Time in the US , Family , Siblings

Time in the US , Family

Time in US - childhood - memories

Time in US - crime - fights - theft - gang affiliation - comradery - arrests - time in the US

Time in US - homelife - caring for siblings - abuse

Reflections, The United States, Worst Parts

Reflections, Identity, Global/Human

Reflections, The United States, Favorite parts/ missing

Return to Mexico, Challenges, Family Separation

Time in the US, Relationships

Time in the US, Drugs, Addiction

Time in the US, Homelife, Parents/ Step-Parents, Expectations

Time in the US, Arrests

Time in the US, Relationships, Creating Families

Time in the US, School, High School, Struggling/ Suspension/ Dropping Out

Time in the US, Gangs, Camaraderie/Family

Time in the US, School, High School, Struggling/ Suspension/ Dropping out

Time in the US, Homelife, Siblings, Caring for Them

Time in US - losing hope loss of dreams

Fitting in - belonging - feelings of inferiority

Gaiman on boredom.

Time in the US, Homelife, Parents

Time in the US, Tattoos, Meaning

Time in the US, School, Middle School, Teachers; Time in the US, Mentors, Teachers

Time in the US - family - father returns for children

Time in the US - mentor - teachers - education

Return to Mexico- Feelings- Dignity

Macheath is mean and bad to these men. It's less comraderie and moreso dictatorship

I used to go out and drink a beer while reading a book or journaling to have an excuse to eavesdrop aggressively. This still felt like an amelioration of my loneliness even if it's obvious that lots of other things would have been better if I could have managed them.

Soul ties

Any thoughts on whether URLs should match the chapter or part title exactly, or whether a shorter URL is better? For instance, if a chapter in a history textbook is titled, "Fur Traders and Indigenous Relations during the 18th Century," would a better URL be "fur-traders-and-indigenous-relations-during-the-18th-century" or "fur-trade-18th-century"? Personally, I like the shorter URL better, but that does create extra work. Plus, there could be a chapter on a similar enough topic elsewhere in the book that a more complicated URL could be required elsewhere, so it's more subjective than just using the chapter title verbatim as the URL.

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

We thank the reviewers for their comments, criticisms and suggestions that will help to improve the quality of our manusrcipt.

Please find enclosed in this initial response our answer to each point raised by the reviewers.

Please note that for several answers normally come along with an additional figure that could be added in the full revised version of the manuscript. However, these additional figures could not be added in the way we have to submit our answers but we are ready to send a pdf file including our answers with the additional figures upon request.

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

The paper by Genest et al. describes the effect of flotillins and sphingosine kinase 2 to stabilize AXL as a mechanism to promote epithelial-mesenchymal transition in breast (cancer) cells. The potential role of vesicles trafficking EMT-promoting proteins is of high interest in the field, also for exploring new opportunities of pharmacological targeting. However, the paper fails to convincingly demonstrate that the proposed mechanism is of real importance to support or promote EMT for the following main reasons:

1-a) The role of flotillins is studied only by overexpression and in the context of non-cancerous MCF10A cells, while breast cancer cells of epithelial-like origin are not analyzed.

Regarding the first part of the point raised here, we are not sure to understand correctly the sentence “[…] while breast cancer cells of epithelial-like origin are not analyzed”. Indeed, we used the breast cancer cell line MDA-MB-231 and a derived cell line that we generated by knocking down flotillin expression (MDA-MB-231shFlot2) in the second part of this study (Figure 6C, F and H and S7A, E and F). This previously characterized cell line allowed us to demonstrate that abolishing flotillin overexpression was sufficient to significantly inhibit the invasive properties of MDA-MB-231 cells (Planchon et al, J Cell Science 2018, https://doi.org/10.1242/jcs.218925

Although flotillin upregulation induces some major mechanisms of the EMT process in MCF10A cells, flotillin downregulation was not sufficient to reverse the EMT phenotype in MDA-MB-231 cells. This could be explained by the fact that EMT is a multifactorial process and that MDA-MB-231 cells went through too many irreversible changes leading to this process. By contrast, when we analyzed EMT markers after SphK2 inhibition or knock down in MCF10AF1F2 and in MDA-MB-231 cells (Figure 6A-C), we could observe a significant decrease in ZEB1 expression.

