem is with the misperception that machines can feel, more than it is a problem with people being "rude" to machines.

This is just really horrible to validate a falsehood to children that Alexa does in fact have feelings. Really warped, really messed up. Of course children should be taught good manners, and by example no less, but I worry for a future where people can be manipulated by suggesting that a non-living thing has feelings, regardless whether it has an AI or not.

Note that a true AI has yet to be created ... only facsimile's exist, mostly of the expert-based AI which Alexa is, which doesn't even fit the definition of a partial AI, it's just a lookup table.

This isn't clear at all. It is non-obvious to consider any multiplicity in subjective experience unless you conflate privileged access with distinct subjectivity.

Continues to refute Fisher's view.

Humans are often the one vulnerable, just like eroding the bulwarks

You have to keep a dozen of your favorite problems constantly present in your mind, although by and large they will lay in a dormant state. Every time you hear or read a new trick or a

Gian-Carlo Rota, Indiscrete Thoughts (Boston: Birkhäuser Boston, 1997), 202.

Richard Feynman indicated in an interview that he kept a dozen of his favorite problems at the top of his mind. As he encountered new results and tricks, he tried applying them to those problems in hopes of either solving them or in coming up with new ideas. Over time by random but combinatorial chance, solutions or ideas would present themselves as ideas were juxtaposed.

One would suspect that Feynman hadn't actually read Raymond Llull, but this technique sounds very similar to the Llullan combinatorial arts from centuries earlier, albeit in a much more simplified form.

Can we find evidence of Feynman having read or interacted with Llull? Was it independently created or was he influenced?

I had an example of this on 2022-05-28 in Dan Allosso's book club on Equality in the closing minutes where a bit of inspiration hit me to combine the ideas of memes, evolution, and Indigenous knowledge and storytelling to our current political situation. Several of them are problems and ideas I've been working with over years or months, and they came together all at once to present a surprising and useful new combination. #examples

Link this also to the idea of diffuse thinking as a means of solving problems. One can combine the idea of diffuse thinking with combinatorial creativity to super-charge one's problem solving and idea generation capacity this way. What would one call this combination? It definitely needs a name. Llullan combinatorial diffusion, perhaps? To some extent Llull was doing this already as part of his practice, it's just that he didn't know or write explicitly about the diffuse thinking portion (to my knowledge), though this doesn't mean that he wasn't the beneficiary of it in actual practice, particularly when it's known that many of his time practiced lectio divina and meditated on their ideas. Alternately meditating on ideas and then "walking away" from them will by force cause diffuse thinking to be triggered.

Are there people for whom diffuse thinking doesn't work from a physiological perspective? What type of neurodiversity does this cause?

In an interview about how she wrote the smash hit “Blank Space,”3 Swift says, “I’ll be going about my daily life and I’ll think, ‘Wow, so we only have two real options in relationships—it’s going to be forever or it’s going to go down in flames,’ so I’ll jot that down in my notes . . . I’ll come up with a line that I think is clever like ‘Darling I’m a nightmare dressed like a daydream’ and I just pick them and

NME, “Taylor Swift—How I Wrote My Massive Hit ‘Blank Space,’ ”NME.com, October 9, 2015, YouTube video, 3:58, https://www.youtube.com/watch?v=8bYUDY4lmls

link to Eminem and "stacking ammo"

motivational...

6 Craig Mod, “Post-Artifact Books and Publishing,” craigmod.com, June 2011, https://craigmod.com/journal/post_artifact/.

It's not just me... I might hope that someone could leverage Hypothes.is' product to create a more explicit digital commonplace book out of their product.

Joint Public Review:

The present manuscript compares the connectomes of a large range of mammal species using diffusion MRI data. The manuscript reports two main findings: (1) connectomes of more related species are generally more similar, as assessed using Laplacian eigenspectra, than of unrelated species; (2) differences between species' connectomes are generally driven by local regional connectivity profiles, whereas global features are generally preserved.

The first finding is comforting, but in a way not extremely surprising. It would be extremely surprising if more related species do not show more similarity in their connectome. Indeed, this is the reason many phylogenetic analyses use statistical techniques that take the relatedness of species explicitly into account. I find the statement that connectome organization recapitulates traditional taxonomies a bit over the top, as this suggests that a phylogenetic tree constructed based on connectomes would be similar to a tree based on other measures, such as morphology or genetics. This will probably be the case, but is not what the authors have tested here.

The second result is in my opinion the key result of the paper. The main novelty of the paper is that -finally, for the field-bridges approaches taken by some researchers in searching for differences across species (these are usually researchers interested in anatomy) and researchers searching for conserved principles across species (usually researchers approaching connectivity from a network or graph theory perspective). By showing what aspects of a connectome are generally conserved and which are changed, this paper starts unifying the two views and this is an important contribution.

It would, however, have been nice if the authors had explored this notion a bit further. Now, they just state that taking certain features into account means the connectomes look more different, but they do not zoom into the specific brains to see what this means at a biological level. Some of the authors have published, for instance, on the unique connectivity profiles of parts of the human brain and it would have been nice to show that these fall under the local regional connectivity profile aspects of the connectomes. This is a missed opportunity to even further unify the different research traditions.

The manuscript suggests that white matter connectivity in mammals is more similar between species within one taxonomic group than across different groups, proposing that the brain's connectome reflects phylogenetic relationships. The manuscript further details which features of the network organisation are associated with larger differences across groups and hence may drive speciation; and which features seem to be a common principle across mammals.

The authors present evidence based on the analysis of diffusion-weighted brain imaging data across 124 species, 111 of which were included in the comparison. The dataset is a great resource to address their research question.

The paper is clear and the evidence compelling. The manuscript adds valuable insights into the connectome architecture across species, potentially opening a new perspective on the link between genetics and behaviour. I would like to point out the great open science practice of the authors - code is available with a great ReadMe to guide potential users, connectivity matrices are available, and all software packages used in the analyses have been cited.

The figures are clear and complement the manuscript.

Technical Comments:

- Spectral approach / Interpretation<br /> It would be good to have more insight into the meaning of the spectral distance results. My understanding is this: the eigenvalues of the normalised Laplacian obviously have a mean of 1 (because their sum equals the trace of the Laplacian, which is equal to N [number of nodes]). Therefore, the distances between the spectra essentially amounts to comparing higher moments, and in particular the variance (as the histograms look quite Gaussian, I am guessing the distances are dominated by differences in the variance). But what does it mean that bats have a higher variance in these Eigenvalues than primates? I know that the authors try to give *some* insight, e.g. that when the distribution is peaky around 1, it means there are more stereotypical local patterns of connectivity. I understand that. But what are these patterns?

- Effect Size / Null Distribution<br /> I like the idea and the ambition of this paper. My main concern is that the differences are very small. Pretty much all the measures (laplacian eigenspectra and network-theoretic measures) are very similar between animals. This can be interpreted in two ways. (1) it may mean that the brain organisation is preserved, which is the interpretation of the authors. But it could also mean that (2) the metrics are not very informative. How do we know if we are in situation (1) or (2)? There is no comparison to a good null model (except in Fig4 but I don't think a random network is a good null). One possible null is two random networks connected to each other with a few random connections (to mimic left-right brains)?

* The authors use cosine similarity to compare the eigenspectra distributions. I think this does them a disservice. cosine similarity normalises the distributions quadratically instead of linearly. But the main thing that is changing is the variance. So normalising quadratically diminishes the dissimilarities between distributions. I have looked at their data (thanks for sharing!) and using multidimensional scaling with Euclidean looks much better than with cosine distance. I would suggest using euclidean.

* The authors use a bootstrapping method to calculate an average distance which they claim is useful because they don't have the same number of animals in each category. I don't think this bootstrapping is useful at all. If anything, it just adds noise. Averaging 10,000 samples with replacement does not change the outcome compared to simply averaging the matrices without the sampling. To test this: vary n and it should converge to the average of the original non-sampled data. (I've tried it!)

* The authors should clarify whether they are using the weighted or binarised connectivity matrices in the spectral approach (and also what threshold). I suspect that they are using binarised matrices, which probably explains why the spectral results fit better with the graph topology results when the latter uses binarised matrices.

- Parcellation.<br /> One main issue is the way in which the connectomes are divided up into 200 regions each, independent of the brain size. This to me seems a confound. I know it's rather standard practise in the field, but I have yet to see a validation that this does not influence the results. Given the enormity of the dataset here I would ask the authors to run their analyses in a way that the number of regions is a function of the size of the brain-this is a much more realistic assumption, as we know that a shrew size brain has about 20 cortical areas, whereas the human has about 180 according to Glasser et al.

Author Response

Reviewer #1 (Public Review):

This paper presents an approach to estimate Rt for 170 countries. While it is an impressive amount of work, I think the pipeline is similar to many currently available frameworks. The paper claims the following novelties over current framework, but more efforts are needed to be done to make it convincing.

1) Obtain stable estimate from multiple types of data:

It turns out the stable estimates just repeatedly use the same approaches to different time series (Figure 3A middle). From the wording I think there should be some methods to combine these time series to have a single estimate of Rt. Overall, the Rt and the time series of infection should be unique. It would be suboptimal, for example if there are big differences in the results from death time series and reported cases time series, which one should I trust?

We think it is a strength to compute different Rt values based on different data, as this allows researchers, policy makers and the public alike to compare the information from different observation types directly. Any discrepancy between two Re trajectories (e.g. between the Re based on cases, Rcc(t), and that based on hospitalisations Rh(t)) is an indication to investigate which external variables (e.g. testing strategy) have changed. We have found it a great advantage when communicating and sharing our results outside of academia that we could point to these separately obtained Re estimates: if the estimates all agreed, more confidence could be given to them.

If one would want to estimate a single estimate, this would require adopting a fundamentally different framework to estimate Re, which exceeds the scope of this work. One could use heuristics (weights representing the trustworthiness of a given source at a given time) to combine the various Re estimates into a single ensemble estimate. Alternatively, one could model the full underlying population dynamics (e.g. with a compartmental model including hospitalization and death) and adopt a fully Bayesian approach to fitting such a model. However, both options require heuristics or priors that will vary substantially through time and per country (as discussed in the Supplementary Discussion), and thus limit how widely the pipeline can be applied.

We have revised the manuscript to make it more clear (early on) that we estimate multiple Re values from separate types of data (see also the response to reviewer 3, item #5). In addition, we now discuss more explicitly what the advantages and disadvantages are of showing these estimates separately (lines 281-290).

2) Adequate representation of uncertainty:

This is the result in Figure 2, suggesting the CI from EpiEstim is too narrow. This would be expected given that EpiEstim assumed the input infection time series is observed and fixed. It would be expected that the proposed approach would provide wider CI and hence the proportion covered would be more. However, I think to validate the wider CI is the correct one, simulation studies are required. I think the most related one would be Figure 1B. The results suggested that the approach works when the Rt is not rapidly changing. However, I have concern on the methods for simulation (details below).

Indeed, the difference in coverage between our method and EpiEstim is due to observation noise. We agree the CI from EpiEstim should be correct assuming that the infection incidence time series can be observed perfectly. However, in reality quite a bit of variability is introduced between infection and case observation: not only due to the delay from infection to observation, but also due to e.g. reduced testing capacity on weekends or reporting errors. To accurately assess the coverage of our method (and whether the CIs are too narrow or too wide) we need to include realistic amounts of observation noise in the simulations. This is why we add autocorrelated noise to our simulated observations, where this noise mimics observed residuals in Switzerland and other countries (Figs. S3, S4, S15, S17).

We have now added explicit comparison to the EpiEstim confidence intervals to supplementary Fig. S4. In addition, we extended the corresponding method section to describe more extensively why and how we added observation noise to our simulations (lines 498-518; see also the detailed response to comment 4 below).

3) Real-time of the Rt

There is no simulation about the real-time property of the Rt. The most related one is still Figure 1B. However, looks at the right-tail of the figure (the real-time performance), the proportion covered the true value is decreasing and more efforts are needed to support the framework can be accurate in real-time. For example, how is the real-time performance when Rt is increasing, or Rt decrease sharply due to lock down?

As suggested, we included an additional simulation study to investigate the accuracy and stability of the last possible Re estimate. We present this analysis in a new results paragraph (subsection "Stability of Re estimates in an outbreak monitoring context"; line 121) and Figure S10. Using this analysis, we highlight the trade-off that exists between the timeliness of the Re estimates and their stability.

4) simulation methods to estimate Rt

Both 2) and 3) need simulation to support the results, and hence the simulation approach would be critical. The first part based on Poisson distribution to generate an infection time series, which is OK. However, the issue is the secondary part about how the authors obtained the time series for death/hospitalization/reported cases. To me, after generating the infection time series, based on the delay distribution from infection to death/hospitalization/reported, we could obtain those time series. I am not clear and sure if the authors approach is correct by using smoothing and fitting ARIMA to get those time series.

We believe there may have been some confusion about how our simulation set-up works, and we provided insufficient detail on the design decisions behind this set-up. We have added more explanation for both points to the paper (lines 503-518; additional supplementary Figs. S15-S17). In brief, our simulation process consists of three parts. We first conduct the two steps the reviewer also mentioned: (i) simulating the infection time series, and (ii) simulating the observed time series by using the delay distribution from infection to death/hospitalisation/case report.

However, we find that the observations simulated this way are too smooth compared to real data (see Figure S17). Possible reasons for this are that the delay distribution does not account for weekend and holiday effects, the random and occasional delay in recording confirmed cases, nor irregular components such as confirmed cases that are imported from abroad. We therefore added a noise term in our simulations, resulting in a third step: (iii) adding noise generated from an ARIMA model.

To obtain a realistic ARIMA model for this third step, we fitted a model based on the confirmed case data for SARS-CoV-2 in Switzerland. Specifically, we first obtained the additive residuals based on the log-transformed confirmed cases. We then fitted ARIMA models of various orders and assessed the resulting ACF and PACF plots of their residuals. Based on this, we chose an ARIMA(2,0,1)(0,1,1) model. We refer to Figure S16 to support this: The first row shows the ACF and PACF plots of the original residuals, showing strong autocorrelation. The second row shows the ACF and PACF plots of the residuals after fitting the ARIMA model. We see that there is little autocorrelation left, indicating that this model is reasonable.

In Figure S17, we present simulated observations based on all three steps, and one can see that they look more realistic than the simulated observations after step (ii).

We would also like to point out that the ARIMA model is only used to obtain simulated observations. Our main method to estimate Re and obtain the related confidence intervals does not require fitting an ARIMA model.

Minor comments:

1) What does near real-time mean? The estimates of Rt are delayed for a few days like other approaches?

Indeed, the estimates of Rt are delayed by the time it takes from infection to a case to be observed. We have replaced the term “near real-time” by “timely” throughout the manuscript, and added this explanation of the delay more explicitly to the text (line 86).

2) For the results in Table 1, I think if there are some results suggesting that other approaches (like EpiEstim) perform worse than the proposed approach, it would be better to illustrate the value of the proposed approach.

We have improved and extended the comparison of our method against others in two ways: (i) we added further comparison of the coverage of our method vs. that of EpiEstim to Fig. S4 (see also the response to major comment 2), and (ii) we added comparison against different commonly used pipelines (see minor comment 3 below). Instead of comparing to other approaches, the analysis in Table 1 was meant to illustrate the use of the Re estimates resulting from our method alone.

3) I think more discussions are needed for the similarity and differences for current approach. For example, Abbott et al (https://wellcomeopenresearch.org/articles/5-112) used a similar pipeline.

We added a section to the results (paragraph starting line 182; Fig. 3), dedicated to comparing our approach with relevant alternatives. We compared some of our empirical results with the estimates published on epiforecasts.io (based on EpiNow2 package from Abbott et al.), as well as official COVID-19 Re estimates for Austria (by AGES) and Germany (by RKI). We find that estimates published by the RKI and AGES health authorities are likely to be overconfident and to suffer from previously-identified biases (notably in Gostic et al., 2020, PLOS Computational Biology). We provide a detailed comparison of the features and approaches of these methods (EpiNow2, AGES, RKI), with the addition of the epidemia R-package (Supp File S2). This comparison highlights the unique features of the method developed: its ability to account for time-varying delay distributions and to combine symptom onset data with case data.

4) Figure S11 is about accounting for known imports. While if the local cases are dominant and hence imported cases would not have a big impact on estimates of Rt. The impact of imported cases on estimates of Rt could be complicated, as suggested in Tsang et al. (https://pubmed.ncbi.nlm.nih.gov/34086944/). In addition to assuming imported cases and 'exported' cases could be canceled, it is also assumed that the imported cases had similar transmissibility to the local cases, which may not be true if there is border control.

We thank the reviewer for this interesting comment and reference. We added a brief discussion in the result section of the manuscript to address this limitation (lines 174-177).

Reviewer #2 (Public Review):

This manuscript describes an algorithm of estimating real time effective reproductive number R_e (t). This algorithm combines several methods in a reasonable way: deconvolution of time series of reported case into time series of infection, a Poisson model for generation of infections, and block-bootstrap of residuals to assess uncertainty. Each component is not necessarily novel, but the performance of this algorithm has been validated using comprehensive simulation studies. The algorithm was applied to COVID-19 surveillance data in selected countries across continents, revealing a great deal of heterogeneity in the association of R_e (t) with nonpharmaceutical interventions. Overall, the conclusions seem reliable.

I have several moderate critiques and suggestions:

1) From a statistical point of view, it seems much more natural to integrate the infection generation process and the delay from infection to reporting, possibly with reporting errors, into the same model, with which you will avoid combining the bootstrap and the credible intervals in a somewhat awkward way. I understand you can take advantage of EpiEstim package, but the likelihood is very simple and easy to program up. Nevertheless, I'm not strongly against the current paradigm.

We agree that such an integrated approach is useful, and makes the uncertainty interval estimation more coherent. However, in such an integrated approach one can not use the analytical solution for the likelihood, and methods that choose this approach (like EpiNow2 and epidemia) tend to pay for it in computational complexity. It also makes it harder to include time-varying delay distributions into the model, one aspect that sets our pipeline apart from existing alternatives.

An additional advantage of our method is that estimates for the infection incidence are not influenced by priors on Re. In case of a bad model fit this allows us to separate more easily which part of the model may be misbehaving; and as such can help as a sanity check.

Lastly, our framework has the advantage of modularity: pieces of the pipeline can be (and were) continuously refined or replaced with better pieces. This continuous improvement process allowed a flexible response to the pressing circumstances (the COVID-19 pandemic), and allowed us to extend it to entirely new types of proxy data (e.g., wastewater viral loads - https://ehp.niehs.nih.gov/doi/10.1289/EHP10050 ).

2) Is there a strong reason to believe the residuals are autocorrelated? The block sampling with block size 10 seems arbitrary. The authors fitted an ARIMA model to the residuals for some countries, how good was the fitting? If the block size doesn't matter, then probably the stronger but simpler assumption of independent residuals may not compromise the estimation of R_e (t) much.

Yes, there is reason to believe the residuals are autocorrelated. New supplementary Figure S15 shows the ACF and PACF of the residuals based on the confirmed cases of Switzerland, China, New Zealand, France and the US, and one can see that for most countries, the obtained residuals are clearly autocorrelated. We added this point to the simulations method section in the paper (lines 503-518). Please also see our response to Reviewer 2, major point 4 above.

Choosing an optimal block size for the block bootstrap method is generally difficult. To capture weekly patterns, we need a block size of at least 7. We tried different sizes and found that 10 tended to work well in a variety of simulation settings (an example is given in Fig. S19).

3) I don't see the necessity of using segmented R_e (t) instead of a smooth curve in the simulation studies. The inferential performance, especially the coverage of the CI's, is much less satisfactory when a segment has a steep slope. The authors may consider constructing splines based on the segments or using basis functions directly.

We started using a segmented Re(t) trajectory to allow for simple parametric generation of different scenarios (e.g. in new Fig. S10), and to specifically study our ability to estimate sudden transitions in Re (discussed wrt. Table 1, Fig. S2). We agree this approach makes our method look worse than necessary, since it is generally difficult to estimate such abrupt changes in Re. However, we thought this would be the more stringent test of our method, as we will perform better on any more smooth trajectory.

4) The authors smoothed the log-transformed observed incidences to come up with the residuals. For Poisson data, a variance-stabilized transformation is taking the square root, not the logarithm. In addition, as you already have bootstrap estimates, why not using quantiles directly for CIs but instead using a normal approximation (asymptotic)? When incidence is low, the normal approximation may be much less satisfactory. Also, when using normal approximation for CI, it's much safer to calculate standard deviation and construct CI at the log-scale, i.e., log(θ ̂^*(t)), and then exponentiate back.

Our goal of transforming the original case observations is to stabilize the variance of the residuals. Indeed, the square root transformation is generally recommended if the data to be transformed is Poisson distributed. In our case, however, the original case observations are not quite Poisson. Specifically, the infection incidence at time t given the past incidence is modelled with a Poisson process (see Section 4.4), but the case observations are modelled with an additional convolution step of the infection incidence with a delay distribution, and there is additional variation due to e.g. weekday effects. It is thus not clear a priory which transformation works best for our data, and we therefore investigated various possible transformations (including the square root transformation). We found that no transformation was uniformly the best for data of different countries, but that the log-transformation tended to perform best overall. This is why we chose the log-transformation. Please see the new supplementary Figure S14, where we show the residuals after the square root transformation and the log transformations for various countries.

Regarding the bootstrap confidence intervals, we also investigated different options. Again it is not clear a priory which bootstrap confidence interval performs best for our data, so we compared common choices like quantile, reversed quantile and normal-based in a simulation study. Specifically, we assessed their coverage and found that the normal-based confidence intervals performed best overall (see Fig. S4).

For low incidence settings, none of the bootstrap methods perform very well (as bootstrap consistency does not apply). We now mention this consideration in the paper (line 442).

Finally, regarding the suggestion to compute exp(SD(log(X)): This quantity is generally different from SD(X), which we need for the confidence intervals. We also refer to the coverage in the various supplementary figures (e.g. S2, S4, S5) to support that our approach works well.

5) The stringency index is a convenient metric for intervention intensity. However, it doesn't reflect actual compliance as the authors admitted. Another likely more pertinent metric is human movement (could be multiple movement indices). Human movement indices may not be available in all countries, but they are available in some, e.g., the US, and first wave in China. In some states of the US, it was clear that human movement decreased substantially even before initiation of lockdown. Lack of human movement metrics most likely has contributed to the difficulty in the interpretation of Figure 4.