1-b) This is contrast with the purpose of the paper (see abstract, introduction, patients' data) which is to study tumors and EMT. Effect of shRNAs is also not reported, making it difficult to estimate the importance on the EMT phenotype.

As we mentioned in our manuscript, previous studies by other groups who downregulated flotillin expression in different cancer cell lines using siRNA approaches or re-expression of miRNAs that inhibit flotillin expression, already showed flotillin participation in EMT (for review please see, Gauthier-Rouvière et al, Cancer Metastasis Review, 2020, **doi: 10.1007/s10555-020-09873-y).

In this context, the novelty and the first goal of our study was to investigate how strong is the contribution of flotillin upregulation to EMT induction. To achieve this goal, we chose on purpose to use non-tumoral epithelial cells that do not harbor the anomalies already favoring EMT, unlike the cancer cell lines used in previous studies. In these non-tumoral models (the human MCF10A and mouse NMuMG mammary epithelial cell lines), we ectopically overexpressed flotillins (MCF10AF1F2 and NMuMGF1F2) to levels similar to what observed in invasive breast cancer cells. Using this approach, we found that flotillin overexpression is enough to induce EMT.

1-c) Then, alteration of EMT should be concluded also from other non-genetic functional parameters, not just by markers. For instance: was morphology of the cells changed? Was cell migration affected with F1F2?

Our conclusion that flotillin upregulation is sufficient to induce EMT in MCF10AF1F2 and NMuMGF1F2 cells is not based only on genetic functional parameters or markers. For instance, Figure S1 (panels H and I) shows a strong modification of the cell morphology and of the actin cytoskeleton organization in NMuMG cells upon flotillin upregulation. NMuMGF1F2 cells became flat and lost their apical F-actin belt and exhibited an increase in stress fibers.

As shown below (Additional Figure 1), similar modifications of the cell morphology and of the F-actin cytoskeleton organization occur also when flotillins are upregulated in MCF10A cells (see below the comparison of MCF10A and MCF10AF1F2 cells) (these data could be added in the manuscript).

ADDITIONAL FIGURE 1 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST

Additional figure 1: Upregulation of flotillins in MCF10A cells leads to changes in the cell morphology and in F-actin cytoskeleton organization. Comparison of the morphology and of the actin cytoskeleton organization in MCF10AmCh and MCF10AF1F2 cells. Confluent cells were fixed and stained for F-actin (green) using Alexa488-conjugated-Phalloidin and for nuclei (blue) using Hoechst (in panel A flotillin2-mCherry signal is shown). (A) Upper panels show the maximum intensity projection images (MIP) of MCF10AmCh (control) and MCF10AF1F2 (flotillin overexpression) cells obtained from a stack of images acquired by confocal microscopy. Lower panels show magnified images from the boxed areas, including one single plane and the x-z and y-z projections along the indicated axes. (B) 3D reconstruction images obtained from the region in the boxed area from the MIP-images shown in A.

These data show that in MCF10AF1F2 cells the apical actin belt is lost and the height of the cellular monolayer is lower compared with control MCF10AmCh cells.

We also analyzed the migration capacity of these cells (shown in Figure 3G of the submitted manuscript). Briefly, using a Boyden chamber assay, we showed that flotillin upregulation significantly increased migration of MCF10A cells (Figure 3G). We previously demonstrated that flotillin upregulation also promotes cell invasion in 3D using a spheroid assay (Planchon et al, J Cell Science, 2018, https://doi.org/10.1242/jcs.218925**). As shown below (Additional Figure 2), using a wound healing assay, we also observed that cell velocity is higher in flotillin-overexpressing NMuMGF1F2 cells than in control NMuMG cells (this could be added to the manuscript).

ADDITIONAL FIGURE 2 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST

Additional figure 2: Upregulation of flotillins in NMuMG cells increases cell velocity in a 2D migration assay. (A) Representative images of NMuMGmCh (control) and NMuMGF1F2 cells during wound healing. The yellow dashed line indicates the leading edge of the migrating monolayer at the indicated times. The trajectory of 60 individual cells was tracked and the cell velocity and persistence of migration were extracted. The histogram shows the velocity quantification (mean ± SEM of 4 independent experiments). (B) Representative trajectories of individual cells.