We have added mobility data (from Apple and Google location data) to our general dashboard, and to the analysis shown in Fig. 5. The mobility traces give more detailed insight in the behavior that may have led to decreases in Re. However, we find similar patterns wrt. decreases in Re as with the stringency index. A more extensive analysis that focuses on different phases of the pandemic may allow for more detailed insights, but we believe this is beyond the scope of our manuscript.

yeah

Hey Ariel, I like the idea that you gave use the option to annotate the document to give Feedback. The tool was easy for me to set up and not complicated to use. However, I think this section of your explanations leads a little to confusion. Maybe it's just me, but I wasn't sure which link to click to annotate your Techshowcase. I wish, you would add this: If you'd like to try annotating my Techshowcase, follow the steps above and then you're most welcome.... It's just a minor detail though, I really enjoyed the clarity of your explanations.

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

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Referee #3

Evidence, reproducibility and clarity

In this manuscript, the authors address the important topic of post-transcriptional gene regulation using the larval nervous system in Drosophila. They utilize a novel approach taking advantage of existing protein trap library, which permits use of the same smFISH probe to detect an array of 200 RNAs and visualize their corresponding protein expression. Furthermore, the authors developed a computational pipeline to visualize and analyze the resulting data, which should enhance the application of this method by other researchers. A major strength of the data comes from the analysis of multiple cell types in distinct compartments of the nervous system, cell types (neuron, glia, neuroblast), and subcellular domains. From the cumulative data, the authors are able to describe several interesting observations relating to cell-specific post-transcriptional regulation, regulation within a central-neuroblast lineage and glial post-transcriptional regulation, among others.

However, in spite of these strengths, there are several concerns related to the organization and interpretation of the manuscript that the authors should address in order to improve the manuscript:

General concerns:

1. The approach relies on gene traps that often fail to be made homozygous, presumably due to deleterious function of the YFP insert. This is an obvious limitation of the study, which the authors address, but do so insufficiently by only analyzing a single case Dlg1. The authors should report how many of the 200 YFP-traps can produce viable homozygous animals, whether phenotypes can be observed, and any other relevant information to assess the functional properties of the tagged genes.
2. The term "discordant" is used for non-congruous RNA/Protein levels in soma and distal processes, and sometimes the two are analyzed in the same figure (e.g Fig 3A). When it is stated that 98% of genes are discordant, this is an over-simplification as what the authors describe as "discordant" is expected to occur frequently in the distal process, but less often in the soma (which is what the authors find when presenting the data for individual compartments - Fig 3B-C). This is confusing because the observation means completely different things in the two compartments, though both are interesting to describe. These analyses, and their interpretation, should be kept separate.
3. There is not enough emphasis placed on the cell-type specific regulation of RNAs. There are very few studies that have investigated how localization of individual RNAs changes in different cell types or regions of the nervous system, and the authors find that this is quite prevalent. Therefore, the rather superficial analysis of these data fails to take advantage of a major strength of the data. For example, for the discordant genes that differ in neuropil localization between different regions of the CNS, what types of molecules do they encode, what is their function in neurons (if known), and why might they be required locally in one region of the CNS but not the other?
4. The authors conclude that mRNA and protein co-localization in glia processes shows that mRNA localization makes a major contribution of the proteome in processes. However, there is not enough evidence for such conclusion since neither translation of these mRNAs nor lack of protein trafficking from the somas was shown.
5. An important caveat of this technique that should be discussed is the lack of knowledge about the translation of these mRNAs, if the mRNA that is being detected is the same as the one that is translated. While the authors emphasize the discordance between mRNA and protein localization, it is not possible to know whether these mRNAs are being translated where they are found, e.g. soma vs neuropil. Moreover, there are many examples (e.g. BDNF) where the isoform influences the subcellular localization of the mRNA. There is no way of studying the isoforms here, and we could be looking for a different mRNA isoform localized to a specific compartment compared to the protein. These points must be discussed.

Minor suggestions:

• The authors should identify GO terms to understand what types of molecules are subjected to RNA regulation. They provide a supplementary table for all genes, but it would be useful to have a chart showing the proportion of different GO terms represented in the overall gene set, genes that show cell-specific regulation, genes that show neuron vs glia specific regulation, etc.
• "However, post-transcriptional regulation can also manifest itself within a cell, so that a protein is localised to a distinct site from the mRNA that encodes it". While subcellular RNA localization may represent a regulatory layer, I do not agree that proteins that function in the cell at a different location than their translation site represents regulation per se. Many such cases exist for proteins that are trafficked!
• "The majority of individual puncta appearing in the dlg1::YFP line (51% in the brain, 64% in larval muscles". Why is the agreement between YFP and endogenous FISH so low? Do many individual RNAs fail to hybridize? This should be discussed.
• "However, one gene, indy, is highly transcribed in neuroblasts and a single ganglion mother cell before it is rapidly shut off (Figure S1A)". This figure does not exist. Where are the data?
• The authors should be consistent about calling perineurial or perineural glia (both correct) in their images and text.
• "We only observe a minority of localised axonal mRNAs that lack the protein they encode at the axon extremities, in contrast to our findings in the mushroom body, optic lobe, and ventral nerve cord neuropils" These results are not contrasted, as in all neuropils the minority of localized mRNAs are those lacking their corresponding proteins. For example, 9% in NMJ vs 7.5% in OL neuropil according to Fig. 1B. What is conflicting with the conclusion?
• "These results suggest that motor axons are more selective than the other neuronal extensions in the mRNAs that are transported over their very long distances from the soma to the neuromuscular synapse" The current literature says that the same mechanism (cis-elements) is used to transport mRNAs to subcellular compartments, which would be inconsistent with the idea of motor axons being "more selective" than other neurons for the same mRNA, but just a result of fewer mRNAs being found in motor neurons: 34.% of the mRNAs are found in motor neurons soma vs 83% in OL soma, 86.5% in VNC soma, and 70.5% in MB soma. To get to this conclusion, the authors should show that mRNAs previously found in the neuronal extensions of other neurons are not found in the axons of motor neurons but are still expressed in thesir somas. They might want to suggest different RBPs involved in the transport or discussing the very long distance they need to travel which can influence their detection in the tips. Figures
• Figure 1. Experimental approach summary
  • Some colors do not show well and should be changed, e.g: grey in Fig. 1A, and Fig. 1B probe sites indicated in light blue and pink within the introns of dlg1.
  • Fig. 1E': There appears to be a large discrepancy in co-detection % for CNS and muscle in the graph judging by the size of circles, yet in the text, it is stated that there is average of 51% and 64% in the two, respectively. I don't see any green circles with over 25% agreement in the graph. Are the colors correct here?
  • Fig. 1D-I: It's difficult to identify where the zoomed panels come from. E has its own square (indicating zoom in E'). Please make this square dashed or a different color in E so it is clear F and G do not come from there.
  • Comparing Fig. 1F vs K: Why does there appear to be so much more dlg1 mRNA in the YFP-tag condition? If this is due to selection of imaging area, please choose a more similar region to image so the RNA levels are comparable. Otherwise it indicates the YFP-tag line has more RNA expression, which is likely not the case.
• Figure 2. Analysis pipeline overview
  • The lines for the first two zoomed panels are switched: The optic lobe is going to VNC and vice-versa.
• Figure 3. Overall summary of results
  • Figure 3A: Soma/Neuropil/muscle should be separate or at least ordered such that they are next to each other to facilitate direct comparison of genes in the same region of the cell in neurons from different CNS areas. Why are glia not included in this summary? A third color should be used to indicate when there is neither mRNA nor protein expression.
  • "Compiling all the information together shows that there are that 196/200 or 98% of the genes show discordance between RNA and protein expression" However, 5 genes shown in Fig. 3A do not show "discordance": CG9650, cup, Lasb, rg, and vsg!!
• Figure 4. Neuroblast lineage analysis
  • Is clustering around the NB sufficient to determine lineage relationship? There seems to be other neurons around the NB.
  • More examples should be shown for the post-transcriptional category, as it is the most interesting category, and there are many different possible outcomes. Are there cases of transcriptional control and post-transcriptional regulation? Are there cases where the youngest neurons (closer to the NB) in the progeny are expressing the protein while the oldest are not? If not, could this be an artifact from a slow translation and the protein being detected only after building up in the cell? Top1 protein (Fig. 4D) seems to be less expressed in the youngest neurons.
  • "The transcription rate of these genes, as indicated by the relative intensity of smFISH nuclear transcription foci, is similar across the neuroblast lineage, however protein signal is only detectable in a minority of the progeny cells (Figure 4E)". Many nuclei lack clear large spots, but have small spots indicative of RNA; how is this interpreted? Do they lack transcription, or is this due failure of the smFISH to capture all transcription sites? Were transcripts actually counted to assess cell-specific differences? This should be possible with smFISH
• Figure 5. RNA synaptic localization
  • A have global analysis comparison of all neuropil areas would be welcome in this figure.
  • "Surprisingly, another 59 transcripts are present at synapses without detectable levels of protein (Figure 5E-H)" This text does not correspond to Fig 5E-H but 5I-L. Where is the text about 5E-H?
  • For Fig. 5J and 5N RNA appears scattered regularly throughout the entire panel area. How sure are the authors that this is not due to poor signal/noise? For example, perhaps too much probe being used for these targets.
  • Fig. 5R is not cited in the text.
• Figure 6. RNA localization in glia
  • For Fig. 6B-G it is hard to tell if there is any overlap of the RNA and Glia. Maybe show multiple zoomed-in merged images and/or highlight the structures with lines that are present in all panels.
  • For Fig. 6L-O: How reproducible is this small amount of RNA puncta in the NMJ glia? Is this possibly biologically important?
  • Why do cartoons labelling subnuclear/perinuclear glia in Fig.6 and Fig.S6 show different localization?
  • The cartoons seem to extrapolate from the data: While in Fig 6B-D, we see neither the big bright spot of transcription in the glial nucleus nor as many transcripts in the neuropil, they are both present in the cartoon. In Fig. 6E-G there is no indication of cortical glia soma nor the transcription spot only in glia nuclei.
  • "To assess glial localisation for the 200 genes of interest, we used a pan-glial gal4 driving a membrane mCherry marker (repo-GAL4>UAS-mcd8-mCherry) to learn the expression pattern of all glial cells, and then classified the pattern in the YFP lines (without the marker) based on knowledge of that expression pattern. We validated this approach by combining the RFP marker" Did the authors use mCherry or RFP for these experiments? Also, the previous sentence is redundant.
• Figure 7. RNA localization at neuromuscular synapse
  • RNA for these genes seems far too spread throughout the muscle to draw any conclusions
  • Also with so many RNAs distributed in the muscle, specific localization of RNA molecule to the precise PSD would have no conceivable benefit
  • I suggest drawing lines around the protein expression to facilitate visualization of the mRNA localization for panels B, F and J. It is especially hard to conclude anything from panels B and F.
  • Light grey with white dots is hard to see in the cartoons
• Figure 8. Role of khc and activity in sgg localization
  • Presumably there is a huge number of developmental problems associated with this mutant that could cause decrease in sgg localization
  • If the authors include this, then they should characterize the mutant NMJs: what is the change in size, synapse number, etc..
  • Is there more sgg accumulated in soma as a result of less transport? Is sgg being expressed at the same level?
  • Fig. 8F-H: Why is Dlg1 accumulated in the entire axon, not just the presume synapse?
  • Fig. 8J: Why is sgg signal occurring in circles disconnected from the main axon? The authors should show a different image

Significance

This is a significant and complex paper that contributes with novel tools to an important issue

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HEHEH HAW

This section on reference intervals and biologic variation is interesting and helpful. But don’t concentrate on the information here. The important thing to remember is that for many of the test such as the enzymes have significant biologic variation. Reference intervals may be wide and therefore we don’t just look at if it is over the reference interval, we actually look at how much increase is there to see if it’s significant.

teacher that would pause it and then have a discussion and then unpause it and of course there's a soon in the classroom I was like oh just keep playing the video but the truth is I understood much better as we had the shared discussion about what was going on in the video then we were just doing something very isolated so when you are Co engaging in media whatever the media is a game a simulation video whatever it might be you're able to have the same content experience you're able to developed a shared understanding you're able to work through misconceptions because sometimes we understand things differently or don't understand something and it's easier to talk through when you're having the shared experience when everybody is doing their own thing with their own separate content one of the dangers in education is that as an educator if I don't know the content because every one thing is still personalized then I can't help them work through the misconception as

Is there really an increase in meta-positioning? Also: would this description of Society At Large resonate with someone who isn't specifically terminally online on Twitter?

Animals ->

Primal motivations (primitive mind) + Environment = Behaviour

La motivacion es tu programacion interna que te dice como sentirte en cada situacion. El entorno es toda la influencia externa que afecta a tu decision.

Sascha Fast analogizes the writing process using a zettelkasten to Henry Ford's assembly line for building cars. Each worker on the assembly line has a limited bit of knowledge for their individual part of the process, but most of the knowledge and value is built into the overarching process itself. This makes the overall system quicker and more efficient.

Similarly with note taking, each individual portion of the process is simple and self-contained, but it allows the writer to create a much more creative and complex piece in the end. Here an individual can accomplish all of the individual steps in a self-contained way while focusing on individual steps without becoming lost in the subsequent steps which would otherwise require a tremendous additional amount of energy.

Dig into research on maintenance rehearsal versus elaborative rehearsal.

This is like what I described above as an "easy but harmful" solution. I first encountered this phrase through an interview about prison abolition on, I believe the "How to be Antiracist" podcast, but I think it's actually a common manner of speaking. I think it's widely applicable.

This sentiment is very similar to one in a recent lecture series I'd started listening to: The Modern Intellectual Tradition: From Descartes to Derrida #.

Lawrence Cahoone specifically pointed out that he would be highlighting the revolutionary (and also consequently the most famous) writers because they were the ones over history that created the most change in their field of thought.

How does the novel and the different manage to break through?

How does this relate to the broad thesis of Thomas S. Kuhn's The Structure of Scientific Revolutions?

The comment Wengrow makes about "remarking on [an anarchist's] politics" as a means of attacking their ideas is quite similar to the sort of attacks that are commonly made on women. When female politicians make relevant remarks and points, mainstream culture goes to standbys about their voice or appearance: "She's 'shrill'", or "She doesn't look very good in that dress." They attack anything but the idea itself.

I was really hoping for so much more in this essay on the combinatorial creativity, espcially since the author threw the idea into the title. The real meat must be in the two linked articles about Seneca and Einstein.

There is a slight mention of combinatorics in the justaposition of pieces within one's commonplace book, and a mention that these books may date back to the 12th century where they were probably more influenced by the combinatoric creativity of Raymond Lull. It's still an open question for me just how far back the idea of commonplaces goes as well as how far back Lull's combinatoric pieces go...

Own your pedagogy. Send just like anything else out there...

One interesting thing here is the rivalry between Gilgamesh and Enkidu. What is interesting about this rivalry is that it's not just a personal rivalry. Enkidu is first mentioned as the child of nature, where gazelles are mentioned, but also threatening lions. In this passage, one can also see Gilgamesh in what he represents, the city. The terms "squares", "districts", "walls" are mentioned.

This first contact between Enkidu and Gilgamesh is a social moment, since it is on the main square that the story takes place.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): Excellent quality of cell biology and biochemistry. the additional supports are needed for the claim of actin elongation using different formin variants.

Reviewer #1 (Significance (Required)): Ingrid Billault-Chaumartin and co-authors described interesting research that provides insights on formin-isoform specific function in fission yeast and a new role of Fus1 FH2 domain in cell-cell fusion event. While three formin isoforms have different localization, research proposed an additional dissection in their functional differences by having different functions in C-terminus, including FH1 FH2 and formin C-terminus. The work also described additional factors that regulate cell fusions from autotrophy effect and formin expression level, in addition to the well-accepted formin biochemical activities. Here are my comments regarding the strengths of the work and improvements that could further strengthen the story.

Major comments 1. Fig.1 shows Cdc12C could recapitulate Fus1 function by ~80% if fused with Fus1C, whereas deletion of the C-terminal tail of Cdc12 following FH2 introduces drastic dysfunction. Together with Fig. 3, these results indicate Cdc12 Cter plays more important roles than Fus1 Cter for there respective functions. Such results suggested a Cter-mediated mechanism that differentiates the functions of three fission yeast formin isoforms. The authors examined contributions from the difference in FH1 (Figs 4,5) and FH2 residues (Fig. 6). Whereas the obvious phenotype of Cter was not further investigated and not much discussed. The Cter of budding yeast formins interacts with nucleation-promoting factors, Bud6 and Aip5. Although S. Pombe does not have orthologs of budding yeast Bud6 and Aip5, I wonder would the author discuss the potential contribution of Cter in differentiating S. Pombe formins.

The reviewer is correct that the C-terminal tail region of Cdc12 beyond the FH1-FH2 domains has a strong influence on the ability of Cdc12C to replace Fus1C. This is one reason why we specifically investigated the possible role of Fus1 C-terminal tail, which is much shorter than that of Cdc12. We found that Fus1 C-terminal tail plays only very minor role in regulating Fus1 function, as described in Figure 3. We note that contrary to what the reviewer states, Bud6 exists in S. pombe and binds the C-terminal tail of the formin For3 (see Martin et al, MBoC 2007), but whether it binds Fus1 is unknown. We have expanded our discussion to include a paragraph on the role of formin C-termini.

Because the manuscript is focused on the function of Fus1 formin, we did not explore further the role of the Cdc12 C-terminal tail. It was previously shown that this region of Cdc12 contains an oligomerization domain that promotes actin bundling (Bohnert et al, Genes and Dev 2013). It is thus likely that this helps Cdc12 FH1-FH2 perform well in replacement of Fus1. In fact, it is likely that oligomerization boosts formin function, as we have discovered that Fus1 N-terminus contains a disordered region that fulfils exactly this function. This is described in a distinct manuscript under review elsewhere and just deposited on BioRxiv (Billault-Chaumartin et al, BioRxiv 2022; DOI: 10.1101/2022.05.05.490810). We have now cited this point in the discussion.

1. Here, the study focuses on the FH1 between Fus1 and Cdc12 to understand their different functions in actin polymerization. FH1 mediated actin elongation through its interaction with profilin via polyP. The transfer rate of G-actin from profilin and profilin sliding depends on the polyP patterns regarding the length of each polyp motif and their distance to FH2 (Naomi Courtemanche and Thomas D. Pollard, JBC, 2012). To better understand the mechanisms by which these engineered FH1 variants on both Fus1 and Cdc12 in Fig. 4, the author may want to list the sequence of these engineered FH1 domains, including the information of the number and length of polyp motifs, and discuss these patterns.

This list and discussion were available in the initial paper that characterized each of the constructs in vitro (Scott et al, MBoC 2011). We have now re-drawn it in a supplemental figure for convenience (as also answered in response to minor point 2), which is already provided in the revised manuscript as Figure S1. (Previous supplementary figures are re-numbered S1>S2, S2>S3 and S3>S4).

1. Figs.4,5 cell biology results do not directly support the point of specific elongation rate unless the LifeAct-labeled actin cable elongation speed could be followed and quantified. The fluorescent tagging of tropomyosin does not show the actin cable pattern, which makes it very difficult to be used to study actin cable dynamics, such as elongation. Therefore, I feel the data in current Fig. 4 and Fig. 5 could not claim the differences in actin elongation without a quantitative comparison of elongation rate. I suggest a CK666 treatment to increase the visibility of the actin cable pattern of LifeAct, used before in both fission and budding yeasts, which would allow the author to quantify the actin cable elongation rate. Another way is to use the TIRF assay used in this study, which would give a better quantitation of formin nucleation and profilin-aided elongation.

We respectfully disagree with the reviewer on this point. All the constructs we use in vivo have been characterized in vitro and their elongation rate carefully measured (Scott et al, MBoC 2011). These values are thus known and can be directly compared to our results in vivo.

Of course, it would be fantastic to be able to directly measure formin elongation rates in vivo, but we are not aware that this has been done in any system. The proxy experiments that the reviewer suggests would be good ones, but each faces technical challenges that make them impossible in our system. First, because the fusion focus is a structure that forms in response to cell-cell pheromonal communication, we cannot add CK-666 or any other drug during this phase, as this perturbs the pheromone signal. Indeed, we had shown that simple buffer wash leads to loss of the fusion focus (see Dudin et al, Genes and Dev 2016). Second, the fusion focus is at the contact site between partner cells, i-e somewhat distant (1-2µm) from the coverslip during imaging. It is thus impossible to use TIRF. Finally, the fusion focus is a tightly packed actin structure. This is the reason why (rather than use of the tropomyosin marker) we cannot image single actin filaments (or even bundles) of which we could follow the dynamics as has been done to measure the retrograde flow of actin cables in yeast.

What we have done is to use a better tropomyosin tag, mNeonGreen-Cdc8, which was just described (Hatano et al, BioRxiv 2022; DOI: 10.1101/2022.05.19.492673) to quantify amounts of linear actin. Although this is not a measure of elongation rate, it would give some sense about amounts of polymer assembled. We have obtained images with mNeonGreen-Cdc8 of all experiments previously conducted with GFP-Cdc8 and have replaced them in Figure 4C, Figure 5E, Figure 6E and Figure S2B. We have also quantified the relevant strains. The relative intensities of mNeonGreen-Cdc8 at the fusion focus at fusion time reflect remarkably well the measured elongation rates of the various formin constructs characterized in vitro. These data are now provided as new panels Figure 4F and Figure 5F.

1. I appreciated the detailed biochemical dissections of multiple aspects of WTFus1 and Fus1R1054E, although the biochemical assays could not identify the mechanism by which R1054E causes the cell fusion. In many cases, the formin functions are diverse in diverse biological processes and sophisticated that cannot be explained well only from its biochemical activities in actin polymerization, such as the bundling, nucleation, and elongation studied in this story regarding fusion. This exciting information allows us to think of more possibilities that might regulate formin function rather than a direct change of formin activities in actin polymerization. I think a discussion of different aspects of functional regulation of formin might inspire society to investigate new possibilities to solve the mysteries. For example, the changes in formin behaviors and functions could be regulated by stress-induced formin turnover by degradation, cell signaling-regulated formin clustering and complex assembly, and their potential relevance to recruit protein constituents for fusion progression.

We have added a paragraph on the role of Fus1 C-terminus. If you feel we should expand more on the diverse modes of regulation of formins, we could, but we have so far kept the discussion centred around the points of investigation in this paper, whose aim was to probe how changes in nucleation and elongation rates, rather than other regulations, affect the in vivo function of Fus1.

Minor comments. 1. There are two types of "C", one includes FH1/FH2 and one following FH2, used in the manuscript, and it is a bit confusing. Better to differentiate them that allows an easy following. Fig. 1 uses Cdc12C-deltaC, Fig. 3 uses Fus1-delta Cter.