2) AXL up-regulation is not very strong (2-fold). What is unclear is if the minimal AXL increase due to F1F2 really provides a significant contribution to the EMT phenotype (as the authors conclude). The siRNA experiment knocks down all AXL, not just the F1F2-induced levels, making it difficult to estimate the real effect of the mechanism proposed.

As shown in figure 3A and D, in MCF10AF1F2 cells compared with MCF10AmCh cells, we measured a significant 2.5 ± 0.7-fold increase in the AXL protein level. We do not think that this can be considered as a minimal increase.

Considering that flotillin upregulation may affect simultaneously different receptors (Figure S2I, Figure S6A-F), we did not expect that downregulating a single receptor would have a major impact on the level of EMT markers and on cell migration. Yet, after knocking down AXL in MCF10AF1F2 cells, we observed a decrease in ZEB1 and N-cadherin expression and the re-expression of E-cadherin (Figure 3D-F) and the inhibition of cell migration (Figure 3G). The fact that we observed such an effect by downregulating AXL, which according to Reviewer #1 is minimally increased, might be explained by its well-known ability to act not alone but through cross-talk with other signaling receptors (Graham et al, Nature Reviews Cancer 2014; Halmos and Haura, Science Signaling 2016; Colavito et al, Journal of Oncology 2020).

As suggested by Reviewer #1, ideally, it would be interesting to bring back AXL to its level in MCF10AmCh cells to better evaluate only the contribution of its increase. However, adjusting so precisely the efficacy of AXL downregulation by siRNA seems quite difficult to achieve.

3) Why didn’t the author focus on EphA4 (or to a lesser extent ALK), which showed better regulation ?

As we mentioned (page 18) “the available tools allowed us to validate this result only for AXL, but not for EphA4 and ALK”**.

Nevertheless, for EphA4, we showed in Figure S6 that it is located in flotillin-positive late endosomes (Figure S6 A and C, for MCF10AF1F2 and NMuMGF1F2 cells, respectively) in a phosphorylated form (using an antibody against P-Y588/Y596-EphA4 that works in NMuMG cells, Figure S6D). However, the signals obtained by western blotting using the same antibody were too low to validate any significant variation of EphA4 Y-phosphorylation status, as suggested by the results from the phospho-RTK array.

Regarding ALK, the increase in its phosphorylation, suggested by the phospho-RTK array, remains puzzling to us. By western blotting of cell lysates and in the presence of positive controls, we did not detect any positive signal for phosphorylated ALK and even for total ALK in MCF10A and MCF10AF1F2 cells. In addition, to our knowledge, ALK expression in MCF10A cells has never been reported in the literature. These observations did not encourage us to pursue our investigations on ALK.

Moreover, several points led us to focus on AXL. Indeed, AXL expression is associated with the acquisition of a mesenchymal cell phenotype, invasive properties, and resistance to treatments and AXL is an attractive therapeutic target against which several inhibitors are in preclinical and clinical development (Shen Y et al. Life Sciences 2018). Moreover, AXL expression in tumors is attributed to post-transcriptional regulation, but the mechanisms are totally unknown. Understanding how its stabilization and signaling can be triggered by flotillin-mediated endocytic pathways is new and of high significance for the cancer field and the trafficking community.

3) The conclusions of the manuscript are contradicted by the reported clinical data. In Figure S4 the authors clearly observe co-expression of Flotillin 1 and AXL prevalently in luminal breast cancers, which is the subtype known to not be driven by EMT. This evidence already indicates that this (otherwise interesting) mechanism is not relevant to EMT in breast cancer. So, the conclusions are not supported by the data, and the experimental setup and model chosen are not appropriate to generalize the findings to cancer.

We acknowledge that flotillin 1/AXL co-expression is highest in the luminal subtype. If this co-expression was observed only in this particular subtype, we would have agreed that it excluded that flotillins and AXL co-overexpression may participate in EMT in tumor cells. However, our results show that flotillin 1 and AXL are co-expressed also in other subtypes that have undergone EMT. Considering this observation and the influence of flotillin upregulation on AXL overexpression we reported here, we believe that the point raised by the Reviewer is not sufficient to exclude that the co-upregulation of flotillins and AXL can participate in EMT induction in breast cancer cells.