We have updated the nomenclature to make this clearer: the C-terminal region beyond the FH1-FH2 domains is now called Cter throughout the manuscript.

1. It's better to specify the amino acid position on the schematic of formins, such as panel A in many figures. It's always more informative to compare formin activities by considering the domain lengths, especially for the C-terminal tail that is variable in lengths and sequences. With similar thoughts, I suggest a supplementary figure that lists the sequence of all FH1 domains variants and Cter domains, such as the FH2 domain in Fig. S1.

We have made a supplementary figure (new Figure S1) listing all constructs with specific aa positions as well as the FH1 domain variants and their sequences (see also answer to point 2 above). We have not added the sequence of the Cter domains in this figure, as these are extremely divergent and not particularly informative at this point.

1. "n" for the statistic needs to be provided for Fig. S3.

We have added the information to the legend of the figure (now Fig S4).

1. The SDS-PAGE staining gel of the purified recombinant proteins for biochemical assays should be provided, particularly for these newly reported mutant variants.

This is now provided as new panel S4C. We show the purified recombinant Cdc122FH1-Fus1FH2 proteins, which are the newly reported ones.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): In this study, Billaut-Chaumartin and colleagues investigate the molecular specialization of the S. pombe formin, Fus1. The authors systematically modulate the actin filament elongation and nucleation activities of Fus1 by expressing chimeric constructs that contain Formin Homology 1 and 2 domains from two other formins with known polymerization activities. By characterizing the architecture of the fusion focus and the efficiency of cell fusion, they find that both the elongation and nucleation properties of Fus1 are specifically tailored for its cellular role. Comparison of formin constructs with similar elongation and nucleation activities also reveals that the Fus1 FH2 domain possesses a specific property that promotes efficient cell fusion. Using sequence alignment and homology modeling, the authors identify R1054 as the residue that confers this novel, fusion-specific activity to Fus1, despite producing no effect on its bundling or polymerization properties in vitro.

Overall, this study is well motivated, and the results support the conclusions that are drawn. I have only minor suggestions, as described below.

Minor comments: (1) The schematic diagrams of the chimeric formin constructs are very helpful. However, it is difficult to distinguish the colors from one another, especially in the case of the Cdc12FH1-Fus1FH2 variant, which requires discernment of the relatively small purple region within the dark blue molecule. Would it be possible to modify the colors to increase their contrast? Similarly, the blue and gray data sets in Figure 3B are very difficult to discern.

We have changed the colours to improve contrasts.

(2) The affinities (Kd) with which the formins bind the barbed ends as described in the second-to-last paragraph on page 8, in Figure Legend 7G, and in the "Analysis of pyrene data" section of the Materials and Methods should be defined as dissociation "constants", rather than dissociation "rates". Also, these affinities are lacking units in the following sentence on page 8.

We have corrected this. The unit is nM.

(3) When comparing the TIRF micrographs in Figure S3A, it looks as though both formins (but especially the R1054E variant) nucleate more filaments in the presence of profilin than in its absence. Is this a reproducible effect? If so, can the authors provide an explanation for this?

There is strong variability in the filament numbers observed by TIRF in replicate experiments, which makes it difficult to use this technique to determine the nucleation efficiency. This may be due for instance to the stickiness of the glass, which may influence the number of observed filaments. We have measured the number of filaments after 130s of polymerization for each condition to test whether there are any significant differences between conditions despite overall variability. The measurements suggest that the addition of profilin increases the number of actin filaments. However, these results should be taken very carefully due to the experimental variations (very large error bars). Additionally, because Fus1-associated filaments are very short in absence of profilin, it is quite likely that this influences their crowding at the glass surface compared to longer filaments (in presence of profilin). Since in TIRF we can only observe the filaments at the glass surface, we may miss a portion of short Fus1-bound actin filaments in absence of profilin.

For these reasons, and because the possible role of profilin in modulating nucleation efficiency by formins is not the object of the work here, would thus prefer not to include this graph in the manuscript.

Reviewer #2 (Significance (Required)): This study contributes a key advancement towards understanding how the polymerization activities of formins are tailored to support diverse and specific cellular functions. The results in this study nicely complement and expand upon similar recent work that dissected the polymerization requirements of the formin Cdc12, which mediates cytokinetic ring assembly in S. pombe, and For2, which drives the assembly of apical networks that are necessary for polarized growth in Physcomitrella patens. As such, this work will likely be of significant interest to scientists who study mechanisms of actin dynamics regulation. The identification of R1054 as a residue that confers a novel regulatory activity to the FH2 domain of Fus1 will also likely be of great interest to biochemists and other scientists who study formins at the molecular level.

My expertise is in the field of formins and actin polymerization.

The author uses of civility is through the depiction of police brutality. They know the emotions this will bring up for some readers and try to feed on that empathy.

See also An app can be a home-cooked meal along with this comment containing RMS's remarks with his code-as-recipe metaphor in the HN thread about Sloan's post:

some of you may not ever write computer programs, but perhaps you cook. And if you cook, unless you're really great, you probably use recipes. And, if you use recipes, you've probably had the experience of getting a copy of a recipe from a friend who's sharing it. And you've probably also had the experience — unless you're a total neophyte — of changing a recipe. You know, it says certain things, but you don't have to do exactly that. You can leave out some ingredients. Add some mushrooms, 'cause you like mushrooms. Put in less salt because your doctor said you should cut down on salt — whatever. You can even make bigger changes according to your skill. And if you've made changes in a recipe, and you cook it for your friends, and they like it, one of your friends might say, “Hey, could I have the recipe?” And then, what do you do? You could write down your modified version of the recipe and make a copy for your friend. These are the natural things to do with functionally useful recipes of any kind.

Now a recipe is a lot like a computer program. A computer program's a lot like a recipe: a series of steps to be carried out to get some result that you want. So it's just as natural to do those same things with computer programs — hand a copy to your friend. Make changes in it because the job it was written to do isn't exactly what you want. It did a great job for somebody else, but your job is a different job. And after you've changed it, that's likely to be useful for other people. Maybe they have a job to do that's like the job you do. So they ask, “Hey, can I have a copy?” Of course, if you're a nice person, you're going to give a copy. That's the way to be a decent person.

I think this is avoiding the real issue

that

the market is actually

not able to be designed enough

to correct for (at the moment) nor does it welcome "help" to make it more anti fragile,

aka i'm not saying we need more regulation, we would just need different regulation

to actually solve for those structural flaws.

— my core issue with this quote is that, trying to posit blatantly, that the market is heavily regulated is pretty far from accurate in my mind... like what regulations is this person referencing specifically?

1. Headline: ...for Shopify *stores
2. CTA should be "Integrate now"
3. We are completely missing the top propositions form our side - Checkout (with all its features incl. industry-leading SR, etc.). Global payments is a nice-to-have IMHO in most cases - we can make that a feature of Checkout like language support.
4. Integration - instead of a video people would be impatient with, can we have a simple animated GIF since it's just a few steps now?

GWG, Some random thoughts:

Your challenge question is tough, not just for the mere discovery portion, but for the multiple other functions involved, particularly a "submit/reply" portion and a separate "I want to subscribe to something for future updates".

I can't think of any sites that do both of these functionalities at the same time. They're almost always a two step process, and quite often, after the submission part, few people ever revisit the original challenge to see further updates and follow along. The lack of an easy subscribe function is the downfall of the second part. A system that allowed one to do both a cross-site submit/subscribe simultaneously would be ideal UI, but that seems a harder problem, especially as subscribe isn't well implemented in IndieWeb spaces with a one click and done set up.

Silo based spaces where you're subscribed to the people who might also participate might drip feed you some responses, but I don't think that even micro.blog has something that you could use to follow the daily photo challenges by does it?

Other examples: https://daily.ds106.us/ is a good example of a sort of /planet that does regular challenges and has a back end that aggregates responses (usually from Twitter). I imagine that people are subscribed to the main feed of the daily challenges, but I don't imagine that many are subscribed to the comments feed (is there even one?)

Maxwell's Sith Lord Challenge is one of the few I've seen in the personal site space that has aggregated responses at https://www.maxwelljoslyn.com/sithlordchallenge. I don't think it has an easy way to subscribe to the responses though an h-feed of responses on the page might work in a reader? Maybe he's got some thoughts about how this worked out.

Ongoing challenges, like a 30 day photography challenge for example, are even harder because they're an ongoing one that either requires a central repository to collect, curate, and display them (indieweb.xyz, or a similar planet) or require something that can collect one or more of a variety of submitted feeds and then display them or allow a feed(s) of them. I've seen something like this before with http://connectedcourses.net/ in the education space using RSS, but it took some time to not only set it up but to get people's sites to work with it. (It was manual and it definitely hurt as I recall.)

I don't think of it as a challenge, but I often submit to the IndieWeb sub on indieweb.xyz and I'm also subscribed to its output as well. In this case it works as an example since this is one of its primary functions. It's not framed as a challenge, though it certainly could be. Here one could suggest that participants tag their posts with a particular hashtag for tracking, but in IndieWeb space they'd be "tagging" their posts with the planet's particular post URL and either manually or automatically pinging the Webmention endpoint.

Another option that could help implement some fun in the system is to salmention all the prior submissions on each submission as an update mechanism, but one would need to have a way to unsubscribe to this as it could be(come) a spam vector.

This aspect of classical music culture has made it very difficult for me as I've struggled with insecurities or feelings of being "behind." It's somewhat reassuring to know that this isn't just a feeling I made up with no reason, but that it comes from this cultural value of "fast progression", rather than what I've been encouraged by my mentors to be on my "own path at my own pace" and not compare myself to others

So much wisdom and stuff to think about here.

(NB: the compilation "object" format here would, much like triple scripts, be another form of human readable source.)

How does the author claim to know that the students weren't listening and were just "venting anger?" There's nothing in the cited text to lead one to believe that ... and it's more than possible to be angry and confrontational about ideas. That's been my experience in classrooms for decades. Could the others have not gotten angry? Of course. And arguably they shouldn't have. But that's a skill that is often acquired with age, and a different thing from the not listening scenario. If they weren't listening, what would they be mad at?

oh, that's right, this author is very fond of reasoning toward his own foregone conclusions.

Memory usage is a concern? wat

It's a problem, sure, if you're programming the way NPMers do. So don't do that.

This is a huge problem I've noticed when it comes to people programming in JS—even, bizarrely, people coming from other languages like Java or C# and where you'd expect them to at least try to continue to do things in JS just like they're comfortable doing in their own language. Just because it's there (i.e. possible in the language, e.g. dynamic language features) doesn't mean you have to use it...

(Relevant: How (and why) developers use the dynamic features of programming languages https://users.dcc.uchile.cl/~rrobbes/p/EMSE-features.pdf)

The really annoying thing is that the NPM style isn't even idiomatic for the language! So much of what the NodeJS camp does is so clearly done in frustration and the byproduct of a desire to work against the language. Case in point: the absolutely nonsensical attitude about always using triple equals (as if to ward off some evil spirits) and the undeniable contempt that so many have for this.

This is really only a problem in languages that make the unfortunate mistake of allowing references to unqualified names that get fixed up as if the programmer had written this.mPoint or Foo.point. Even if you're writing in a language where that's possible, just don't write code like that! Just because you can doesn't mean you have to.

The only real exception is distinguishing locals from parameters. Keep your procedures short and it's less of a problem.

This seems like both a very philosophically interesting and cathartic activity, as well as something that could be turned into a very bold art project.

Author Response

Reviewer #1 (Public Review):

1) The connectivity patterns along the anterior-posterior hippocampal axis broadly follow an anterior-posterior cortical bias, such that posterior regions, e.g. the visual cortex, are preferentially connected to the hippocampal tail, and anterior regions, e.g. the temporal pole, are preferentially connected to the hippocampal head. The authors focus on the twenty regions with the highest connectivity profiles, which appears to capture the majority of all connections. However, some of the present structural connectivity patterns differ in interesting ways from previously described cortical networks reported in resting-state fMRI studies. Most notably, the medial PFC and orbitofrontal regions combined account for less than 1% of all connections in the present investigation (Table S1 & S2). This is an interesting contrast to functional investigations which tend to find that these regions cluster with the aHPC (e.g., Adnan et al. 2016 Brain Struct Func; Barnett et al. 2021 PLoS Biol; Robinson et al. 2016 NeuroImage). In contrast, the present DWI results suggesting preferential pHPC-medial parietal connectivity dovetail with those observed in fMRI studies. It seems important to discuss why these differences may arise: whether this is a differentiation between structural and functional networks, or whether this is due to a difference in methods.

We thank Reviewer 1 for making this important point and agree that these observations are deserving of further expansion. We have now included additional text where we place the surprising observation of sparse connectivity between PFC regions and the hippocampus more firmly in the context of recent evidence and argue that these observations suggest a potential differentiation between structural and functional networks.

We have included the following text in the discussion (pp. 16-17, lines 439-457);

“While many of our observed anatomical connections dovetail nicely with known functional associations, patterns of anatomical connectivity strength did not always mirror well characterised functional associations between the hippocampus and cortical areas. For example, a surprising observation from our study was that only weak patterns of anatomical connectivity were observed between the hippocampus and the ventromedial prefrontal cortex (vmPFC) and other frontal cortical areas. This lies in contrast to well documented functional associations between these regions (46-48). Our observation, however, supports a growing body of evidence that direct anatomical connectivity between the hippocampus and areas of the PFC may be surprisingly sparse in the human brain. For example, Rosen and Halgren (49) recently reported that long range connections between the hippocampus and functionally related frontal cortical areas may constitute fewer than 10 axons/mm2 and more broadly observed that axon density between spatially distant but functionally associated brain areas may be much lower than previously thought. Our observation of sparse anatomical connectivity between the hippocampus and PFC mirrors this recent work and suggests a potential differentiation between structural and functional networks as they relate to the hippocampus. It remains possible, however, that methodological factors may contribute to these differences. We return to this point later in the discussion. A future dedicated study aimed at assessing whether the well characterised functional associations between the hippocampus and vmPFC are driven by sparse direct connections or primarily by intermediary structures is necessary to address this issue in an appropriate level of detail.”

2) While the analytic pipeline is described in sufficient detail in the Methods, it is somewhat unclear to a non-DWI expert what the major methodological advance is over prior approaches. The authors refer to a tailored processing pipeline and 'an advance in the ability to map the anatomical connectivity (p. 5), but it's not immediately clear what these entail. It would be useful to highlight the key methodological differences or advances in the Introduction to help with the interpretation of the similarities and differences with previous connectivity findings.

We have now included a brief description in the Introduction highlighting the key methodological advances used in the current study.

We have included the following text in the Introduction (pp. 4-5, lines 130-144);

“In typical fibre-tracking studies, we cannot reliably ascertain where streamlines would naturally terminate, as they have been found to also display unrealistic terminations, such as in the middle of white matter or in cerebrospinal fluid (39). While methods have been proposed to ensure more meaningful terminations (40), for example, with terminations forced at the grey matter-white matter interface (gmwmi), this approach is still not appropriate for characterising terminations within complex structures like the hippocampus. A key methodological advance of our approach was to remove portions of the gmwmi inferior to the hippocampus (where white matter fibres are known to enter/leave the hippocampus). This allowed streamlines to permeate the hippocampus in a biologically plausible manner. Importantly, we combined this with a tailored processing pipeline that allowed us to follow the course of streamlines within the hippocampus and identify their ‘natural’ termination points. These simple but effective methodological advances allowed us to map the spatial distribution of streamline ‘endpoints’ within the hippocampus. We further combined this approach with state-of-the-art tractography methods that incorporate anatomical information (40) and assign weights to each streamline (41) to achieve quantitative connectivity results that more faithfully reflect the biological accuracy of the connection’s strength (39).”

3) Related to the point above, it was a bit unclear to me how the present connections map onto canonical white matter tracts. In Fig., 4A, the tracts are shown for a single participant, but it would be helpful to map or quantify know how many of the connections for a given hippocampal subregion are associated with a given tract to provide a link to prior work or clarify the approach. A fairly large body of prior research on hippocampal white matter connectivity has focused on the fornix, but it's a little difficult to align these prior findings with the connectivity density results in the current paper.

We thank Reviewer 1 for this comment and agree this would be an interesting avenue to pursue. However, the reliable segmentation of white matter fibre bundles is currently an area of contention in the DWI community. This pervasive and problematic issue was highlighted in a recently published large multi-site study that revealed a high degree of variability in how white matter bundles are defined, even from the same set of whole-brain streamlines (Schilling et al., 2021, Neuroimage. Nov; 243:118502. https://pubmed.ncbi.nlm.nih.gov/34433094/). This means that, even if we were to choose a particular method to segment white matter bundles, our results would not be readily translatable to those reported in previous DWI studies. This significantly limits meaningful comparison and/or interpretation. Indeed, such an approach may paradoxically take away from the detailed characterisations we have achieved in the current study. As highlighted in that study, it is now paramount that consensus is reached in this field to define criteria to reliably and reproducibly define white matter fibre bundles. Once that is achieved, we plan to conduct a follow-up study to characterise this in more detail, with bundles that will be able to be reliably reproduced by others.

4) Finally, on a more speculative note: based on the endpoint density maps, there seems to be a lot of overlap between the EDMs associated with different cortical regions (which makes sense given the subregion results). Does this effectively mean that the same endpoints may be equally connected with multiple different cortical regions? Part of the answer can be found in Fig. 3D showing the combined EDM for three different regions, but how spatially unique is each endpoint? This is likely not a feasible question to address analytically but it might be helpful to provide some more context for what these maps represent and how they might relate to differences across individuals.

The primary aim of the current analysis was to characterise broad patterns of endpoint density captured by our averaged group level analysis. However, Reviewer 1 is astute in assuming that, although there is overlap in the group averaged endpoint density maps (EDMs) associated with different cortical areas, at the single participant level, there are both overlaps and spatial uniqueness in the location of individual endpoints. For example, while group level analysis revealed that area V1 and area V2 showed preferential connectivity with overlapping regions of the posterior medial hippocampus, when visualising individual endpoints associated with each of these areas at the single participant level, we can see that some endpoints overlap while others display spatially unique patterns (see image below). Although a more in-depth analysis of individual variability in these patterns was beyond the scope of this investigation (as noted on Page14; Lines 379-381), we agree with Reviewer 1 that this is an important point to note in the manuscript. We have, therefore, included additional text touching on this and have included a new Supplementary Figure (Page 42; also see below) to emphasise that, at the single participant level, different cortical areas display both overlapping and spatially unique endpoints within specific regions of the hippocampus (using areas V1 and V2 as an example).

We have included the following text in the Results section (pp. 14, lines 370-379);

“Finally, while we observed clear overlaps in the group averaged EDMs associated with specific cortical areas, a closer inspection of individual endpoints at the single participant level revealed that endpoints associated with different cortical areas displayed both overlapping and spatially unique characteristics within these areas of overlap. For example, at the group level, areas V1 and V2 showed preferential connectivity with overlapping regions of the posterior medial hippocampus (see Supplementary Figure S5) while, at the single participant level, individual endpoints associated with each of these areas display both overlapping and spatially unique patterns (see Supplementary Figure S6). This suggests that, while specific cortical areas display overlapping patterns of connectivity within specific regions of the hippocampus, subtle differences in how these cortical regions connect within these areas of overlap likely exist.”

Reviewer #2 (Public Review):

Dalton and colleagues present an interesting and timely manuscript on diffusion weighted imaging analysis of human hippocampal connectivity. The focus is on connectivity differences along the hippocampal long axis, which in principle would provide important insights into the neuroanatomical underpinnings of functional long axis differences in the human brain. In keeping with current models of long-axis organisation, connectivity profiles show both discrete areas of higher connectivity in long axis portions, as well as an anterior-to-posterior gradient of increasing connectivity. Endpoint density mapping provided a finer grained analysis, by allowing visualisation of the spatial distribution of hippocampal endpoint density associated with each cortical area. This is particularly interesting in terms of the medial-lateral distribution with hippocampal head, body and tail. Specific areas map to precise hippocampal loci, and some hippocampal loci receive inputs from multiple cortical areas.

This work is well-motivated, well-written and interesting. The authors have capitalised on existing data from the Human Connectome Project. I particularly like the way the authors try to link their findings to human histological data, and to previous NHP tracing results.

Many thanks.

1) There are some important surprises in the results, particularly the relatively strong connectivity between hippocampus and early visual areas (including V1) and low connectivity with areas highly relevant from functional perspectives, such as the medial prefrontal cortex (rank order by strength of connectivity 7th and 78th of all cortical structures, respectively). This raises a concern that the fibre tracking method may be joining hippocampal connections with other tracts. In particular, given the anatomical proximity of the lateral geniculate nucleus to the body and tail of the hippocampus, the reported V1 connectivity potentially reflects a fusion of tracked fibres with the optic radiation. In visualizing the putative posterior hippocampus-to-V1 projection (Figure 4B, turquoise), the tract does indeed resemble the optic radiation topography. Although care was taken to minimise the hippocampus mask 'spilling' into adjacent white matter, this was done with focus on the hippocampal inferior margin, whereas the different components of the optic radiation lie lateral and superior to the hippocampus.

We agree with Reviewer 2 that our observations relating to area V1 could be the result of limitations inherent to current tracking methodology. Indeed, probabilistic tracking can result in tracks mistakenly ‘jumping’ between fibre bundles. Unfortunately, primarily due to limitations in image resolution, we do not believe that we can categorically rule this possibility out in the current dataset beyond the measures we have already taken in our analysis pipeline. We have now included additional text in the Discussion acknowledging and emphasising this possible limitation of our study.

We have included the following text in the Discussion section (Page 25; Lines 694-699);

“Also, we cannot rule out that some connections observed in the current study may result from limitations inherent to current probabilistic fibre-tracking methods whereby tracks can mistakenly ‘jump’ between fibre bundles (e.g. for connections between the posterior medial hippocampus and area V1 due to the proximity to the optic radiation), especially in “bottleneck” areas. Again, future work using higher resolution data may allow more targeted investigations necessary to confirm or refute the patterns we observed here.”

Beyond the possibility of tracks jumping between fibre bundles, we feel it is important to emphasise that an integral part of our analysis was the detailed attention we took to minimise mask ‘spillage’ of the entire hippocampus mask. It is not the case that we primarily focussed on inferior portions of the hippocampus as stated by Reviewer 2. Equal focus was paid to medial, lateral and superior portions of the mask which lie adjacent to visual thalamic nuclei, the optic radiation posteriorly and a number of other structures. We can see that our description relating to this lacked the necessary detail to convey this important point clearly and we apologise for the confusion. We have, therefore, included additional text in the Methods section clarifying this further.