**Minor (here the most important):**

4) The point of the Figure 2 is not clear. Why this part should have such a central role in the story? The entire data presented are not followed up in the rest of the paper. Moreover, in some cases upregulations also questionably significant (like RAS and STAT3 are not even 2 fold).

Moreover, the error bars are so small that it seems unrealistic that the plots indicate three independent experiments.

Because the activation of oncogenic signaling pathways is crucial to promote EMT, we think that analyzing these pathways in the context of flotillin upregulation is coherent with the message of the paper.

To our knowledge, the amplitude of up- or down-regulation has nothing to do with its significance. The amplitude also depends strongly on the context (stimulation with an agonist, overexpression of GEF, etc). For instance, increases lower than 2-fold are frequently reported (Bodin and Welch, Mol Biol Cell, 2005; Miura SI et al, Arteriosclerosis, Thromb and Vasc Biology, 2003; Matsunaga-Udagawa R et al, J Bio Chem 2010)** when assessing the activity of Ras or small GTPases, but they represent real upregulations. Furthermore, Ras activation is supported by the downstream 4-fold activation of ERK that we measured (Figure 2C).

In Figure 2, panels B, C, E, F and J, considering the amplitude of the mean increases shown, the error bars corresponding to SEM do not seem disproportionately small.

As the Reviewer seems to insinuate that we have not performed independent experiments, we are presenting in the table below the detailed results all obtained from independent experiments.

Panel

Parameter measured

Number of independent experiments

Fold of increase value in MCF10AF1F2 cells compared with MCF10AmCh cells in each experiment

Mean

SEM

p-value

B

Ras-GTP

5

1.95 ; 1.96 ; 1.18 ; 1.67 ; 1.86

1.72

0.14

0.001

C

Phospho- ERK

5

1.24 ; 5.43 ; 3.22 ; 6.11 ; 3.52

3.71

0.73

0.0042

E

Phospho-AKT

4

2.29 ; 6.54 ; 3.76 ; 2.6

3.8

0.97

0.0276

F

Phospho-STAT3

4

1.63 ; 1.63 ; 2.42 ; 1.60

1.82

0.20

0.0066

J

Phospho-SMAD3

8

4.1 ; 5.12 ; 6.29 ; 1.82 ; 2.58 ; 6.66 ; 2.82 ; 5.40

4.35

0.64

0.0001

In the legend to figure 2 panels C, E, F, J, “The histograms show […] with control MCF10AmCh **cells calculated from 4 independent experiments” was corrected by “The histograms show […] with control MCF10AmCh cells calculated from at least 4 independent experiments” as data shown in panel J were actually calculated from 8 independent experiments.

5) More robust statistical analysis should be provided in the Figure 1 to support that EMT is suppressed with F1F2 overexpression. For instance a more standard GSEA on hallmark signatures.

To avoid confusion, we understand that Reviewer #1 meant “… that EMT is induced with F1F2 overexpression” and not “… suppressed …”.

As recommended by Reviewer #1, we performed a GSEA on the hallmark signature and the results are already included in the current revised version of our manuscript (figure 1C).

6) In Figure 3 E-Cadherin is rescued with siAXL in the IF but not in the western blot.

Using siRNA transfection, we can have a mosaic effect due to the fact that not all the cells of the sample are transfected and thus efficiently knocked down. This mosaicism was clear when we analyzed E-cadherin by immunocytochemistry. Indeed, in some cells, probably the ones that have been more efficiently transfected with the AXL siRNA, E-cadherin expression is clearly seen. By western blotting, which provides a global analysis in which transfected and non-transfected cells are mixed, this was not significantly higher than in MCF10AF1F2 cells transfected with a control siRNA, although there was a trend towards increased E-cadherin expression in MCF10AF1F2 transfected with the AXL siRNA.

For the revised version of our manuscript we will try to improve the efficacy of the AXL siRNA and test whether we can fully rescue E-cadherin expression. The corresponding panel could be modified according to the data we will obtain.