We have included the following text in the Methods section (Page 26; Lines 751-755);

“We took particular care to ensure that all boundaries of the hippocampus mask (including inferior, superior, medial and lateral aspects) did not encroach into adjacent white or grey matter structures (e.g., amygdala, thalamic nuclei). This minimised the potential fusion of white matter tracts associated with other areas with our hippocampus mask.”

These points notwithstanding, our results support recently observed structural and functional associations between the posterior hippocampus and early visual processing areas. We agree that these findings are potentially of great conceptual importance for how we think about the hippocampus and its connectivity with primary sensory cortices in the human brain and we have now included a brief comment relating to this in the Discussion.

We have included the following text in the Discussion (Page 23-24; Lines 638-644);

“However, this observation supports recent reports of similar patterns of anatomical connectivity as measured by DWI in the human brain (38) and functional associations between these areas (43, 60). Collectively, these findings are potentially of great conceptual importance for how we think about the hippocampus and its connectivity with early sensory cortices in the human brain and open new avenues to probe the degree to which these regions may interact to support visuospatial cognitive functions such as episodic memory, mental imagery and imagination.”

2) A second concern pertains to the location of endpoint densities within the hippocampus from the cortical mantle. These are almost entirely in CA1/subiculum/presubiculum. It is, however, puzzling why, in Supp Figure 2, the hippocampal endpoints for entorhinal projections is really quite similar to what is observed for other cortical projections (e.g., those from area TF). One would expect more endpoint density in the superior portions of the hippocampal cross section in head and body, in keeping with DG/CA3 termination. I note that streamlines were permitted to move within the hippocampus, but the highest density of endpoints is still around the margins.

We agree with Reviewer 2 that, in relation to the entorhinal cortex, we would expect to see more endpoint density in areas aligning with the dentate gyrus (DG) and CA3 regions of the hippocampus. We noted in the discussion that “Despite the high-quality HCP data used in this study, limitations in spatial resolution likely restrict our ability to track particularly convoluted white-matter pathways within the hippocampus and our results should be interpreted with this in mind”. We believe that this limitation applies to pathways between the entorhinal cortex and DG/CA3. We have now included additional text specifically noting that this limitation likely affects our ability to track streamlines as they relate to DG/CA3. A targeted investigation of this effect using higher resolution diffusion MRI data may help address this issue, and this will be the subject of future work.

We have included the following text in the Discussion (Page 25; Lines 690-693);

“Indeed, this may explain the surprising lack of endpoint density observed in the DG/CA4-CA3 regions of the hippocampus where we would expect to see high endpoint density associated with, for example, the entorhinal cortex which is known to project to these regions. Future dedicated studies using higher resolution data are needed to assess these pathways in greater detail.”

3) On a related point, the use of "medial" and "lateral" hippocampus can be confusing. In the head, CA2/3 is medial to CA1, but so are subicular subareas, just that the latter are inferior.”

We agree that applying the terms ‘medial’ and ‘lateral’ to our three-dimensional representations can lead to some ambiguities and confusion. We have included a new description defining our use of these terms in the Results section.

We have included the following text in the Results section (Page 10; Lines 268-273).

“In relation to nomenclature, our use of the term ‘medial’ hippocampus refers to inferior portions of the hippocampus aligning with the distal subiculum, presubiculum and parasubiculum. Our use of the term ‘lateral’ hippocampus refers to inferior portions of the hippocampus aligning with the proximal subiculum and CA1. In instances that we refer to portions of the hippocampus that align with the DG or CA3/2 we state these regions explicitly by name”.

Discussion, revision and decision

Decision

Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

Dr. Bañuelos: Verified manuscript

Dr. Morris: Verified with reservations

Revision

Response to Reviewer 1 (Dr. Bañuelos)

1. Most importantly, I would like to see an introduction that explains the authors’ general arguments about grading changes – including the trajectory of these changes at Dalhousie and why this arc contributes to our knowledge of the history of higher education more broadly. Then, the authors might continually remind us of the arc they present at the outset of their paper – especially when they are highlighting a piece of evidence that illustrates their central argument. To me, the quotes from students and faculty responding to grading changes are among the most interesting parts of the paper and placing these in additional context should make them shine even more brightly!

Our Response: Thank you so much for your thoughtful review. We have added a larger new introduction section of the paper (paragraphs 1-5 in the latest draft are new) that outlines the general importance of the topic, the Canadian context, details on Dalhousie University, and our overall thesis statement (i.e., most decisions were to improve the external communication value of grades). Moreover, we have added three new student quotes form the Dalhousie Gazette to build a stronger picture for student reactions, and to build a better case for our overall thesis statement (i.e., that changes in grading were often to increase the external communication value of grades). Moreover, throughout we have added some details on the overall funding trajectory for institutions in Canada that created some pressure to standardize grading. We think that these changes have improved the manuscript.

1. I’d like to read a little more about Dalhousie itself – why it is either a remarkable or unremarkable place to study changes in grading policies. Is it representative of most Canadian universities and thus, a good example of how grading changes work in this national context? Is it unlike any other institution of higher education and thus, tells us something important about grades that we could not learn from other case studies? I don’t think this kind of description needs to be particularly long, but it should be a little more involved than the brief sentences the authors currently include (p.3, paragraph 1) and should explain the choice of this case.

Our Response: This comment revealed that two additional pieces of context were needed for the introduction: (a) some national context for higher education policy in Canada and (b) some extended description of Dalhousie University when compared to other universities in Canada. To this end, two new paragraphs have been added to the paper (paragraphs 2 & 3 in the current draft).

Notably, Jones (2014) notes that “Canada may have the most decentralized approach to higher education than any other developed country on the planet” (pg 20). With this in mind, any historical review of education policy is by necessity specific to province and institution – that is, the information can be placed in its context, but resists wide generalization to the country as a whole. In the newest draft, we tried to describe the national, provincial, and institutional context in some more detail in paragraphs 2 & 3.

1. I’d also like to know more about the archival materials the authors used. The authors mention that they drew from “Senate minutes, university calendars, and student newspapers” (p. 3), but what kinds of conversations about grades did these materials include? At various points, the authors engage in “speculation” (e.g. p.4) about why a particular change occurred. This is just fine and, in fact, it’s good of the authors to remind us that they are not really sure why some of these shifts happened. But, they might go one step further and tell us why they have to speculate. Were explicit discussions of grading changes – including in inter- and intradepartmental letters and memo, reports, and other documents – not available in these archives? Why are these important discussions absent from the historical record?

Our Response: We have added a new paragraph (paragraph 4) to the paper discussing the sources in some more detail. It is true that the verbatim discussions are frequently absent from the record, especially earlier in history – or if they exist, we have not found them! Instead, we frequently are reviewing meeting minutes or committee reports, which are summaries of discussions. As we now note in the paper, “Thus, the sources used showed what policy changes were implemented, when they were implemented, and a general sense of whether there was opposition to changes; however, there were notable gaps in faculty and student reactions to grade policy changes, as these reactions were frequently not written down and archived.”

This gap was most apparent in the Senate minutes around the 1940s, where I (the first author) could not find any direct discussions of why changes were implemented. Under the 1937-1947 heading, we more clearly indicate that the rationale for the changes was absent from the Senate minutes during this period. I add some further speculation on why these records might be absent, based on summaries from Waite (1998b); specifically, the university president of the time often made unilateral decisions, circumventing Senate, which might account for why the changes are absent from the records.

This will hopefully make the limitations of what can be learned from this approach more apparent.

1. At various points, the authors make references to the outside world – for example, WWII (p. 5), the Veteran’s Rehabilitation Act (pp. 6-7), and British versus American grading schemas (p. 6). But, these references are brief and seem almost off-handed. I know space is limited, but putting these grading changes in their broader context might help make the case for why this study is interesting and important. Are the changes in the 1940s, for example, related to the ascendance of one national graduate education model over another (e.g. American versus British)? Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time? If so, I would share any information you might have on these broader trends.

Our Response: To our knowledge, there isn’t any comparable report to what we’ve written here documenting the transition from British “divisions” to American “letter grades” in Canadian Universities, making our report novel in this regard. It might well be that a similar historical arc exists in many of the 223 public and private universities in Canada, but we don’t believe such data exists in any readily accessible way – excepting perhaps undergoing a similar deep dive into historical documents at each respective institution! So, we do not have the answer to your question: “Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time?” However, we did add one reference which provided a snapshot point of comparison in 1960, noting in the paper “Baldwin (1960) notes that the criteria for “High First Class” grades in the humanities was around 75-80% at Universities of Toronto, Alberta, and British Columbia in 1960, suggesting that Dalhousie’s system was similar to other research-intensive universities around this time.” That said, there are a few major national events related to the funding of universities in Canada that we have elaborated on in the text to address the spirit of your recommendation for describing the national context:

a) In the “Late 1940s” section of the paper, we added: “Though Dalhousie had an unusually high proportion of veterans enrolled relative to other maritime universities during this period (Turner, 2011), the Veteran’s Rehabilitation Act was a turning point for large increases in enrollment and government funding Canada-wide, at least until the economic recession of the 1970s (Jones, 2014).”

b) In the 1990s, there were major government cuts to funding, creating challenging financial times for the university. We discuss the funding pressures that likely contributed to standardization of grading during this time by saying the following in the 1980s-2000s section: “Starting in in the 1980s-1990s there were major government cuts to university funding nation-wide, with the cuts becoming more severe in the 1990s (Jones, 2014; Higher Education Strategy Associates, 2021). Because of the nature of the funding formulas, cuts in Nova Scotia were especially deep. Beyond tuition increases, university administrators knew that obtaining external research grants, Canada Research Chairs, and scholarship funding was one of the few other ways for a university to balance budgets, so there was extra pressure to be competitive in these pools. […] The increased standardization was likely related to increased financial pressures at this time – standardization is an oft-employed tool to deal with ever-increasing class sizes with no additional resources.”

c) In the 2010s section of the paper, we added context to how universities in country-wide have become increasingly dependent on tuition fees for funding: “Following the 2008 recession, federal funding decreased again (Jones, 2014; Higher Education Strategy Associates, 2021); however, this time universities tended to balance budgets by increasing tuition and international student fees. This trend towards increased reliance on tuition for income is especially pronounced in Nova Scotia, which has the highest tuition rates in the country (Higher Education Strategy Associates, 2021). Thus, the university moved closer to a “consumer” model of education, so it makes sense that a driving force for standardization was student complaints.”

1. This is a very nitpicky concern that doesn’t fit well elsewhere, so please take it with a grain of salt. I was surprised at the length of the reference list – it seemed quite short for a historical piece! I wonder, again, if more description of the archival material - including why you looked at these sources, in particular, and what was missing from the record – would help explain this and further convince the reader that you have all your bases covered.

Our Response: In the introduction section, paragraph 4, we describe our sources in more detail including what is likely missing from the record and why we used them. Regarding the length of the reference list, we did add ~12 new references to the list in the course of making various revisions, which partially addresses your concern. Beyond this though, it’s worth noting that some of the sources more extensive than they seem, even though they don’t take up much space in the reference list (e.g., there is one entry for course calendars, but this covers ~100 documents reviewed!). Moreover, there were many dead-ends in the archives that are not cited (e.g., reviewing 10 years of Senate minutes in the 1940s produced little of relevance), so the reference list is curated to only those sources where relevant materials were found.

Reviewer response to revisions

The new introduction to the piece addresses many of my previous questions about the authors’ general arguments, the Dalhousie context, and the source material. Thank you for addressing these! Reading this version, it is much clearer that the key argument is that standardized, centralized grading practices were “to improve the external communication value of the grades, rather than for pedagogical reasons” (p. 6). I also really enjoyed the added quotes from students in the Dalhousie Gazette.

The authors’ response to Reviewer 2 really gave me a better sense of why they wrote this piece and also helped me to more clearly put my finger on what was troubling me in the first round. It still reads a little like a report for an internal audience – which is just fine and, in fact, can be extremely useful for historians of the future. But, as Reviewer 2 notes, this means it does not really seem like a piece of historical scholarship. I do worry that shaping it into this form would take an extensive revision and might not be in the spirit of what the authors intended to do.

A different version of this article might start with this idea that grades were standardized for external audiences and in response to financial pressures. It would then develop a richer story behind the sudden importance of these external audiences and the nature (i.e. source, type) of financial pressures Dalhousie was facing. It would highlight the impact such changes had on students and their future careers/graduate experiences. It could then connect these trends to other similar changes for external audiences and the increasing interconnectedness of American, Canadian, and British systems through graduate education. It might even turn to sociological theories of organizational change and adaptation and make an argument for when (historically) similar forms of decoupling were likely to occur in the Canadian higher education system. Finally, it might connect these grading changes to current trends – including accusations of grade inflation and accepted best practices for measuring learning outcomes.

But, it doesn’t seem that the authors necessarily want to do this, which I can understand and respect. I think there is enormous value in a piece of scholarship like this existing – both for internal audiences and for future historians. Indeed, imagine if every university had a detailed history of its grading policies like this available somewhere online! Comparing such practices across institutions would certainly tell us a lot about why grading currently looks the way it does.

Decision changed

Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.

Response to Reviewer 2 (Dr. Morris)

The authors dove headfirst into Dalhousie’s archives, unpacking the subtle shifts in grading policy. Their work seems to be comparable to archaeologists, digging deep beneath mountains of primary sources to find nuggets of clues into Dalhousie’s grading evolution. I particularly liked when the authors were able to link these changes to student voices, as seen in moments when they referenced student publications.

Ultimately, I kept coming back to one main comment that I wrote in the margins: “So what?” I would humbly suggest that the authors reflect on why this history matters to them. Granted, they do this in the conclusion, where they touch on Schneider & Hutt’s argument that grades evolved to increasingly be a form of external communication with audiences beyond school communities. Sure. But I want more. I wanted to see a new insight that this microhistory of Dalhousie significant to the history of Canada or the history of education more generally.

If the authors are so inclined, there might be several approaches to transform this manuscript. I would suggest the following. First, instead of tracing the entire history of grading at the institution, choose one moment of change that you think is the most important. Perhaps in the 1920s and the lack of transparency in grading, or the post-war shift toward American grading. Second, show me – don’t tell me – what Dalhousie was like at this moment. Paint a picture of the institution with details about student demographics, curriculum, educational goals, the broader town, etc. Make the community come alive. Show me what makes Dalhousie unique from other institutions of higher ed. Once you establish that picture, perhaps you could link the change in grading practices to subtle changes at the university community, thereby establishing a before and after snapshot. This will require considerable amounts of work, and the skills of a historian. You will have to find primary and secondary sources that go far beyond what you’ve relied on thus far.

In the end, I found myself wanting the authors to humanize this manuscript, meaning I wanted them to show me that changes in grading practices have tangible effects on real-life human beings. A humanization of their research would mean going narrower and deeper; or, in other words, eliminating much of what they have documented.

However, if that is too tall of an order, I would ask that the authors clarify for themselves who this manuscript is for. Is this a chronicling of facts for an internal audience at Dalhousie’s faculty, alumni, and students? Fine. But my guess is that even members of the Dalhousie community want to read something relatable.

I am suggesting revisions, although not because of objective errors. History is more of an art, in my opinion. With that in mind, I would suggest that the authors paint a more vivid picture (metaphorically) of Dalhousie, showing me how changes one moment of change in grading practices impacted the lives of human beings.

Our Response: Thank you very much for taking the time to read our paper and provide your thoughts and recommendations. It may be helpful to begin by describing why I (the first author) decided to write this paper. Ultimately, I wrote this paper to satisfy my own personal curiosity and to connect with other people at my own place of employment by exploring our shared history. At present day, Dalhousie has a letter grading scheme with a standardized percentage conversion scheme that all instructors used. I wanted to know why this particular scheme was used, but I quickly realized that nobody at Dalhousie really knew how we ended up grading this way! There was an institutional memory gap, and a puzzle that was irresistible to me. So, I wrote this paper for the most basic of all academic reasons: Pure curiosity. I do very much recognize that the subject matter is very niche, perhaps too niche for a traditional journal outlet. Thus, my publishing plan is to self-publish a manuscript to the Education Resources Information Center (ERIC) database and a preprint server as a way of sharing my work with others who might be interested in what I found. Nonetheless, I believe in the importance and value of peer review, especially since I am writing in a field different than most of my scholarly work. That is why I chose PeerRef as a place to submit, so that I could undergo rigorous peer review to improve the work while still maintaining the niche subject matter and focus that drives my passion and curiosity for the project. Of course, if you feel the whole endeavor is so flawed that it precludes publication anywhere, then we can consider this a “rejection” and I will not make any further edits through PeerRef.<br /> The core of your critique suggested that I should write a fundamentally different paper on different subject matter. While I don’t necessarily disagree that the kind of paper you describe might have broader appeal, it would no longer answer the core research question I wanted an answer to: How has Dalhousie’s grading changed over time? So, I must decline to rewrite the paper to focus on a single timeframe as recommended. All this said, I did try my best to address the spirit of your various concerns to improve the quality of the manuscript. Below, I will outline the various major changes to the manuscript that we made to improve the manuscript along the lines you described, while maintaining our original vision for the structure and focus of the paper. The specific changes are outline below:

a) Two new paragraphs (now paragraphs 1-2 of the revised manuscript) were added to explain the “so what” part of the question. Specifically, we describe why we think the subject matter might be of interest to others and summarize the general dearth of historical information on grading practices in Canada as a whole.

b) Consistent with recommendations from the other reviewer, we now state a core argument (i.e., that most major grading changes were implemented to improve the external communication value of the grades) earlier in the introduction in paragraph 5 and describe how various pieces of evidence throughout the manuscript tie back to that core theme.

c) In an attempt to “humanize” the manuscript more, we added more student quotes from the Dalhousie Gazette throughout the paper so that readers can get a better sense of how students thought about grading practices at various times throughout history. Specifically, three new quotes were added in the following sections: 1901-1936, late 1940s, 1950s-1970s. We also added this short note about the physical location where grades used to be posted: “Naturally, this physical location was dreaded by students, and was colloquially referred to as “The Morgue” (Anonymous Dalhousie Gazette Author, 1937).”

d) Early in the paper, we describe why we chose Dalhousie and the potential audience of interest: “As employees of Dalhousie, we naturally chose this institution as a case study due to accessibility of records and because it has local, community-level interest. The audience was intended to be members of the Dalhousie community; however, it may also be a useful point of comparison for other institutions, should similar histories be written.”

e) We have described some of the limitations of our sources in paragraph 4, which may explain why the manuscript takes the form it does – it has conformed to the information that is available!

f) We have linked events at Dalhousie to the national context in some more detail, by detailing some national events related to the funding of universities in Canada. See our response to Reviewer 1, #4 above for more details on the specific changes.

g) Consistent with your stylistic recommendations, we have changed various spots throughout the paper from the present tense (e.g., “is”) to the past tense (e.g., “was”), and were careful in our new additions to maintain the past tense, when appropriate. If there are any spots that we missed, let us know the page number / section, and we will make further changes, as necessary.

h) We retained the first person in our writing – this may be discipline-specific, but in Psychology (the first author’s home discipline), first person is acceptable in academic writing. If you feel strongly about this, we can go through the manuscript and remove all instances of the first person, but we would prefer to keep it, if at all possible.

Hopefully this helps address the spirit of your concerns, and I look forward to hearing your thoughts in the second round of reviews.

Decision changed

Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

Peer review report

Reviewer: Nidia Bañuelos Institution: University of Wisconsin-Madison email: nbanuelos@wisc.edu

General comments

Questions of how and why grading practices change over time, how students, faculty, and administrators respond to grades, and the external pressures on grading practices (e.g. war, graduate school requirements) are inherently interesting! The authors have clearly done a careful job of tracking these – often minute, and likely, difficult to follow – changes at Dalhousie University. The manuscript is well-written and relatively easy to follow.

My biggest concern, reflected in the more detailed comments below, is that the authors could do a better job of explaining to the reader why these changes are interesting, important, and relevant to historians of higher education more broadly – even those who aren’t at Dalhousie. They do some of this at the very end of the paper and, indeed, this summing up of their findings and explanation of their relevance was my favorite part of the manuscript. I would suggest reorganizing the paper so that these bigger takeaways appear in the introduction and so that the reader is reminded of them at each major section break of the paper. For example, when the authors present a quote from a student who is concerned that grades have little to do with learning outcomes, they might remind us that one of their main arguments is that “decisions about university grading schemes had very little to do with actual pedagogy” (p. 15).

As it is written, the manuscript sometimes reads like a list of facts about grading changes. But, I think a reframing that focuses on the general importance of these changes could make the entire piece more engaging. More on this below…

Section 1 – Serious concerns

• Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No

• Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

Section 2 – Language quality

• How would you rate the English language quality? High quality

Section 3 – validity and reproducibility

• Does the manuscript contain any objective errors, fundamental flaws, or is key information missing?

While I don’t notice any “objective errors”, I do think the paper has a major flaw (i.e. little explanation of the broader significance of this case study) and could benefit from additional information about the institutional context, the archival material, and external influences on grading trends. (Please see below.)

Section 4 – Suggestions

• Based on your answer in section 3 how could the author improve the study?

I. Most importantly, I would like to see an introduction that explains the authors’ general arguments about grading changes – including the trajectory of these changes at Dalhousie and why this arc contributes to our knowledge of the history of higher education more broadly. Then, the authors might continually remind us of the arc they present at the outset of their paper – especially when they are highlighting a piece of evidence that illustrates their central argument. To me, the quotes from students and faculty responding to grading changes are among the most interesting parts of the paper and placing these in additional context should make them shine even more brightly!

II. I’d like to read a little more about Dalhousie itself – why it is either a remarkable or unremarkable place to study changes in grading policies. Is it representative of most Canadian universities and thus, a good example of how grading changes work in this national context? Is it unlike any other institution of higher education and thus, tells us something important about grades that we could not learn from other case studies? I don’t think this kind of description needs to be particularly long, but it should be a little more involved than the brief sentences the authors currently include (p.3, paragraph 1) and should explain the choice of this case.

III. I’d also like to know more about the archival materials the authors used. The authors mention that they drew from “Senate minutes, university calendars, and student newspapers” (p. 3), but what kinds of conversations about grades did these materials include? At various points, the authors engage in “speculation” (e.g. p.4) about why a particular change occurred. This is just fine and, in fact, it’s good of the authors to remind us that they are not really sure why some of these shifts happened. But, they might go one step further and tell us why they have to speculate. Were explicit discussions of grading changes – including in inter- and intradepartmental letters and memo, reports, and other documents – not available in these archives? Why are these important discussions absent from the historical record?