7) Some sentences require clarifications. The authors should be more clear on why ZEB2 antibody was not available or what they mean with "Unfortunately the available tools..".

Page 7: we wrote «no anti-Zeb2 antibody is available». We should have said: «none of the anti-Zeb2 antibodies tested worked in MCF10A cells». We decided to remove “no anti-Zeb2 antibody is available” from the sentence to avoid confusion in the revised version of our manuscript.

Page 19: we wrote «unfortunately the available tools» to refer **the available tools against EphA4 and ALK that did not allow us to validate the data obtained using the phospho-RTK array showing that the Y-phosphorylation of these two RTK is increased in cells with upregulated flotillins. (see also our answer to major point 2).

8) Western blot from the CHX experiment should be shown, at least in the supplements. Again, the standard deviation in this experiment is minimal, was this really an average of three independent experiments (and not three western on the same lysates)?

As asked, a representative western blot is now shown in Figure 3C in the current revised version of the manuscript.

As indicated in the legend to the figure already in the initial version of our manuscript: “**The results are the mean ± SEM of 6 to 8 independent experiments depending on the time point, and are expressed as the percentage of AXL level at T0”. We wish to reassure Reviewer#1 that the results are really based on western blots performed on different lysates obtained in independent experiments. We can show the Reviewer these data obtained from independent experiments if necessary.

9) All conclusions are derived from one single cells MCF10a. NMuMG cells are shown at the beginning but not used for the rest of the paper. Anyway, if this wants to be a cancer research paper, then cancer cells needs to be used.

It is true that we did not use a cancer cell line at the beginning of the paper because, as expected, flotillin knock-down did not allow to revert the mesenchymal phenotype of MDA-MB-231 cells toward an epithelial one. If this had been obtained, we would have used these cells from the beginning of the paper. The lack of reversion of the mesenchymal phenotype after flotillin knock-down was expected. Indeed, the EMT process is multifactorial and the decrease of flotillins alone is obviously not sufficient to reverse it in a tumor cell line bearing multiple oncogenic mutations. Moreover, because we wanted to assess whether flotillin upregulation is sufficient in normal cells to acquire the properties of tumor cells and particularly to induce EMT, we used human MCF10A and murine NMuMG cells, two non-tumoral epithelial cell lines. Until now, the studies carried out on the effects of flotillin overexpression have used tumor cells that already harbor pro-oncogenic perturbations, preventing to show that flotillin overexpression alone activates oncogenic processes leading to EMT, and to identify the downstream mechanisms.

Nevertheless, we have used the MDA-MB-231 cell line in several experiments to analyze: i) AXL distribution and internalization following the knock-down of flotillins (Figures 4 and S5), ii) SphK2 and flotillin 2 co-localization and co-endocytosis (Figures 5A and D and S7A), iii) the impact of SphK2 inhibition on AXL expression level distribution and endocytosis (Figure 6), iv) SphK2 expression level upon flotillin knock-down (Figure S7E) and AXL expression level upon SphK1 inhibition (Figure S7F). With these experiments performed in MDA-MB-231 cells, we showed that AXL and SphK2 colocalize in flotillin-positive late endosomes and are co-endocytosed from the plasma membrane containing flotillin-rich domains to flotillin-positive vesicles. We also demonstrated that flotillins and SphK2 control the rate of AXL endocytosis and its stabilization.

We recently obtained additional data with HS578T cells, another triple negative breast cancer cell line, on the co-trafficking of AXL and flotillins as well as the co-trafficking of SphK2 and flotillins (Additional Figure 3, this data could be added in the fully revised version of our manuscript).

In addition, we observed that inhibiting SphK2 also decreased the level of AXL in HS578T cells. This data could be added in the revised version of the manuscript (see data in our answer to Point #1 from Reviewer #3).

• ADDITIONAL FIGURE 3 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST*

Additional figure 3: Co-trafficking of SphK2 and AXL with flotillin 1 in intracellular vesicles in HS578T cells. HS578T cells co-expressing Flot1-mCherry with SphK2-GFP (A) or AXL-GFP (B) were monitored by time lapse spinning disk confocal video-microscopy. On the right of each panel are shown still images at different time points (min) of the boxed area. The colored arrows allow following three distinct vesicles that are positive for Flot1-mCherry and Sphk2-GFP, or AXL-GFP.