IV. At various points, the authors make references to the outside world – for example, WWII (p. 5), the Veteran’s Rehabilitation Act (pp. 6-7), and British versus American grading schemas (p. 6). But, these references are brief and seem almost off-handed. I know space is limited, but putting these grading changes in their broader context might help make the case for why this study is interesting and important. Are the changes in the 1940s, for example, related to the ascendance of one national graduate education model over another (e.g. American versus British)? Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time? If so, I would share any information you might have on these broader trends.

Similarly, the authors make brief mention of the internal reaction to grade changes – quoting students or faculty minutes. But, it would be wonderful (space permitting) to have even more information the internal impact of these changes. Did they change faculty instructional practices? Did they seem to have any effect on students’ orientation to their learning? Did standardization reflect an increasing interdependence of departments, or did it contribute to their lessening autonomy? If the archival record doesn’t permit us to know these things, then this might be a limitation the authors note at the end of the manuscript. I noticed that the authors reference a secondary source on Dalhousie student experiences repeatedly (Waite, 1998). Even a little more from this text or another secondary source like it could help the reader better understand the impact of grade changes.

• Do you have any other suggestions, feedback, or comments for the Author?

This is a very nitpicky concern that doesn’t fit well elsewhere, so please take it with a grain of salt. I was surprised at the length of the reference list – it seemed quite short for a historical piece! I wonder, again, if more description of the archival material - including why you looked at these sources, in particular, and what was missing from the record – would help explain this and further convince the reader that you have all your bases covered.

Section 5 – Decision

Requires revisions: The manuscript contains objective errors or fundamental flaws that must be addressed and/or major revisions are suggested.

Sorry, just seeing this now. It's assuredly a sliderule, which would have been a common tool for engineers and mathematicians of his era to have had. They became less common with the advent and proliferation of calculators.

There's plenty of irrationality to be found in opposing camps, too. I won't say that it's actually worse (because it's not), but it's definitely a lot more annoying, because it usually also carries overtones that there's a sort of well-informed moral and technological high ground—when it turns out it's usually just a bunch of second panel thinkers who themselves don't even understand computers (incl. compilers, system software, etc.) very well.

This is what makes it hard to have discussions about reforming the practices in mainstream Web development. The Web devs are doing awful things, but at least ~half of the criticism that these devs are actually exposed to ends up being junk because lots of the critics unfortunately just have no fucking idea what they're talking about and are nowhere near the high ground they think they're standing on—often taking things for granted that just don't make sense when actually considered in terms of the technological uppercrust that they hope to invoke. Just a kneejerk "browser = bad" association from people who can't meaningfully distinguish between JS (the language), browser APIs, and the NPM corpus (though most NPM programmers are usually guilty of exactly the same...).

It's a very "the enemy of my enemy is not my friend" sort of thing.

What's the Best Way to Teach Science?

0:31 What's the best way to teach science in my opinion? It's to do science. And to summarize my motto it's this: Don't Kill the Wonder! (and don't hide the practices)

0:42 The wonder is a look that you see on a person's face when they're REALLY interested in a problem but they don't know the answer.

1:13 And so the WORST way to teach science is to start by explaining! You want them to have that curiosity and then follow that curiosity.

3:16 When you're teaching science it's not the content ... what is the most important thing. It's the actual practices of doing science.

3:41 ...We live in an ironic time. At a time where people are so excited about science and new discoveries but students are not excited about their classroom. And I think one of the reasons why, is that what we do, is we tend to just explain all the time. When you explain all the time what you lose is the Wonder. —

https://youtu.be/TzoIz2W-gLQ

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

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2021-01219

Corresponding author(s): Rajan, Akhila

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.

The goal of this study is to:

• Define how prolonged exposure to a high-sugar diet (HSD) regime alters both the lipid landscape and feeding behavior.
• Determine how changes in lipid classes within the adipose tissue regulates feeding behavior. Key findings:

In this study, by taking an unbiased systems level and genetic approach, we reveal that phospholipid status of the fat tissue controls global satiety sensing.

Impact of Key findings:

By uncovering a critical role for adipose tissue phospholipid balance as a key regulator of organismal feeding, our work raises the possibility that the rate-limiting enzymes in phospholipid synthesis, including Pect, are potential targets for therapeutic interventions for obesity and feeding disorders.

Peer review comments:

This study has immensely benefited from the thoughtful peer-review of three reviewers. As per their recommendations, we have performed a major revision by performing additional experiments (see summary table below in next section) and strived to address the major concerns raised. Based on our reading, there were two major concerns that overlapped between all three reviewers raised. They are as follows:

• Does the genetic disruption of Pect in fly fat body alter phospholipid levels? Two reviewers (#2 and #3) recommended that we perform lipidomic analyses on adult flies with adipose tissue specific knockdown of For the revised version, we have completed this lipidomic experiment, and present results as a new main Figure 6, Supplemental S7 and S9.
• Is the dampened HSD induced hunger-driven feeding (HDF) behavior because of increased baseline feeding (#1 and #3)? In addition, reviewer #1, asked us whether HSD flies experience an energy-deficit? In other words, we were asked to uncouple whether what we observed was HSD-driven allostasis or indeed, as we had interpreted, that HSD dampened hunger-driven feeding response.

Hence, they recommended that we:

1. Re-analyze our hunger-driven feeding datasets and present non-normalized data (also requested by Reviewer #3) and show baseline feeding behavior on HSD. To address this, we have completed this analysis and present our results in Figure 1B-D and S1.
2. Determine whether the HSD fed flies display an energy deficit on starvation. To this end, we performed an assayed starvation-induced fat mobilization on HSD, results for this are now presented on Figure 1E-G and S2. Conclusions after the revision:

First, it is important to note here that the additional experiments have not caused a significant revision of the major conclusions of the original version of our study. In fact, we hope that the revised version provides clarity and further substantiation to our original arguments.

• The lipidomics experiments on Pect fat-specific knock-down flies show that reducing Pect in fat-body causes a significant reduction in certain PE lipid species (PE 36.2 specifically- Figure 6B). This is consistent with a prior report on lipidomics of the Pect null allele by Tom Clandinin’s group (PMID: 30737130). Furthermore, we note that when Pect is knocked down in the fat body, there is a significant increase in two other classes of phospholipids LPC and LPE (Figure 6A). Together, this suggests that an imbalance in phospholipid composition in the absence of Pect activity in fat.
• The starvation-induced fat mobilization experiments show that despite being fed a prolonged HSD, adult flies sense starvation and effectively mobilize fat stores, at a level comparable to Normal food (NF) fed adult flies, suggesting that even despite HSD exposure, adult flies experience an energy deficit on starvation.
• In our non-normalized data, we find that the baseline feeding events are not significantly altered between HSD and NF-fed flies (Figure 1D). This suggests that the effects we observe are not due to an increase in the “denominator”, but a dampening of hunger-driven feeding on HSD. With regard to our original version, all three peer-reviewers found that the study was interesting, significant, important, and novel – Reviewer #1: “The work is potentially novel and interesting”; #2 : “I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing”; #3: “This manuscript demonstrates how fat body Pect levels affect HSD induced changes in hunger-driven feeding response. I agree with all the reviewers points; potentially very interesting”. But had requested that we provide further substantiation and clarification.

We sincerely hope that the peer-reviewers find that our revised version with additional new experimental datasets, improved data visualization, and the presentation of non-normalized raw data points, makes this study clear, compelling, and well-substantiated.

• Point-by-point description of the revisions This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

Below we summarize in Part A, the key experiments that were performed to address the major concerns. In Part B, we provide a point-point response to each reviewer with embedded datasets.

Part a:

We performed several new experiments, including:

• To address the primary concern of Reviewer #1 regarding whether the HSD flies have a similar energy deficit to Normal food (NF) fed flies, we performed analysis of stored neutral fat Triacylglycerol (TAG) reserves and how HSD fed flies mobilized fat stores on starvation. We present these results in Figure 1E-G, S2. These results show that HSD-flies despite accumulating more TAG (S2), breakdown a similar amount of fat reserves as NF-fed flies on starvation at any time-point (Figure 1E-G). This suggests that HSD-fed flies do sense and respond to energy deficit.
• To address concerns of reviewer #2 and #3 on whether Pect genetic manipulation affects specific phospholipid classes, we performed lipidomic analyses. The table below summarizes the new 3 new figures and 4 supplemental figures (blue text are all new figure numbers and figure panels) and three new Supplementary files as per reviewer’s request.

Figure #

Main point

New datasets in revision

Companion Supplement

1

HSD alters feeding behavior, but flies still breakdown TAG on starvation.

TAG storage and breakdown over longitudinal HSD shows that HSD and NF fed flies show similar levels of TAG breakdown on starvation, despite consistently elevated TAG on HSD. This supports the idea that flies do sense starvation even on HSD, but there is a uncoupling of the feeding behavior after Day 14. Revised the data representation of Figure 1 to show non-normalized data over time. S1 and S2 companions are new in the revision. Panels 1D to 1E are new for the revision.

S1- Raw data of feeding events plotted.

S2 Elevated TAG at all time points.

2

HSD causes insulin resistance

S3A added to show that insulin transcript levels remain the same in response to reviewer #3’s concerns.

S3

3

Phospholipid concentration raw data from lipidomic on Day 7 and Day 14 HSD suggest that PC, PE levels are increased on Day 14 HSD.

Figure 3 revamped to show new data visualization and non-normalized raw data to address Reviewer #2’s major concerns. S4A and S4B added. In addition Supplementary File 1 and 2 provided with raw lipidomics data as per reviewer #2’s request.

S4.

S4A- non normalized raw data of all other lipid classes on HSD.

S4B- fatty acid species data on Day 14 added as per request of rev.#2.

4

HSD regulate Apo-I levels in the IPCs and phenocopies Pect KD.

Added Figure 4A to show that HSD phenocopies Pect-KD in terms of delivery to brain

S5 showing the validation of the Apo-I antibody.

S6 validation of Pect KD and over-expression and Pect mRNA levels dysregulation on HSD.

5

Pect RNAi is insulin resistant

N/A

N/A

6

Pect knockdown shows significant increase in LPC and LPE, and a non-significant reduction in PC, PE levels. Specifically, the PE lipid class PE36.2 is downregulated.

Fig 6, S7, S9 are completely new based on reviewer #2 and #3 requests. In addition Supplementary File 3 provided with raw lipidomics data as per reviewer #2’s request

S7, S8, S9#.

S7- new Pect KD other classes

S8- new PE classes for day 14 and Pect associated classes.

S9- Pect OE lipidomics

7

Pisd and Pect activity in adipocytes are required for hunger-driven feeding behavior in normal diets

Pisd RNAi data was moved from supplement to main figure.

N/A

Note on revised text: We have revised text not only in the results section, but also as per reviewer #2’s recommendation, we have revamped our introduction and discussion as well. Since the manuscript has been significantly revised to include a main figure 6, fully altered Figure 1 and 3, multiple new supplemental figures, the changes in text are extensive. Hence, they are unmarked in the main text. Nonetheless, we hope that the reviewers will be able to evaluate these changes, as we have provided the specific locations in text and embed key figures in the point-point response below.

__Part B: __Point-Point responses to reviewer comments.

Reviewer #1 comments in Blue, author response in black.

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

In this manuscript, Kelly et al. show that the difference between the feeding behavior of fed and starved flies (hunger-driven feeding; HDF) is absent in animals fed a high-sugar diet (HSD) for two weeks or more. The disappearance of HDF with HSD coincides with changes in phospholipid profiles caused by HSD. Furthermore, RNAi-mediated downregulation of Pect in the fat body-a key enzyme in the PE biosynthesis pathway-phenocopies physiological effects of HSD. Moreover, downregulation or overexpression in the fat body abolishes or induces HDF, respectively, abolishes or induces HDF, respectively, independent of HSD treatment.

Overall, the manuscript is well-written and the phenotypes are clear. However, I have major concerns regarding the authors' interpretation of the data and their conclusion. Most importantly, while it is clear that the authors' high-sugar dietary treatment affects feeding behavior and physiology, I am not convinced that the changes can be considered "hunger-driven"-which is central to the main point of the manuscript. Therefore, it is my recommendation that the authors substantially revise the manuscript by either showing additional/re-analyzed data that rule out alternative hypotheses, or rewriting the manuscript keeping alternative interpretations in mind.

We are thankful to this reviewer for their thoughtful critique, and constructive and specific suggestions on how we can redress these concerns. We have taken on board the concerns of this reviewer regarding our interpretation of whether the changes in feeding behavior can be considered hunger-driven or not. Based on their advice, we have made significant changes by addressing: i) does HSD increased baseline feeding- we now show non-normalized raw data and data supports conclusion that baseline feeding is not higher; ii) whether HSD- fed flies can sense an energy deficit at levels similar to NF fed flies- we show that HSD flies sense energy deficit. We have provided detailed response below, and we hope the reviewer finds the additional datasets and re-analyzed data are consistent with the interpretation that prolonged HSD dampens starvation induced feeding. In addition to this key concern this reviewer has made a many other salient points that we have addressed with additional data or by clarifying the text.

Major comments: 1) The data do not sufficiently show that the long-term HSD regime disrupts "hunger-sensing." The manuscript should address alternative hypotheses by showing raw instead of normalized data, rewriting the manuscript with a new central conclusion, or running additional experiments that actually show a defect in hunger-driven response. a. The main results that the authors rely on for the argument is that the ratio of feeding events that the starved and non-starved flies eat is different between the groups fed normal or HSD. However, because the authors only show normalized data (normalized to non-starved flies; Fig. 1), it is difficult to tell whether the change is due to a chronically increased feeding in non-starved HSD flies-maybe in perpetual hunger-like allostasis-or dampened starvation response. Indeed, the data shown in Fig S1 show that flies fed HSD for as short as 5 days show more frequent feeding events compared to age-matched controls fed normal food. It is possible that because the HSD-fed flies eat more than NF-fed flies, even without being starved, the ratio of starved/non-starved feeding is lower in the HSD-fed group-due to changes in the denominator, rather than the numerator.

We have taken onboard this concern regarding presenting only normalized data, and that clouded the interpretation and left open other possibilities. In the completely revised figure 1 and S1. We now show non-normalized data, as a function of time. First we note that HSD-fed flies, do not show higher baseline feeding that NF fed flies, except on Day 10 of HSD, when there is a modest but significant elevation (Figure 1D).

Nonetheless, on Day 10 HSD, flies still display increased hunger-driven feeding HDF (Figure 1C), it is only after Day 14 HSD that HSD dampens the starvation induced feeding.

1. It is also possible that the HSD-fed flies are simply not in as big an energy deficit physiologically, due to the increased fat deposits they've accumulated (as the authors show later in the manuscript). It may take longer for the fat HSD flies to reach substantial energy deficiency than the NF flies, but they still may eventually be able to appropriately respond to hunger, just like NF flies. In such case, it would be a misnomer to call this behavioral change a 'defect in hunger-driven feeding behavior.' Maybe an experiment with a dose-response curve of "hunger driven feeding response" as a function of duration of starvation would help? Prompted by this reviewers question, we asked whether HSD fed flies, that have a higher baseline neutral fat store (Triacylglycerol-TAG) level, and if HSD-fed flies can sense energy deficit. For this, we revisited the longitudinal assays for neutral fat triacylglycerol (TAG) storage that our lab had generated, along with the HSD-HDF studies. We now present this evidence as Figure 1E-1G and Figure S2. Overall, our experiments point to the idea that adult flies fed HSD, are able to sense and mobilize TAG stores effectively throughout the 28-day time point that we analysed.

First as shown in Figure S2, flies fed HSD display an increase in TAG levels. But it is to be noted that while TAG stores increase, the increase is not linear with time. This suggests that adult flies exposed to HSD store excess energy as TAG, but the increased TAG stores stay within a certain range despite the length of HSD exposure. This suggests that adult flies on HSD still display TAG homeostasis.

Next, to directly address the reviewers point about HSD fed flies not sensing an energy deficit, we subject HSD-fed flies to an overnight starvation, same regime as used in the overnight feeding experiments, and asked whether they mobilize TAG. We noted that flies exposed to HSD breakdown TAG throughout the 28-day exposure at statistically significant levels for Day 3- Day 28, except on 14 and 21 days (Figure 1F). While there is TAG mobilization on Day 14 and 21, the difference is not statistically significant. Nonetheless, we note the same levels TAG breakdown for normal lab food (NF) fed flies on Day 14 and 21 (Figure 1E). Overall, HSD fed flies sense and display energy deficit, as measured by TAG store mobilization, throughout the 28 days of HSD exposure, at levels comparable to NF-fed flies (Figure 1G).

Taken together, these results suggest that while HSD-fed flies experience an energy deficit on starvation, at levels comparable to NF-fed flies, throughout the 28-day time point assayed. But, their starvation driven feeding-response is dampened by Day 14 and by Day 28, the HSD-fed flies display more feeding events than HSD starved flies. These results are consistent with the interpretation that in HSD-fed flies the starvation-induced feeding behavior becomes desynchronized from the starvation induced TAG-mobilization, suggesting that there is an absence of hunger-driven feeding.

2) How can you be sure that lower Dilp5 immunofluorescence is indicative of increased Dilp5 secretion? Wouldn't decreased production of dilp5 also have the same results?

It has been shown previously in HSD fed larvae are hyperinsulinemic, i.e., they have 55% increase in circulating Dilp2 ( PMID: 22567167). Additionally, we have shown that ectopic activation of the insulin-producing neurons by expressing TRPA1, an ion channel that activates neurons, reduces Dilp5 accumulation without a change in Dilp5 mRNA levels (PMID: 32976758), suggesting that reduced Dilp5 accumulation, without alterations to mRNA levels is a proxy for increased secretion. Now, in response to this concern, in the revised manuscript, we have added qPCR data of Dilp2 and 5 (Figure S3A), which show no difference in expression levels after 14 days on HSD. Therefore, there is no dip in Dilp5 mRNA production. Given that Dilp2 and Dilp5 mRNA levels remain the same, but we see reduced Dilp5 accumulation, we interpret this to mean that Dilp5 secretion is increased.

1. Also, the authors should state in the main text that it is Dilp5, not just any Dilp. Thanks for this suggestion and we have fixed this and referred to Dilp5 specifically throughout the text in the results section.

3) Data presentation: a. Sometimes the data are normalized to NF (Fig 4B-C), sometimes not (ex. Fig 4A, S4C). Unless there is a specific rationale for the data transformation, it would be more appropriate to show untransformed data (ex. Fig 4A, S4C), especially as the authors use two-way ANOVA to determine significance. Only showing the differences implies comparison against a hypothetical mean (i.e. μ0=0), not between two group means.

We thank the reviewers for bringing this issue to our attention. We updated all the figures to show untransformed data in the revised manuscript.

1. Some figures show both individual data points and summary statistics (mean, SD, ... ex. Fig 2A)-which I believe is ideal-but some show only one or the other (ex. Fig 2B, no summary statistics; Fig. 3, no data points. The manuscript would read more convincing if data visualization is consistent across figures. We thank the reviewers for their feedback. We have made changes to all the figures in the revised manuscript to improve visual consistency.

Minor comments: 1) High sugar diet: what is the actual sugar concentration in the NF v. HSD diets? The authors write that the HSD diet contains "30% more sugar" than the NF, but providing the final sugar concentrations-sucrose or others-would be informative for other scientists studying the effect of high sugar diets.

We thank the reviewer for their suggestion and now we have updated the methods to include this sentence. “After 7 days, flies were either maintained on normal diet or moved to a high sugar diet (HSD), composed of the same composition as normal diet but with an additional 300g of sucrose per liter”.

1. Additionally, the definition of HSD is inconsistent. Main text (Page 5, line 17) states that their HSD is "60% more sugar than normal media," whereas the figure legend (Fig 1) and the Methods state that the HSD contains "30% more sugar." We apologize for this egregious typo in the figure legend! We have now fixed this to say 30% HSD. Only 30% HSD was used throughout this study.

2) Starvation medium: please provide justification for why the authors used 1% sucrose/agar for starvation medium, instead of plain agar/water that most labs use. At least clarify and provide a reference for the claim that the 1% sucrose/agar "is a minimal food media to elicit a starvation response."

We are very grateful for this reviewer identifying this this methods description error and bring it to our attention. We used 0% sucrose agar for overnight starvation in this study as most labs do. The error occurred because we were using another manuscript from the lab to help draft the methods section (PMID: 29017032). In that study, where we assayed the effect of chronic starvation our lab used: “1% sucrose agar for 5 days at 25C”. However, in this current study, because we are testing acute effects of overnight starvation, we are using 0% sucrose agar.

3) Pect mRNA level is higher with HSD. This is surprising because not only, as authors mention, is increased PC32.2 with HSD suggests lower Pect activity, but also because Pect RNAi phenocopies long-term HSD in HDF behavior, lipid morphology, FOXO accumulation in fat body. The authors speculate that the data "likely shown an upregulation in an attempt to mediate the Pect dysregulation occurring at the protein level." If that were true, a western blot may be informative. Zhao and Wang (2020, PLoS Genetics) generated a Pect antibody that seems compatible with western blot applications. That being said, I don't think such data is critical for the manuscript. I mention this simply as a suggestion for the authors. a. page 8, line 22-23, did you mean to write "Given how PC32.2 is elevated after 14 days of exposure to HSD, we assumed that Pect levels would be low for flies under HSD," not "high?" Otherwise the subsequent 2 sentences don't make sense.

We agree that the most confusing aspect of the study was that Pect mRNA levels being very high on Day 14 HSD, but nonetheless the effects of Pect-KD phenocopied HSD. To resolve this, we have now performed lipidomic analyses on whole adult flies, when Pect is knocked-down (KD) by RNAi in the fat tissue. We now present a new dataset in Figure 6. Two striking changes occur. They are:

1. Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3).
2. Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding increase in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). In contrast, PE 36.2 trends upwards on 14 day HSD (Figure S7C) though not significantly. On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

We agree that a western blot would be informative as well, but we were unable to obtain the reagent from Dr. Wang’s group, precluding us from performing this request. See email snapshot.

To ensure that we appropriately discuss and clarify this issue, we have now included a section in the discussion - Page 14 Lines 26-34- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

“Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9) , but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

Reviewer #1 (Significance (Required)):

The work is potentially novel and interesting, but at this stage it's difficult to interpret what the phenotype signifies. Although the manuscript could be revised simply by modifying the text, experimentally addressing the concerns would significantly improve the work.

In sum, we hope we have addressed the key concern for Reviewer #1 as to whether the behavior we report here is indeed a dampening of starvation-induced feeding, or an effect of increase in baseline feeding. We hope that by reviewing our non-normalized data, they can appreciate that it is the former. Also, we hope that Reviewer #1 appreciates that we have strived to address the concerns by additional experiments, to clarify our findings and improve the impact of the work.

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

This intriguing manuscript by Kelly and colleagues uses the fruit fly Drosophila melanogaster as a model to understand how diet-induced obesity alters the feeding response over time. In particular, the authors findings indicate that chronic exposure to a high-sugar diet significantly alters the starvation-induced feeding response. These behavioral studies are complemented by a lipidomics approach that reveals how a chronic high sugar affects many lipid species, including phospholipids. The authors then pursue mechanistic studies that indicate phospholipid metabolism within the fat body appears to remotely affect insulin secretion from the insulin producing cells. Moreover, the changes in phospholipid abundance are associated with changes in insulin-signaling, including increased insulin secretion from the IPCs and elevated levels of FOXO within the nucleus.

I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The conclusions are mostly convincing, but a few follow-up experiments are required:

We are grateful for the reviewers constructive, detail-oriented, and balanced feedback, and their recognition of the value of this study. Now, we have performed additional experiments to address the key concerns raised by all reviewers. We hope that on reading the revised version of our study, that the reviewer continues to feel positive about the message of this study and its potential impact.

1. The key conclusions from the manuscript assume that manipulation of Pect expression levels alters phosphatidylethanolamine (PE) levels. However, the authors make no attempt to verify that the genetic experiments described herein actually affect PE levels. At a minimum, changes in PE levels should be verified for the Pect knockdown and overexpression lines. Similarly, there is no evidence that manipulation of either EAS or Pcyt2 induces the expected metabolic effects. I'm not asking that the longitudinal feeding experiments be repeated, simply that the authors measure the relevant lipid species, preferably with a targeted LC-MS approach.

Prompted by this reviewer, we performed targeted LC-MS on whole adult flies, on normal diet, to assess lipid levels for fat-specific Pect-KD and overexpression. We decided to focus on Pect, as its knock-down even on normal diet causes a dampened hunger-driven feeding behavior (Figure 7A) and phenocopied a 14-day HSD feeding phenotype.

We now present a new dataset in Figure 6. Two striking changes occur:

They are:

Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding decrease in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). It is to be noted that though overall levels of all PE species trend downwards, like the Clandinin lab study on Pect (PMID: 30737130), we did not find a significant change in the overall PC and PE levels.

• Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3). On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

Finally, fat-specific Pect-OE did not cause significant changes to lipid species (Figure S9). This could either be due to the fact that in fat-specific Pect-OE flies under normal food and that we were assaying whole body lipid levels and not fat-specific lipid changes. But to counter that, even a 60% reduction in Pect mRNA levels (Figure S6A), was sufficient to produce an effect on whole body phospholipid balance (Figure 6). Hence, we speculate that by maintaining a basally higher (7-fold higher Pect mRNA level Figure S6A), might allow 14-day HSD-fed flies to buffer the negative effects of HSD and we predict that it might take longer to disrupt the phospholipid balance and HDF response.

We have now included a section in the discussion - Page 14 Lines 26-34- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

“Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9), but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

A central hypothesis in the study is that the HSD over a period of 14 days results in insulin resistant and that these changes are leading to changes in hunger dependent feeding. I would encourage the authors to determine if Foxo mutants are resistant to these HSD-induced effects on HFD.

We thank the reviewers for this suggestion. However, given that dFOXO nuclear localization rather than expression levels regulate insulin sensitivity, we feel that disrupting dFOXO levels via mutation or knockdown will produce a plethora of indirect effects including developmental abnormalities (PMID: 24778227, PMID: 16179433, PMID: 29180716, PMID: 12893776). Our data suggest that chronic HSD treatment and Pect affect insulin sensitivity in fat tissue. However, we feel that investigating whether insulin sensitivity/FOXO signaling in fat tissue regulates feeding behavior is outside the scope of our work.

1. In lines 25-30, the authors draw the conclusion that an increase in unsaturated fatty acid species is associated with the HSD and that these changes results in a more fluid lipid environment. While I agree with the model, the manuscript contains no evidence to support such a model. Either test the hypothesis or move the last line of the section to the discussion.

We thank the reviewer for this important and insightful comment. We agree that the data we presented and discussed in the original version is at the moment speculative. Addressing the hypothesis that increase in unsaturated fatty acid species result in a more fluid lipid environment will require us to build tools and expertise. Hence, this hypothesis is better suited for exploration in a future study. Given this, we have moved this out of the results section into the Discussion section titled “HSD and fat-specific PECT-KD causes changes to phospholipid profile” (See excerpt below from page 13, lines 24-35).

“In addition to changes in phospholipid classes, we found that HSD caused an increase in the concentration of PE and PC species with double bonds (Figure S4C and S4D). Double bonds create kinks in the lipid bilayer, leading to increased lipid membrane fluidity which impacts vesicle budding, endocytosis, and molecular transport14,92. Hence it is possible that a mechanism by which HSD induces changes to signaling is by altering the membrane biophysical properties, such as by increased fluidity, which would have a significant impact on numerous biological processes including synaptic firing and inter-organ vesicle transport.”

Also, as per the reviewer’s guidance, given that we are speculating here, we have also shifted this dataset from Main figure 4 to supplement S4C and S4D.

In addition, lines 25-30 state that FFAs are increased after 14 days of a HSD. Figure 3A shows the exact opposite - FFAs are significantly decreased in 14 day fed animals despite being elevated in the 7 day fed animals. This is an interesting result that warrants discussion. Moreover, I would encourage to examine the lipidomic data more carefully to ensure that the text accurately portrays the lipid profiles.

We apologize for misstating that FFAs are decreased on 14-day HSD in the lines 25-30. It was an error and we have corrected this. We agree with the reviewer that the reduction of FFA on Day 14-HSD is an intriguing and unexpected observation that needs to be emphasized and further discussed. To this end, we have added figure S4B, wherein we have provided the difference in FFA concentration (by species) after days 7 and 14.

Furthermore, we have discussed what the potential meaning of reduced FFA at Day 14 implies in page 12, lines 19-27 of the Discussion section titled “HSD and fat-specific PECT-KD causes changes to phospholipid profile”. We have stated the following-

“We speculate that this reduction in FFA maybe due to their involvement in TAG biogenesis (PMID: 13843753). We were interested to see if the decrease in FFA correlated to a particular lipid species, as PE and PC are made from DAGs with specific fatty acid chains. However, further analysis of FFAs at the species level did not reveal any distinct patterns. The majority of FFA chains decreased in HSD, including 12.0, 16.0, 16.1, 18.0, 18.1, and 18.2 (Figure S4B). This data was more suggestive of a global decrease in FFA, likely being converted to TAG and DAG, rather than a specific fatty acid chain being depleted.”

The processed lipidomics data should also be included as supplementary data table so that they can be independently analyzed by the reader.

We thank the reviewer for this suggestion. As per the reviewers request, we have included the raw data as an attachment in our supplementary material (Supplementary Files 1-3.), so that interested readers can use the datasets generated in this study for future work and further analysis.

Beyond these experimental suggestions, the manuscript needs significant editing for clarity. While I won't provide a comprehensive list, the authors need to provide accurate descriptions and annotation of genotypes (including w[1118], which is written as W1118), typos, and formatting. I've listed a few examples below:

1. Page 3, Line 1 and 2: "...have been shown to impact feeding behavior and metabolism that leads to..." This is an awkward and grammatically incorrect sentence.
2. Page 3, Lines 7-32 is one very large paragraph but contains concepts that should be broken down over at least three paragraphs.
3. Page 3, Line 25: A description of the reaction catalyzed by Pect would be helpful for a manuscript focused on Pecte activity.
4. Page 4, Line 10: "previously characterized method of eliciting diet induced feeding behavior." As stated in the text, the method is previously described yet the manuscript characterizing the method isn't cited.
5. Figure legend 3 contains a random assortment of capitalized lipid species. Also, the names of lipid species are inappropriately broken into multiple names. Please use correct nomenclature throughout the manuscript.

The list above is nowhere near comprehensive. The manuscript requires significant editing.

We are grateful to the reviewer for drawing our attention to these errors. We have made significant edits to the revised manuscript to address the above-mentioned concerns, as well as made additional textual changes throughout and copyedited it. We hope that the reviewer will find the manuscript reads better and the clarity and preciseness is significantly improved.

Reviewer #2 (Significance (Required)):

I find the study to be potentially very important - the authors combine a longitudinal study that would be difficult in any other model with the powerful genetic tools available in the fly. The findings will significantly advance our understanding of how lipid metabolism links dietary nutrition with feeding behavior.

Once again, we are grateful for this reviewer’s thoughtful critique and encouraging words regarding our work and its potential impact.

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

Summary: This manuscript uses Drosophila to investigate how diet-induced obesity and the changes in the lipid metabolism of the fat boy modulate hunger-driven feeding (HDF) response. The authors first demonstrate that chronic exposure (14 days) of high sugar diet (HSD) suppresses HDF response. Through lipidome analysis, the authors identify a specific class of lipids to be elevated upon chronic HSD feeding. This coincided with the changes in expression of Pect, an enzyme that regulates the biosynthesis of these lipids. Modulating the expression of Pect specifically in the fat body affected HDF response.

We thank this reviewer for their rigorous and thoughtful critique and for identifying a key issue with our original study pertaining to a gap in how Pect mRNA levels on 14-day HSD are elevated but the Pect-KD phenocopies the HDF. Now by performing whole-body adult fly lipidomic on fat-specific Pect-KD we have resolved this issue and provided clarity on role of Pect in maintaining phospholipid homeostasis and thus subsequently impacts hunger-driven feeding. We hope the reviewer finds that the revised manuscript provides further clarity to the functional link between Pect’s role in fat-body and hunger-driven feeding.

Major comments: The author claim that the HDF response in HSD is distinct between early (5d, 7d) and chronic (day 14) HSD feeding. However, the data seem to indicate that HDF response is significantly decreased at all time points in HSD. For example, at day 5 HDF response was increased only 3-fold in HSD (Figure 1C) compared to around 50-fold increase in NF (Figure 1B). The scale of the Y-axis in Figure 1B and 1C is an order of magnitude different. Including the starved data (NFstv and HSDstv) in Figure S1, normalized to NF fed group, would better visualize the overall trends. Related to this, having the source data for the actual number of feeding events would be useful (e.g., to see the baseline changes in feeding in different time points in Figure 1 and the effect of genetic manipulations in Figure 7).

As per the reviewers request, we now have modified our graphs to show source data (Figure S1) and show the raw feeding events.

Then in the non-normalized graphs we plot, over a longitudinal time course, baseline and hunger-driven feeding events (Figure 1B-D). We also show that HSD fed flies do not display increased baseline feeding (Figure 1D) suggesting that the effect we see on HDF are no clouded by increased baseline feeding.

Yes, the reviewer makes an important point that HDF response on HSD fed flies is of a lower magnitude than NF fed flies. We think that is a biologically meaningful observation, as it suggests that flies have a remarkably fine-tuned ability to coordinate food-intake with nutrient store levels.

­­Now we have included a paragraph in the Discussion, Page 11 Lines 23-27, that say the following to ensure the readers appreciate this salient point raised by this reviewer.

*It is to be noted that the HDF response of HSD-fed flies (Figure 1C, Days 3-10) is of lower order of magnitude than the NF-fed flies. This suggests that that in addition to sensing an energy deficit and mobilizing fat stores (Figure 1F, 1G, S1), HSD fed flies calibrate their starvation-induced feeding to compensate only for the lost amount of fat. Overall, this suggests that flies have a remarkably fine-tuned ability to coordinate food-intake with nutrient store levels. *

The association between fat body Pect level and phospholipid levels is not clear. Day 14 of HSD feeding shows high expression of Pect in the fat body and elevated levels of PC32.0 and PC32.2. The authors assume the high expression of Pect in the fat body is due to the compensatory response, but there are no data indicating downregulation of Pect levels at the earlier time points of HSD feeding. A previous study demonstrated that Pect mutant flies have lower levels of PC32.0 but higher PC32.2 (PMID: 30737130).

We agree that one puzzling aspect of the original version of this study was that Pect mRNA levels being very high on Day 14 HSD, but nonetheless the effects of Pect-KD phenocopied HSD. To resolve this, prompted by Reviewer #2 and #3 concerns, for this revised version we have now performed lipidomic analyses on whole adult flies, when Pect is knocked down (KD) by RNAi in the fat tissue. We now present a new dataset in Figure 6. Two striking changes occu. They are:

1. Pect-KD shows increase in the phospholipid classes LPC and LPE (Figure 6A). In contrast, LPE is significantly downregulated on HSD Day 14 (Figure 3).
2. Pect-KD shows a significant reduction in specific class of PE 36.2 (Figure 6B). Our data regarding increase in PE 36.2 agree with a previous lipidomic analyses of Pect mutant retina (PMID: 30737130). In contrast, PE 36.2 trends upwards on 14 day HSD (Figure S7C) though not significantly. On 14-day HSD consistent with extreme upregulation of Pect mRNA fed flies (Figure S6A; Pect mRNA 200-250 fold), PE trends upwards on 14-day HSD (Figure 3) and PE 36.2 trends higher (Figure S7C). We note that on the surface of it PE and LPE per se are contrasting between 14-day HSD lipidome and fat-specifc Pect-KD. But there is a significant commonality that under both states there is an imbalance of phospholipids classes PE and LPE. Hence, we propose that maintaining the compositional balance of phospholipid classes PE and LPE is critical to hunger-driven feeding and insulin sensitivity. Hence, either increase or decrease, of these key phospholipid species, may lead to abnormal hunger-driven feeding.

On day 14, HDF response was increased 70-fold in w1118 flies in NF (Figure 1B; w1118), but only 2.5-fold in lpp>LucRNAi control flies in NF (Figure 7A). This suggests that lpp-gal4 driver lines have a significant effect on HDF response. Using a different fat-body specific Gal4 line would be necessary to validate conclusions.

Regards reduced HDF magnitude, in our experience using UAS-Gal4 reduces HDF response magnitude consistently and cannot be compared to w1118 which is more robust. To account for background differences, we use Uas-Gal4 with control RNAi. It clearly shows differences in HDF response on starvation, but Pect and Pisd RNAi does not (Figure 7A). Hence, given that this experiment internally controls for any changes in HDF response for UAS-Gal4>RNAi, we conclude that HDF response in disrupted in Pect and PISD KD (Figure 7).

We only presented the Lpp-driver in our study, as this driver is the only fat-specific driver that has no leaky expression in other tissues, and is specific to fat as apolpp promoter used to generate this Gal4 line is only expressed in fat tissue (Eaton and colleagues, PMID: 22844248). Other widely used fat-specific drivers, including the pumpless-Gal4 (ppl-Gal4) driver has leaky expression in gut or other tissues (See Table 2 of this detailed study by Dr. Drummond- Barbosa https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642949/). If the reviewer is aware of a fat-specific Gal4 line, other than Lpp-Gal4, which has a highly specific expression in the fat tissue without leaky expression in other tissues, then we are happy to take onboard the reviewer’s suggestion and try that fat-specific Gal4 that they suggest.

HSD feeding promotes Pect expression (Figure S3C) and global changes in phospholipid levels (Figure 3, 4). Therefore, shouldn't Pect overexpression (not Pect RNAi) in a normal diet mimic HSD feeding state and promote loss of HDF response? Conversely shouldn't knockdown of Pect in HSD rescue loss of HDF response?

We agree that a puzzling aspect is that Pect mRNA levels are significantly elevated in HSD Day-14, but Pect-KD showed displays the inappropriate HDF response. As we have described in our response to this reviewer on Page 19, we believe that Pect-KD and HSD disrupt PE and LPE balance overall but in different ways. Whereas Pect-OE using cDNA expression in fat body does not cause a significant change to any lipid class (Figure S9), and our results suggest that basally higher level of PECT is likely to be protective on HSD with respect to HDF(Figure 7B).

To ensure that we appropriately discuss and clarify this issue, we have now included a section in the discussion - Page 14 Lines 26-33- under the subtitle “The implications of relationship between Pect levels and HSD”. We have pasted an excerpt from that subsection below for this reviewers assessment.

“Also, we note that over-expression of Pect cDNA in the fat-body does not alter phospholipid balance (Figure S9) and indeed improves HDF on HSD (Figure 7B). While this may appear inconsistent, it is critical to note that over-expression of Pect cDNA using UAS/Gal4 only increases Pect mRNA expression by 7-fold (Figure S6A), whereas HSD causes its upregulation by 250-fold (Figure S6B). Hence, we speculate that an increased ‘basal’ level of Pect such as by that provided by a cDNA over-expression in fat, may be protective to the negative effects of HSD (Figure 7B) without affecting overall phospholipid levels (Figure S9) , but extreme upregulation Pect on HSD affects the PE and LPE balance (Figure 3).”

We would have liked to test Pect protein expression on HSD, but since we were unable to access antibodies for Pect published in a prior study (PMID: 33064773) from Dr. Wang’s lab (see Page 10-11, of response to Reviewer #1). Hence, we were unable to test how the proteins levels of Pect correlate with the 250-fold increase mRNA expression.

In conclusion, we hope the reviewer appreciates that our results regarding Pect function are consistent with the main conclusion that achieving the right phospholipid balance between PE and LPE, is critical for an organism to display an appropriate HDF response.

Minor comments: All graphs should plot individual data points and showed as box and whisker plot as much as possible.

Thanks for this suggestion, we have added individual data points to the vast majority of figures in the paper. We have made exceptions to graphs such as seen in figure 1 and FigureS4B-D where we find individual data points add an unnecessary layer of complexity. We hope these changes provide additional clarity and strength to the claims made in this manuscript.

Data for day 14 missing in Figure S4A and S4B.

We have provided Day 14 for the PC composition and PE composition, due to changes in Figures, they are now S7A and S7B.

Reviewer #3 (Significance (Required)):

The interactions between diet-induced obesity, peripheral tissue homeostasis and feeding behavior is an interesting topic that can be addressed using Drosophila. This manuscript demonstrates how fat body Pect levels affect HSD induced changes in hunger-driven feeding response. However, at this point, the functional association between fat body Pect level, global phospholipid level, and loss of hunger-driven feeding response in chronic HSD feeding is not clear.

We hope the revised data, and discussion of the paper, provides well-substantiated functional association on the importance of maintaining phospholipid balance, driven by Pect enzyme, as a critical regulator of hunger-driven feeding behavior. As stated in the revised discussion, the key take home message of our manuscript is that on prolonged HSD exposure PC, PE and LPE levels are dysregulated, the loss of phospholipid homeostasis coincided with a loss of hunger-driven feeding. Following this lead on phospholipid imbalance, we then uncovered a critical requirement for the activity of the rate-limiting PE enzyme PECT within the fat tissue in controlling hunger-driven feeding.

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

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, Kelly et al. show that the difference between the feeding behavior of fed and starved flies (hunger-driven feeding; HDF) is absent in animals fed a high-sugar diet (HSD) for two weeks or more. The disappearance of HDF with HSD coincides with changes in phospholipid profiles caused by HSD. Furthermore, RNAi-mediated downregulation of PECT in the fat body-a key enzyme in the PE biosynthesis pathway-phenocopies physiological effects of HSD. Moreover, downregulation or overexpression in the fat body abolishes or induces HDF, respectively, abolishes or induces HDF, respectively, independent of HSD treatment.

Overall, the manuscript is well-written and the phenotypes are clear. However, I have major concerns regarding the authors' interpretation of the data and their conclusion. Most importantly, while it is clear that the authors' high-sugar dietary treatment affects feeding behavior and physiology, I am not convinced that the changes can be considered "hunger-driven"-which is central to the main point of the manuscript. Therefore, it is my recommendation that the authors substantially revise the manuscript by either showing additional/re-analyzed data that rule out alternative hypotheses, or rewriting the manuscript keeping alternative interpretations in mind.

Major comments:

1. The data do not sufficiently show that the long-term HSD regime disrupts "hunger-sensing." The manuscript should address alternative hypotheses by showing raw instead of normalized data, rewriting the manuscript with a new central conclusion, or running additional experiments that actually show a defect in hunger-driven response.
   • a. The main results that the authors rely on for the argument is that the ratio of feeding events that the starved and non-starved flies eat is different between the groups fed normal or HSD. However, because the authors only show normalized data (normalized to non-starved flies; Fig. 1), it is difficult to tell whether the change is due to a chronically increased feeding in non-starved HSD flies-maybe in perpetual hunger-like allostasis-or dampened starvation response. Indeed, the data shown in Fig S1 show that flies fed HSD for as short as 5 days show more frequent feeding events compared to age-matched controls fed normal food. It is possible that because the HSD-fed flies eat more than NF-fed flies, even without being starved, the ratio of starved/non-starved feeding is lower in the HSD-fed group-due to changes in the denominator, rather than the numerator.
   • b. It is also possible that the HSD-fed flies are simply not in as big an energy deficit physiologically, due to the increased fat deposits they've accumulated (as the authors show later in the manuscript). It may take longer for the fat HSD flies to reach substantial energy deficiency than the NF flies, but they still may eventually be able to appropriately respond to hunger, just like NF flies. In such case, it would be a misnomer to call this behavioral change a 'defect in hunger-driven feeding behavior.' Maybe an experiment with a dose-response curve of "hunger driven feeding response" as a function of duration of starvation would help?
2. How can you be sure that lower Dilp5 immunofluorescence is indicative of increased Dilp5 secretion? Wouldn't decreased production of dilp5 also have the same results?
   • a. Also, the authors should state in the main text that it is Dilp5, not just any Dilp.
3. Data presentation:
   • a. Sometimes the data are normalized to NF (Fig 4B-C), sometimes not (ex. Fig 4A, S4C). Unless there is a specific rationale for the data transformation, it would be more appropriate to show untransformed data (ex. Fig 4A, S4C), especially as the authors use two-way ANOVA to determine significance. Only showing the differences implies comparison against a hypothetical mean (i.e. μ0=0), not between two group means.
   • b. Some figures show both individual data points and summary statistics (mean, SD, ... ex. Fig 2A)-which I believe is ideal-but some show only one or the other (ex. Fig 2B, no summary statistics; Fig. 3, no data points. The manuscript would read more convincing if data visualization is consistent across figures.

Minor comments:

1. High sugar diet: what is the actual sugar concentration in the NF v. HSD diets? The authors write that the HSD diet contains "30% more sugar" than the NF, but providing the final sugar concentrations-sucrose or others-would be informative for other scientists studying the effect of high sugar diets.
   • a. Additionally, the definition of HSD is inconsistent. Main text (Page 5, line 17) states that their HSD is "60% more sugar than normal media," whereas the figure legend (Fig 1) and the Methods state that the HSD contains "30% more sugar."
2. Starvation medium: please provide justification for why the authors used 1% sucrose/agar for starvation medium, instead of plain agar/water that most labs use. At least clarify and provide a reference for the claim that the 1% sucrose/agar "is a minimal food media to elicit a starvation response."
3. PECT mRNA level is higher with HSD. This is surprising because not only, as authors mention, is increased PC32.2 with HSD suggests lower PECT activity, but also because PECT RNAi phenocopies long-term HSD in HDF behavior, lipid morphology, FOXO accumulation in fat body. The authors speculate that the data "likely shown an upregulation in an attempt to mediate the PECT dysregulation occurring at the protein level." If that were true, a western blot may be informative. Zhao and Wang (2020, PLoS Genetics) generated a PECT antibody that seems compatible with western blot applications. That being said, I don't think such data is critical for the manuscript. I mention this simply as a suggestion for the authors.
   • a. page 8, line 22-23, did you mean to write "Given how PC32.2 is elevated after 14 days of exposure to HSD, we assumed that PECT levels would be low for flies under HSD," not "high?" Otherwise the subsequent 2 sentences don't make sense.

Significance

The work is potentially novel and interesting, but at this stage it's difficult to interpret what the phenotype signifies. Although the manuscript could be revised simply by modifying the text, experimentally addressing the concerns would significantly improve the work.

The co-reviewer and I have expertise in Drosophila neurobiology and behavior.

Referees cross-commenting

Hi all, although the reviews hit upon some overlapping, but mostly different points, I agree with all of the concerns raised. There's some really interesting stuff here but some of the results, as presented, don't make sense. It's possible this will be clarified by revising the text, although I suspect it's more likely that the authors will have to add a number of the experimental suggestions made by the reviewers.

It's interesting to see how these gaps can form - and without background knowledge on the history of that period, the gap would have no context or meaning. It would just be a blip. But that gap has significant historical importance, particularly how information about the revolt was suppressed at the time.

Author response

• A comment on the overall organization of the paper. Figure 2 has a major location in the paper, but it seems that its main takeaway is that these MAPs aren't really involved in the main process this paper is probing. While these are important findings, it might be more satisfying to move some of the central results earlier.

We agree that this figure displays mostly negative results. However, most work on anaphase B microtubule dynamics from our group and others has focused on the effect that motors and MAPs may have on microtubule dynamics (EB1 and kinesin-8 in budding yeast, klp9 in fission yeast). Therefore, we consider it is important to clearly show that previously proposed candidates are not required for the observed decrease in microtubule growth speed, prior to introducing the unexpected effect of the membrane.

*A model schematic might drive home the main finding of the paper, and be particularly useful for readers who are not experts in microtubule or spindle dynamics. That said, the Discussion does an excellent job of summarizing the findings and explaining the takeaway message(s), even for the non-expert.

We have added a model schematic and we have referred to it in the main text.

Specific comments

• ‘In higher eukaryotes’ - Suggest avoiding the terms higher and lower when describing organisms, and instead, directly defining which organisms, for instance in animals/metazoans that would be a better description.

We have removed this terminology.

• Figure 1 E-F - It is hard to see the difference in the distribution, maybe a different color could be used instead of stars.

We have used a different color.

• Figure 1 Data shown in pink in G comes from 832 midzone length measurements during anaphase, from 60 cells in 10 independent experiments - The pink here does not correspond to the pink coding in D, consider colour choice for clarity across panels.

We have changed this.

• Finally, yeasts undergo closed mitosis - How does this relate to the findings in the Dey paper (cited here) which shows it was somewhat semi-closed or semi-open. According to the Dey paper, the membrane disassembles locally twice, at the SPB and the bridge.

Membrane disassembly at the nuclear membrane bridge occurs at late anaphase, and leads to the disassembly of the spindle, presumably by the action of cytoplasmic factors (Dey et al. 2020). We do not believe the membrane disassembly itself has a role in spindle elongation or microtubule dynamics, as when it happens the spindle is then disassembled. However, the fact that les1D reduces the decrease in microtubule growth speed associated with internalisation of microtubules in the nuclear membrane bridge suggest that the organisation of the nuclear membrane bridge required for its local disassembly at late anaphase might affect microtubule growth (see section “Formation of Les1 stalks […]”).

• ‘vertical comets in kymographs (Fig. 1C) do not correspond to non-growing microtubules, but rather microtubules that grow at a speed matching the sliding speed’- For clarity, it might be nice to add: "(as the SPB moves away from the plus end in the kymograph)".

We have included this useful clarification.

• ‘significantly shorter than in interphase, where growth events last more than 120 seconds on average [42, 43]. Microtubule shrinking speed did not change during anaphase either (Fig. 1-Supplement 1D), and was on average 3.56±1.75 μm/min, also lower than in interphase (~8 min/μm)’ - This comment concerns the comparison of growth and shrinking rate as well as growth duration. The authors did not measure microtubule dynamics in interphase in this manuscript but compared their numbers to literature values. The comparison raises some questions for three reasons: 1) the microscopy method used is different in this paper and the two references provided, 2) the sample is mounted differently compared to the two references provided - 1) and 2) combined could lead to different levels of stress on the cells which could affect MT dynamics-, 3) (probably the most important caveat) the experiments are done at different temperatures: 27C in this paper versus 25C in the references provided. Microtubule dynamics are sensitive to temperature so this could explain part of the differences observed. Also, there are multiple values published for MT dynamics in interphase depending on the strain used and the microscopy method used. Suggest that the authors measure microtubule dynamics in interphase cells at 27C in SIM to ensure that the differences are not due to the technical parameters employed. Small item - should ‘8 min/μm’ read “8 μm/min"?

We have measured microtubule growth speed and growth event duration using GFP-Mal3 during interphase and anaphase B in the same conditions as proposed (see Figure 1 – Supplement 2). Unfortunately, shrinkage speed cannot be measured using GFP-Mal3, so we cannot confirm that the difference between our measurements and the literature values would be observed.

• ‘we observed two populations of microtubules (fast and slow growing)’ - Does this statement about thistle fast and slow growing populations refer to the data in Fig. 1C and 2A?

Yes, we have added reference to this figures in the next sentence (mentioned below).

• ‘In some cells, all microtubules seemed to switch to the slow growing phase simultaneously (Fig. 1C), while in others fast and slow growing microtubules co-existed (Fig. 2A)’ - This is a very interesting observation, could we know how many cells (%) were detected in each case? Is it that in 90% of the cells the switch is simultaneous, and hence the microtubule growth is somehow synchronized? Or is it more random, e.g. around 50%?

This was just to point the reader to two kymographs and show that a clear point where all microtubules change speed is not present in all kymographs, as one may think from Fig. 1C. Later in the paper, we show that the change in growth depends on whether the microtubule rescue occurs inside or outside the nuclear membrane bridge, so it is a matter of where microtubules are rescued once the dumbbell transition occurs, which is a stochastic process. We have added another sentence pointing the reader to examples in the kymograph (see line 152, This representation captures…).

• ‘On such a plot, the data points visibly cluster in two separate clouds and the variation of growth speeds can be fitted by an error function (Fig. 1F)’ - It is unclear that there are two distinct clusters, maybe the assertion should be toned down, or some sort of cluster analysis provided.

We acknowledge that the data is widely spread across the y axis, and given that the magnitude “distance to the closest pole at rescue” is continuous the transition is not a clear cut. However, we consider the fact that the averaged curve closely matches the error function fit to be sufficient evidence for the existence of two populations of microtubule growth. Additionally, R2 of the fit is ~0.5 indicating that half of the variance is explained by this model. In any case, we show later that these two populations do exist (Fig. 3D), and why plotting microtubule growth against distance to the closest pole at rescue is a good way to segregate them (Fig. 3E).

• ‘speed of interphase microtubules (~2.3 μm/min)’ - It would be interesting to see the dynamics in a les1 mutant (Dey Nature 2020) paper. Just as a control for presence/absence of the bridge?

We thank the reviewers for kindly suggesting this interesting experiment. We have included it after the ase1 section. Les1 forms stalks at the edges of the nuclear membrane bridge that restrict nuclear membrane disassembly to the center of the bridge at the end of mitosis (Dey at al. 2020). While les1 deletion does not prevent the formation of the nuclear membrane bridge, it has been proposed that Les1 stalks may constitute sites of close interaction between the nuclear membrane and the spindle. Therefore, these sites may influence microtuble growth. Indeed, we have found that removing these Les1 stalks by either deleting les1 or nem1 leads to a smaller decrease in microtubule growth speed when plus ends enter the nuclear membrane bridge (see section “Formation of Les1 stalks […]”)

*‘Figure 2, Transition from fast to slow microtubule growth occurs in the absence of known anaphase MAPs’ - It looks like the overlap zone is larger on the mal3 kymograph. Is the size of the midzone changed in some of the mutants? It could be important to report. Related to it, is the spindle length changed in some of the mutants? (It does not look like it from the kymographs displayed).

The midzone is indeed longer in mal3D strains, now this can be seen in Fig. 2 – Supp. 2 and it is mentioned in the main text in line 272. As for the spindle length, diverse kinds of alterations in spindle length have been previously reported for the mutants that we used in this study. For instance, ase1D /cls1off cells have shorter spindles at anaphase onset (Loiodice et al. 2005 and data not shown), and klp5Dklp6D have longer spindles at anaphase onset (Syrivatkina et al. 2013). klp9D / clp1D / dis1D cells have lower spindle elongation velocity and may not reach the wild-type spindle length by the end of anaphase (Kruger et al. 2019). Despite these differences, the decrease in microtubule growth as a function of distance to the closest pole has a similar tendency across conditions, suggesting that the mentioned differences in spindle length are unlikely to have an important effect.

• Additionally, adding the data about rescue localization in the mutant (equivalent of Fig 1 G) would be interesting to better describe the role of these different proteins. Figure 2, Panel G to L - Could the authors indicate the value for the average +/- error in each bin for the WT and the mutants? Also, it is hard to say from the plots, but it looks like the WT average speed in the first bin is different in every panel, that would be good to know to have an idea of the reproducibility/variability.

We have added a figure with the rescue distribution (see Fig. 2 – Supp. 2). This apparent difference in the wt speed in different experiments might have come from looking at normalised data. The new way of representing the data in fig. 2H and J shows that the microtubule growth velocity in the wild-type is very consistent across experiments. We have added a table with microtubule growth velocity values (Table 1), and the source data is available.

• The dots making up the "thick lines" are centered on 1.5/2.5/etc.. in some panels (G and K) and centered on 1/2/3/etc.. the others (I,J,L). Could the authors provide some clarification?

We have fixed this inconsistency across the paper.

• Figure 3 - Can the authors indicate the average values +/- error for each of the distributions in Fig. 3D? Maybe on the plot itself, in the legend or as a table. This would make them easily available without having to infer them from the Y axis. This comment is also valid for Fig 4I and 4J.

We have added tables with average values and confidence intervals in the appendix.

• Figure 3E ‘Distance from the plus-end to the nuclear membrane bridge edge at rescue as a function of distance from the plus-end to the closest pole at rescue’ - The Y axis reads as "distance to the bridge edge" but it shows negative values, could this be "position to the bridge edge" instead? (same item throughout the text).

We have fixed this.

• Figure 3 ‘Number of events: 442 (30 cells) wt, 260 (27 cells) klp9OE, 401 (35 cells) cdc25-22, from 3 independent experiments’ - P values this small raise a concern. Presumably the number of degrees of freedom in the regression analysis should not exceed the number of independent experiments. Instead, the DoF listed under "error" in the analysis output is hundreds or thousands instead of 3. To address this, the regression analysis should use either the "Error" function in R or a linear mixed-effects model to account for the nesting of the repeated measurements within each independent experiment. Alternatively, it is also possible to just calculate summary means for each independent experiment, and calculate p values based on that N=3. See: Lazic. Experimental Design for Laboratory Biologists. p. 157. and the supplemental file of: https://doi.org/10.1371/journal.pbio.2005282 and the additional file 1 of: https://doi.org/10.1186/s12868-015-0228-5 and this for an alternative plotting approach: https://doi.org/10.1083/jcb.202001064 Recommend either recalculating the p values by one of the methods above or removing the reported p values from the paper. The large effects observed in many cases are self-evident without a significance metric, so eliminating the p values would be acceptable here. (This comment applies to other figures through the paper that report p values based on number of cells or number of measurements instead of number of independent samples/experiments.)

We thank the reviewers for suggesting the improvements to the statistical analysis, as well as for pointing us to useful resources that described the statistical methods and their implementation in detail. We have followed Aarts et al. 2015 and used a linear mixed effects model (see Methods>Statistical Analysis)

Due to the change in statistical analysis method, to show that some of the differences we had reported previously were significant, we included more cells in the analysis from our existing data. We did this for klp5Dklp6D kymographs (Fig. 2I and Fig.2 – Supp. 1). Spindle dynamics in ase1D (Fig. 5D and Fig. 5 – Supp. 1) and klp9D (Fig. 2 – Supp. 3 A, C). Cell length (Fig. 3 – Supp. 1A).

For the same reason, we measured anaphase spindle elongation velocity (Fig. 3 – Supp. 1C) from kymographs instead of measuring them from the 1 minute interval movies that we had used previously (from Fig. 3 – Supp 1B). We have reflected this in the methods (see added text in line 800 and deleted text in line 809 in the document with changes highlighted).

None of these changes has altered our conclusions.

• Figure 4 - Nice experiment. It brings the question of how cell-shape affects all these dynamics (probably out of the scope of this work). But a for3 mutant for example?

This is an interesting suggestion, to be tested in the future. Furthermore, we believe that nuclear shape should also have an important effect, since the spindle is confined inside the nuclear membrane. We would expect that mutants that perturb nuclear shape might have effects on microtubule growth. We have observed that the decrease in growth speed associated with internalisation of microtubules in the nuclear membrane bridge is reduced upon nem1 deletion, which increases nuclear membrane surface, and produces membrane ruffling (Fig. 4-Supplement 2). However, nem1 deletion also removes les1 stalks from the nuclear bridge (Dey et al. 2020). It would be interesting to find a perturbation of the nuclear membrane that does not remove the les1 stalks.

• ‘Ase1 is required for microtubule growth speed to decrease during anaphase B, this is unlikely to be a direct effect’ - If it is unlikely to be a direct Ase1 effect is the title of the section accurate? "Ase1 is required for normal rescue distribution and for microtubule growth speed to decrease in anaphase B"

Ase1 recruits multiple proteins to the spindle midzone, so the fact that ase1 deletion produces a given phenotype does not necessarily mean that this phenotype results from the absence of Ase1 protein activity. For instance, deleting ase1 perturbs rescue distribution, but it does not mean that Ase1 acts as a rescue factor itself, or at least to a relevant extent, given that deletion of cls1 completely prevents rescue, but ase1 deletion does not. In the discussion we propose some indirect effects of ase1 deletion that may produce this effect. In any case, upon more careful analysis we have found that ase1 deletion does not prevent the decrease in microtubule growth speed during anaphase B, but rather makes it smaller (see section “The decrease in growth speed associated with internalisation of microtubules in the nuclear membrane bridge is reduced upon ase1 deletion”).

• Figure 5 - What about an ase1 lem1 double mutant?

We suppose that the intended gene is les1. We have studied the effects of les1 deletion in the new version of the manuscript. However, we do not see the information we would obtain from a double deletion ase1D les1D.

• ‘In summary, Ase1 is required for rescue organisation and for microtubule growth speed to decrease during anaphase B ‘- In this context it could make sense to discuss the observations from this paper (doi:10.1371/journal.pone.0056808) about the role of Ase1 ortholog's MAP65-1 in coordinating MT dynamics within bundles.

In the mentioned paper, the authors showed that the presence of PRC1 (ase1 orthologue) in bundles increases microtubule rescue rate, and that it slightly reduces microtubule growth speed.

We observe a small increase in microtubule growth speed throughout anaphase upon ase1 deletion (Fig. 5), which is consistent with the in vitro observation that PRC1 decreases microtubule growth. However, once more this might not be a direct effect of Ase1, since less Cls1 is recruited if ase1 is deleted, and Cls1 reduces microtubule growth speed (Fig. 2). In addition, this can also be a result of higher concentration of tubulin / MAPs resulting from less polymerised tubulin in ase1 deleted cells, which have less spindle microtubules on average.

Regarding the increase in rescue rate produced by PRC1 in vitro, it is possible that Ase1 contributes to microtubule rescue in the spindle. However, given that no rescues occur upon inactivation of cls1 (Bratman et al. 2007), we believe Cls1 is the dominant factor, and Ase1 contribution is likely negligible.

• ‘We initially set the microtubule growth velocity to 1.6 μm/min (early anaphase speed, Fig. 1F), and aimed to reproduce the experimental distribution of positions of rescue and catastrophe at early anaphase (spindle length < 6 μm’ - Kudos to the authors for detailing the model and its parameters in a way that even non-modelling experts can understand.

Discussion - ‘Our data suggests that microtubule growth speed is mainly governed by spatial cues’ - Is it right to assume that in the cases where fast and slow growing microtubules were simultaneously observed, the fast microtubules were not/had not yet reached the midzone?

Our data suggests that it’s not about being inside the midzone, but rather inside the nuclear membrane bridge formed after the dumbbell transition. We have elaborated more on this in the main text, pointing the reader to examples in the kymograph, and giving a quantitative argument for distance to the closest pole being a better predictor than anaphase progression or position with respect to the center (which is equivalent to distance to the midzone), see line 152.

• Methods - ‘PIFOC module (perfect image focus), and sCMOS camera’ - Is this Nikon's "Perfect Focus" autofocus, or some other manufacturer's system? And back-thinned sCMOS.

We have clarified this in the Methods section.

this is losing heat, since the heat in reverse and does not go back to it's initial state that would mean it would be the same going forward and that would mean the system will have more entropy to give to the surrounding. so we add the minus here to indicate that this is just going to lose heat.

I removed a paragraph in which I talked about an Investment Club social and networking event because it didn't connect to my focus on the false narratives I pressure myself to fufill. In this paragraph, I added more concrete and specific details to illustrate another one of my false narratives, living in New York City. I wanted to include this false narrative so that the reader could understand how much I want to live in New York and work at a bulge bracket bank because it would be such a great professional experience. But I also wanted the reader to see the pressure I was placing on myself by comparing my professional situation to Annabel because she will be living out what I imagine for myself.

While I made progress in my revisions by adding specific details, I definitely could have expanded on what "my picture perfect life" is and how at surface level Annabel's living and working situation seems "picture perfect" to me because she is going to be living in New York and working for a great firm which is what I see for myself in my daydreams. In looking back at the revisions I made and taking into account more feedback, I still have more to unpack with that statement.

I don't know exactly what to take from this but I'm dead sure it's really important to understand, even for us internet nobodies.

It's amazing how easily they can switch the subject. It could just be me, but I feel like there should be more focus on the fact that Miss Julie likes to train people. The fact that Jean doesn't draw a line in the sand and consider that maybe Miss Julie may not make for the best partner after this is astounding. Forget not having money, she's just plain weird.

I really felt this statement right here because I also went through the same thoughts when I was in high school. I always considered myself as being very shy and introverted but now that I am in college I reflect back and note that that was not the case at all. I was just not that comfortable expressing my true wild side due to the people in my classrooms.

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Reply to the reviewers

1. General Statements

We thank the reviewers for their careful and constructive analysis of our work. Our manuscript aims to exemplify the use of cryo-soft-X-ray tomography (cryoSXT) as a technique to study the dynamic changes to host-cell morphology that accompanies virus infection. This emerging method has several strengths when compared to other ultrastructural analysis techniques. Specifically, cryoSXT does not require the addition of contrast agents and therefore samples can be prepared via plunge cryopreservation alone, allowing us to capture them in a near-native state. Furthermore, the penetrating power of soft X rays and large field of view in cryoSXT allow rapid data acquisition, facilitating quantitative analysis of 10s to 100s of individual cells. We combined high-throughput cryoSXT data collection with semi-automated tomogram segmentation and fluorescence cryo-microscopy to study a recombinant herpes simplex virus (HSV)-1 that produces a pattern of fluorescence indicative of the stage of the infection in a single cell (‘timestamp’ HSV-1) and quantitatively monitored changes in lipid droplet, vesicle and mitochondrial morphology as HSV-1 infection progresses. In response to the reviewers’ comments, we have expanded our analysis of lipid droplet morphology, identifying a transient increase in the size of lipid droplets at early stages of HSV-1 infection, and completed additional fluorescence microscopy analysis to support our statements about the changes to microtubule, mitochondrial and Golgi morphology that accompany infection. Furthermore, we have included additional discussion on the relative merits of cryoSXT versus other ultrastructural analysis techniques like transmission electron microscopy, electron cryo-microscopy and electron cryotomography. We believe that our study serves as a powerful example of how cryoSXT can be used for quantitative cell biology and will be of broad interest to an audience of cell biologists and colleagues who study infection processes.

1. Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity):

Summary

The authors have performed an explorative study, investigating morphological changes that occur in cells upon infection with Herpes Simplex Virus 1 (HSV-1) by the use of cryo soft X-ray tomography (cryoSXT). cryoSXT is an emerging technique for imaging of biological material, that allows for 3D imaging of significant volumes of cells under near-native conditions, without the need for sectioning or sample preparation other than rapid freezing. Reference (Groen et al. 2019) provides a nice list of examples from various biological samples. By the use of cryoSXT, the authors confirm findings that they have previously published by use of light and expansion microscopy (ref 16 from manuscript), namely an enrichment of small vesicles close to the nucleus and elongation and branching of mitochondria into interconnected networks in infected cells.

Infection experiments were done in two different cell types in this study (HFF and U2OS), and a timestamp reporter virus that allows to distinguish between early and late stages of infection was used to provide more context to the observed morphological changes in the cells.

Major comments

It is a bit difficult to follow the main message throughout the manuscript, as the topics brought up in the introduction, results and discussion sections are not very coherent. The introduction gives some background on the virus and the timestamp reporter system, and further focuses on cryoSXT as a method and how this can overcome sample preparation artefacts that might be introduced by chemical fixation and sample processing. The results do not contain any direct comparisons between cryoSXT and other methods or sample preparations (light microscopy or EM-based), and the discussion only to a small extent comes back to the advantages brought by cryoSXT compared to other methods. Rather the discussion largely revolves around the possible involvement of microtubules in generating the observed morphological changes, and the possible meaning of elongated mitochondria in infected cells. Both of these topics are barely introduced, and not at all experimentally interrogated in the case of microtubules. There is also some discussion about Golgi fragmentation, although this is also not directly interrogated by cryoSXT in the current manuscript.

We thank the reviewer for these comments. We have: - Updated the introduction to enunciate more clearly the aims of our study - Included a substantial comparison of the relative merits of cryoSXT versus other ultrastructural analysis techniques (TEM, cryoEM and cryoET) in the discussion - Updated the introduction to introduce the concepts of microtubule and mitochondrial morphology changes during infection that are covered in depth in the discussion - Included additional microscopy experiments, including super-resolution structured illumination microscopy (SIM), to demonstrate the changes in Golgi (Figures 6 and 7), microtubule (Figure 8) and mitochondrial (Suppl. Figure 4) morphology that accompany HSV-1 infection. These additional experiments support the hypotheses presented in the submitted manuscript, namely that microtubule organising centres are disrupted, Golgi membranes dispersed, and mitochondria redistributed as HSV-1 infection progresses.

The authors perform imaging with a 40nm or a 25nm zone plate, where the 25nm zone plate provides improved resolution of a smaller volume compared to the 40nm zone plate. The authors do not really make use of the improved resolution offered by the 25nm zone plate in the results, so the motivation for turning to this (and therefor also changing cell line) is a bit unclear. The reason for the U2OS cell line to better preserved during X ray imaging is also not discussed, maybe it has to do with the thickness of the cells (as the U2OS cells are very flat). Furthermore, images from the 25 nm zone plate are not compared side by side to neither the 40nm zone plate nor standard TEM, which makes it hard to judge what the increased resolution really brings.

Only one zone plate can be installed at any one time in the microscope and altering the zone plates requires extensive hardware changes that are outside the control of beamline users. We agree that this was not clearly discussed in the text. We have included additional text in the results (lines 207–208) and methods (lines 633–638) explaining this operational limitation and clarifying which zone plate was used for which experiment. In this study we observed that tomograms acquired with the 25 nm zone plate did not provide significantly more biological information than with the 40 nm zone plate, and thus both are suitable for characterisation of overarching cellular ultrastructural changes that accompany infection. We have added a sentence to this effect to the discussion (lines 410–412). Like U2OS cells, HFF-hTERT cells are also very flat. They appear more robust compared to HFFs when used for protracted exposures to soft X-rays and less likely to suffer from heat deposition after an extensive data collection round. We can speculate at this point that this could conceivably be due to the particular chemical composition of the intracellular environment in different cell lineages but it is impossible to offer anything other than speculation and therefore we have refrained from commenting further on this in the manuscript.

The switch from a 40 to a 25nm zone plate required a switch in the model system, as mentioned above. The chosen cell types are not linked to biological relevance however (neurons and epithelial cells are mentioned as relevant cell types in the introduction), and it is therefor a bit unclear what the relevance is of keeping results from both cell types and comparing the two, rather than sticking to the one that works with cryoSXT. The results from the U2OS cells could still be compared by LM to the HFF cells if this contributes to the aim of the study.

U2OS cells were chosen because they have been used previously for studies of HSV-1 infection (references 55–56) and are known to be well suited to cryoSXT analysis (references 32–33). We have added a sentence to this effect to the results (lines 208–211).

The distribution of the viral proteins of the timestamp reporter virus is used to categorize infected HFF cells into 4 infection stages. In the U2OS cells the protein distribution is a bit different, which only allows them to be categorized into early (stage 1+2) and late (stage 3+4) stage of infection. Although this is what the authors state in the text, all 4 stages are included in Fig.2 for the U2OS cells, so it is not clear how this subdivision is performed and it does not seem like an accurate representation of the data. Furthermore, the uninfected population is not included in the timecourse, and there is not really a gradual change in infection states over the different timepoints as one could have expected. Therefor it is a bit hard to see the relevance of the timecourse. In the paper where the reporter virus is published (ref 16), shorter infection times were used, which leads to a more gradual change in infection stages.

We thank the reviewer for pointing out these omissions. We have updated Figure 2A to only show the categories early (stage 1+2) and late (stage 3+4) for the U2OS cells. Furthermore, we have repeated the infection time course experiment, quantitating uninfected cells in addition to infected cells and including additional time points (2-, 4- and 6-hours post-infection). This new data (Figure 2B) demonstrates that the temporal profiles of infection progression are similar in HFF-hTERT and U2OS cells. Furthermore, it supports our choice of 9 hours post-infection as a suitable time point for plunge freezing of samples in order to obtain a mixture of cells at early and late stages of infection.

There is a lot of importance given to the morphological changes of mitochondrial networks in infected cells. However, the quantification represented in Fig.5B is a bit unclear. The mitochondria are classified into different groups, but there is no specific description of the definition and cutoff values of each group. The name of some groups is also confusing, such as "short and long" mitochondria. Furthermore, there are large differences between replicates (suppl. fig. 2). The authors state that some mitochondria are swollen, which they interpret as a sign of apoptosis. They find these swollen mitochondria in 75% of the tomograms of uninfected cells in replicate number 3. If this is indeed cell death this replicate is not healthy.

We apologise that the categorisation of mitochondria was not sufficiently clear in the submitted manuscript. The categories were percentage of tomograms that had the different mitochondrial morphologies present, not percentages of mitochondria. Thus, tomograms with both short and long mitochondria were classified as “short and long”. We have re-generated Figure 5C and Suppl. Figure 2C as a Venn diagram to illustrate this point more clearly. We have also updated the legend of Figure 5C (lines 845–850) to state clearly that the diagram shows percentage of tomograms with the relevant mitochondrial morphologies. The categorisation was performed manually and we have included examples of each category in Figure 5A. Manual classification can be subjective but, given the large number of tomograms analysed and the clear distinction between morphology in uninfected vs early- and late-stage infected cells, we are confident that our results are robust. We note that we have deposited all of the source tomograms in the Apollo repository at the University of Cambridge (https://doi.org/10.17863/CAM.78593); the data we used for this analysis are thus freely available for inspection and re-analysis by interested colleagues. We note that the swollen mitochondria were observed in multiple samples of uninfected and infected cells. This suggests that, regardless of infection, this is a common phenotype of U2OS cells. Others have observed this morphology by EM in the context of apoptosis and suggest it may represent porous mitochondria (reference 61). Although the proportion of tomograms containing these swollen mitochondria were higher in the uninfected sample of replicate 3, the other 25% contained typical mitochondrial morphologies that we could include in our analysis. The presence of inter-cell morphological variability such as this highlights the importance of imaging multiple cells within a population and performing several distinct biological replicates, as we have done in this study, to ensure project-relevant information is captured and delineated from the background structural variability inherent within a cell population. Previous cryoSXT studies had observed (but did not specifically comment on) a similar swollen mitochondrial morphology (reference 59). However, out of an abundance of caution we excluded all tomograms with swollen mitochondria from our analysis of mitochondrial branching (Figure 5C). Moreover, Tukey tests were performed per replicate for each pair of conditions in Figure 5C and statistical significance was reported only if it was observed independently in all three replicates. We are thus confident that any sampling error in replicate 3 that may arise from excluding tomograms will not have meaningfully altered our conclusions.

Minor comments

Results section 1, line 115-117: Where the authors state that it is unclear whether "naked" HSV-1 capsids would be visible by cryoSXT, it would be useful to refer to literature where these are observed by TEM, or to compare to TEM in their own experiments.

We have included references to previous TEM studies in the results (lines 128–129), as requested. However, we note that TEM and cryoSXT are fundamentally different as TEM uses contrast agents whereas contrast in cryoSXT arises from differential elemental densities (in particular the density of oxygen versus carbon or phosphorous). We have updated the results (lines 129–131) to clarify this point.

Results line 143: The authors state that it's hard to observe the perinuclear viruses with TEM, but there are several examples of this in the literature that could be referenced, e.g. (Skepper et al. 2001; Leuzinger et al. 2005; Baines et al. 2007; Johnson and Baines 2011), although this does not mean that they are not hard to find or that 3D is not advantegous.

We thank the reviewer for these references and we have added them to the manuscript.

Fig.4: It is unclear why all the vesicles are open-ended

This is due to the differential path-length of carbon rich (and thus high contrast) membrane traversed by the X-rays for the membranes normal or parallel to the incident X-ray beam. We have clarified this point in the results (lines 290–301).

Some places in the manuscript PFU per cell is used, other places MOI

Thank you for pointing this out. For consistency, we have changed all instances of PFU per cell to MOI.

If some specific adjustments to the methods had to be implemented for bio safely reasons (virus work), this should be stated in the methods.

We have added a section on biosafety measures to the methods (lines 562–568).

Access to the synchrotron should also be described

We have expanded the synchrotron access attribution the Acknowledgments section (lines 737– 738).

Discussion line 320: "consistent with previous research" - there is a reference missing.

Thank you for spotting this. We have now added the reference.

The quantifications are based on a limited number of tomograms, but there is no statement as to how the specific tomograms were selected. With a variability between replicates and tomograms, a random selection is important.

We included all tomograms collected for the relevant experimental condition in all our analyses unless otherwise stated. For the vesicle segmentation we chose four reconstructed tomograms from each condition at random (lines 690–691). For lipid droplet volume analysis and mitochondrial branching analysis we included all tomograms that matched our quality-control criteria. We have added a few sentences to the Segmentation and Graphs and Statistics sections of the methods (lines 691–694 and 724–733) describing our selection criteria for the lipid droplet, vesicle and mitochondrial branching analysis, respectively.

If gold fiducials are visible in the tomograms it could be useful to indicate, as they can look similar to lipid droplets to a non-expert reader.

We have indicated gold fiducials Figure 1 H, the only figure in which they are visible, with a gold star as requested.

Suppl. Fig.2: For clarity it would be good not to use the same color arrows to indicate different things in A and B.

Suppl. Figure 2B has been removed in response to another reviewer request.

Reviewer #1 (Significance):

The authors of this study demonstrate that cells infected by HSV-1 virus can be investigated by the use of cryoSXT, and use this to show that infected cells have more elongated and interconnected mitochondria, and an enrichment of small vesicles close to the nucleus. They thereby also show that cryoSXT offers a nice resolution for characterizing morphological changes in significant volumes of near native-state cells, and that the method offers a promising throughput for screening of large amounts of cells. However, the study does not really present new biological or technical advances compared to previously published literature, see e.g. Müller et.al. 2012, Duke et.al 2014, Perez Berna et.al. 2016, Groen et.al. 2019, Weinhardt et.al. 2020, Loconte et.al. 2021 (not cryo but demonstrates the advantage of capillaries), Kounatidis et.al. 2020, Scherer 2021 (ref 16 from paper), some of which are also referenced in the current study. The study could thus have profited from a more defined focus and possibly further experiments (live-cell imaging, CLEM, TEM, microtubules or more mechanistically focused) depending on the main interest of the authors. The advantage with the current broad focus (assuming that the main concerns are addressed) is that the study could interest a larger audience, ranging from virology, cell biology and immunology to microscopy and methods development.

We thank the reviewer for recognising the broad audience that will be interested in our manuscript. We believe that our analysis highlights the broad applicability of cryoSXT for analysing cell ultrastructure and changes that occur in response to infection. Furthermore, we think that our use of robust numerical analysis to quantitate the phenotypes we observe highlights the strength of cryoSXT as a high throughput technique for ultrastructural analysis. Our study is the first to investigate HSV-1 infection using cryoSXT and, in addition to confirming previous ultrastructural changes observed using other methods, we present new biological insight in organelle architecture and distribution such as that lipid droplets undergo a transient size increase during early stages of infection. We believe that we have demonstrated the robust utility of cryoSXT as a tool to study ultrastructural changes in response to insults, such as infection by intracellular pathogens, and hope that our manuscript will act as inspiration for others seeking to use cryoSXT to image cellular ultrastructure.

Reviewer #2 (Evidence, reproducibility and clarity):

The authors use soft X-ray tomography to examine cell structure following infection by herpes simplex virus-1 (HSV-1). This imaging method can provide 3D images of cryo-preserved intact cells without chemical fixation or staining. The authors find several morphological differences between uninfected and infected cells, including changes in the number and size of vesicles and in the size and shape of mitochondria.

This is a well-done study with careful and extensive analysis that in general produces convincing images to support the authors' conclusions. The procedures are clearly described and reproducible, and the authors have examined an impressive number of images and have performed appropriate statistical analyses.

We thank the reviewer for their positive comments.

I had two comments / suggestions regarding the findings about changes in morphology after infection. First, in the Discussion, the authors consider the possibility of Golgi fragmentation. Can the authors test this by counting Golgi before and after fragmentation?

We did not frequently observe well-defined Golgi apparatuses in our tomograms, consistent with previous cryoSXT studies (reference 61). We therefore performed new experiments using SIM microscopy to demonstrate the disruption of Golgi apparatus and trans-Golgi network in fixed U2OS cells stained with the markers GM130 and TGN46, respectively. These new results are presented in Figures 6 and 7 and in the results (lines 342–355).

Second, in the Results the authors report that they did not observe a change in lipid droplets after infection. However, the late-stage image in Fig. 5A seems to show such a change, with the lipid droplets becoming larger and darker relative to the early stage or uninfected cells. Maybe this is just the particular image that was selected, but perhaps it is worth looking at more images by eye just in case the segmentation procedure somehow missed this change.

We thank the reviewer for suggesting we re-visit the properties of lipid droplets. Based on this suggestion we segmented the lipid droplets from 94 tomograms and found a robust change in the median volume of lipid droplets at early stages of infection. We have included this new data in Figure 4C, Suppl Figure 2 and the text of the results (lines 302–312). The observation that lipid droplet volumes change is particularly interesting as another group recently observed similar changes in lipid droplets in response to HSV-1 infection of astrocytes and they postulate that this may modulate the cellular immune response (reference 85). Our data support and extend their conclusions, as described in the discussion (lines 476–494).

Minor comments:

Line 127 - As I understand it, the alignment by fiducial markers corrects primarily for small inaccuracies in tilting of the stage. Hopefully there are not significant vibrations in the microscope because this would also lead to loss of resolution during the exposure of each tilt angle.

Thank you, we have corrected “vibrations” to “small inaccuracies in tilting of the microscope stage”.

Line 145 - "electron light" Is this common usage? To me it seems more accurate to just say electrons because light to me means photons.

Thank you, we have corrected “electron light” to “electrons”.

Line 390 - detection OF ("of" is missing)

Thank you, we have made the correction.

Line 564 - Fig. 2 legend. "partial retention in the nucleus of U2OS cells". I am not sure where the nucleus is in the images. To me, it looks like there is almost no stain for ICP0 in hTERT at stage 1 and stage 3, and then cytoplasmic stain at stage 2 and stage 4. In contrast, for U2OS, the stain looks mostly nuclear until stage 4 when it is partially cytoplasmic. This all needs to be better explained, and perhaps arrows added to the images such that the reader does not have to guess.

We agree and have added a silhouette around each nuclei in Figure 2 to make this clearer. We have also added arrows to indicate the gC-mCherry enriched juxtanuclear compartment in cells at stage 3 (HFF-hTERT) or a late stage (U2OS) of infection.

Line 585 - The authors could consider rotating the images by 180{degree sign} in panel A (late) in order to maintain the same orientation of nucleus and cytoplasm. This would make it easier for readers to see the point.

Done as requested.

Line 614 - I could not find the length of the scale bar in the legend.

We apologise for omitting this – is has now been added.

Reviewer #2 (Significance):

The significance of the study is two-fold. First, it is a nice technical demonstration of what can be accomplished using soft X-ray tomography. I am qualified to evaluate this, since my expertise is in biological applications of this technique. The second significant aspect of the study is the demonstration of morphological changes in mitochondria and vesicles. I am not a virologist, so I do not know the literature on this point with regard to virus infection, but I find it interesting that the authors were able to detect such changes.

We thank the reviewer for their positive assessment of our work.

I believe the authors should cite a couple of papers:

10.1016/j.cell.2015.11.029 which looks at HSV infection and reports viral particles between the inner and outer nuclear membrane.

We have included a citation to this work as requested (lines 162–165).

10.1016/j.jsb.2011.11.025 which also reports nuclear membrane separations or bulges by soft X-ray tomography.

We have elaborated on this section and incorporated the reference as requested (lines 265– 276).

Regarding these nuclear membrane bulges, there are a number of papers that show they can also arise from mutations in nuclear-lamin associated proteins like nesprin and SUN (see for example https://doi.org/10.1093/hmg/ddm338). This is perhaps something interesting for the authors to think about, but not necessary for the current manuscript.

Thank you for this comment. We did consider studying the breakdown of the nuclear lamina during HSV-1 infection, as this has been shown in previous studies [e.g. 10.1101/2021.06.02.446771]. However, we could not robustly resolve the nuclear lamina from the nuclear envelope in uninfected cells. The nuclear lamina is quite thin (30–100 nm in width) and this may have confounded its identification.

Reviewer #3 (Evidence, reproducibility and clarity):

Summary:

The manuscript by Nahas et al. describes the structural studies performed in U2OS cells infected with a recombinant HSV-1 virus that enables tracing the stage of the infection using fluorescent markers. This system was used to determine major structural changes in HSV-1 infected cells using cryo-soft X ray tomography (cryo-SXT) on near native-state samples. The data presented complement previous studies (particularly ref.16) using similar reagents but different microscopy techniques. While the data are generally well presented and discussed, they do not provide any substantially novel information on the structural changes in HSV-1. Nevetheless, they constitute an interesting technical achievement.

We thank the reviewer for supporting the technical quality of the analysis. In response to the comments of another reviewer we have extended our analysis and documented new biological information for this system relating to lipid droplet re-shaping and distribution in response to HSV-1 infection; all our new findings are included in the updated manuscript.

Major comments:

There are no major concerns on the data, although some of the statements could be revised for a more realistic interpretation of the results.

• In Figure 1F and lines 152-156 it is stated that a bulging of the nuclear envelope occurs around some of the putative particles, while in lines 243-244 and lines 625-628, it is stated that bulging occurs both in mock and infected cells. This should be clarified to avoid confusion. It is possible that authors differentiate both situations and this should be more clearly stated.

Many thanks for identifying a possible area of confusion. We have updated the results to clearly distinguish the expansion of the perinuclear space that accompanies virus nuclear egress (lines 160–175) from the bulges of the nuclear envelope that are observed in uninfected and infected cells (lines 265–276).

• The statistical tests are different for different hypothesis testing throughout the manuscript. The authors should justify in the methods section the use of one or another test. This will contribute to clarity in the hypothesis that is being test and will clarify the reason for the selected test.

We have significantly expanded the Graphs and Statistics section of the methods (lines 703– 734) to further justify the statistical tests used throughout our study.

• Sentence: "Our observation..." in lines 349-352. Even though the sentence is in the Discussion it is wildly speculative. The authors could use different approaches to tackle experimentally the question of whether active fusion or faulty fission is involved, but this is not the main subject the manuscript. Please revise the sentence or address experimentally, this would provide new insight into the impact of HSV-1 infection on mitochondrial network morphology. This sentence could be qualified as "speculative".

We agree that this section of the discussion strayed into speculative territory and have removed it from the updated manuscript.

• Although ref.16 provides evidence supporting Golgi fragmentation and mitochondrial elongation after HSV-1_timestamp virus infection in HFF cells, it would be important to show confocal microscopy data in U2OS cells, which were used for cryo-SXT, particularly since the authors refer differential virus kinetics and subcellular distribution of viral antigens in these cells. These would greatly contribute to support the statements regarding these two phenomena. It is very likely that the authors already have the data and could easily show them.

We have included new microscopy experiments to demonstrate changes in mitochondrial (Suppl. Figure 4) and Golgi (Figures 6 and 7) morphology that accompany HSV-1 infection, and these new experiments are now included in the results (lines 335–310 and 342–355).

-Line 269: Apposition of lipid droplets and mitochondria is not thoroughly described. This statement requires quantitation. Optimally, confocal imaging using Mitotracker and bodipy493/503 or superresolution imaging using specific antibodies may also contribute to strengthen the statement.

We agree with the reviewer that we do not at this stage have adequate data to support this assertion and have therefore removed it from the manuscript.

• It would be of great interest to document the budding events observed by cryo-SXT using higher resolution techniques and the kinetic resolution provided by the fluorescent infection fiducials. This would confirm the nature of the particles (using immunogold) and would demonstrate the the usefulness of the cryo-SXT data. This by itself would justify the use of cryo-SXT to temporally locate events that are difficult to visualize otherwise (as stated by the authors).

We agree with the reviewer that a correlative imaging strategy involving cryoSXT and fluorescence microscopy could aid in identifying features of infection, and have highlighted this interesting future direction in the discussion (line 406–409). However, performing such analysis will be a substantial experimental commitment in its own and is outside the scope of our current manuscript.

Minor comments:

• Given that the software used for segmentation (Contour) is not published, a minimal comparative description between manual and semi-automated segmentation may be shown in the supplementary, to illustrate the robustness of the new method and the reliability of the measurements.

We have now published a preprint (recently accepted in the journal Biological Imaging) that describes Contour in detail, which we have referenced in the updated manuscript: Nahas, K. L., Ferreira Fernandes, J., Crump, C., Graham, S. C. & Harkiolaki, M. (2021) Contour, a semi-automated segmentation and quantitation tool for cryo-soft-X-ray tomography. http://biorxiv.org/lookup/doi/10.1101/2021.12.03.470962

• Lines 278-280: statistical test and p value are not shown.

We have updated the text to include details of the statistical test and p value as requested (lines 326–330 of the updated manuscript).

• After line 376: It would be interesting to mention that transient elongation of mitochondria is observed during dengue virus infection (https://doi.org/10.1016/j.chom.2016.07.008) and that this has also consequences for innate immunity against viruses.

We thank the reviewer for this suggestion, which we have incorporated into the discussion (lines 522–523).

• Given that HSV-1 is a BSL-2 level virus and that a recombinant version (GMO) has been used in the study, the authors should describe the biosafety measures taken to image non-inactivated infectious samples by cryo-SXT. The authors should state that a biosafety committee has reviewed these activities.

We have included a Biosafety Measures section to the methods (lines 562–568) that details the biosafety measures used and their approval by the relevant committees.

Reviewer #3 (Significance):

This study constitutes an incremental technical advance in the study of HSV-1 infection. The broad context and the quasi-native structure of the cells enables documenting events that are difficult to observe thin sections for TEM.

This study is one of the few examples of the use of cryo-SXT for infected cell imaging. Other examples of the literature are cited as well as previous structural studies performed with higher resolution techniques.

The manuscript may be suitable for HSV-1 specialists and cell biologists interested in using near-native samples for gross cellular imaging and documentation of low-resolution maps revealing alterations in large subcellular structures.

We thank the reviewer for highlighting that ours is one of only a few comprehensive studies using cryoSXT, illustrating how it can be used to image cellular processes that are hard to ‘catch’ using techniques that require ultra-thin sectioning, and as such that it will be of interest to cell biologists studying infection processes in cellulo.

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