10) The methods section contains inconsistent data about patients' samples (9 are indicated, but the Figure S4 features 37). Then, where those other 527 come from?

We corrected the manuscript and added all characteristics regarding the 37 patients in the “Supplementary information” section.

The 527 patients are from another cohort and were used for the analysis of the correlation between the mRNA levels of FLOT1 and p63 in breast cancer biopsies from 527 patients (Figure 2I). This cohort was described in our previous study (Planchon et al. J Cell Science 2018, https://doi.org/10.1242/jcs.218925). In the revised version of our manuscript, we now refer to this previous article in the “Result” section and in the legend to figure 2I to explain the origin and characteristics of this cohort.

11) Some figures do not match with the legends or with the description in the text. It has not been easy to review this paper.

We apologize as we indeed made one mistake in figure 2 that was inserted into the manuscript and that was actually figure S2 (that appeared twice). However, the correct figure 2 was uploaded on the website of Review Commons and BioRxiv. Regarding the comments made in point 4, it seems that Reviewer #1 examined the correct figure 2 that was uploaded and that matches the legend indicated in the manuscript.

Besides this mistake, we do not see any other mismatch between figures and legends.

Reviewer #1 (Significance (Required)):

I am a cancer biologist working on EMT.

**Referee Cross-commenting** I have nothing to comment on other's reviews.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Genest and co-authors present in this paper new fascinating evidence on how intracellular trafficking can modulate oncogenic signalling.

First of all, they show how overexpression of Flotillin1 and 2 in non-cancerous breast lines can induce a strong reprogramming towards an EMT phenotype. They analyse mRNA and protein expression, intracellular distribution of activated proteins, cell phenotypes to demonstrate a strong activation of oncogenic signalling pathways. They then identify AXL as a key player in this process and show how this protein is stabilised upon Flotillin expression. The authors use an amazing variety of approaches to study the endocytosis and the trafficking of endogenous, GFP-tagged, Halo-tagged and Myc-tagged AXL in different cell lines and their data are strong and very convincing, the images are of very high quality and the analysis rigorous. Their data strongly support the hypothesis that high Flotillin levels triggers AXL endocytosis and accumulation in non-degradative late endosomes where signalling remains active. The authors then show how SphK2 has a key role in AXL stabilisation, it colocalises with Flotillin, AXL and CD63 and its activity (which they block by using inhibitors or siRNA) is necessary for flotillin-induced AXL stabilisation and EMT induction. The paper is extremely well written, the data flow logically and they are appropriately presented and analysed. I don't have any major comment and I believe the paper is suitable for publication.

We thank the Reviewer for the positive appreciation on our manuscript.

I have only some minor comments/questions: 1) did the authors try to colocalise AXL with endogenous Flotillin in MDA-MB-231 cells? They could use the antibodies used in Fig S1B. Of note, the authors have shown it in luminal tumours in Fig S4C.

We performed co-immunofuorescence experiments to detect endogenous AXL with endogenous Flotillin in MDA-MB-231 cells. As shown below (Additional Figure 4), we could find AXL and Flotillin being present in the same intracellular endosomes. Images could be added in the revised version of the manuscript.

ADDITIONAL FIGURE 4 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST

Additional figure 4: Endogenous AXL and flotillin 1 are found in the same in intracellular vesicles in MDA-MB-231 cells. MDA-MB-231 cells were fixed and labelled with relevant antibodies directed against Flotillin1 and AXL. Scale bar in the main image : 10 µm. Scale bars in the magnified images from the boxed area : 1 µm. Arrows indicate flotillin and AXL positives vesicles

2) In Fig6G, it appears that AXL-Flotillin colocalization is lost upon SphK2 inhibition. Is this the case? It could be that the correct lipids are necessary for the formation of Flotillin-positive internalisation domains and this could be very interesting and reinforce the model proposed in the paper.

In figure 6G, cells were not permeabilized. Thus, only AXL at the cell surface was labelled using an antibody against the extracellular domain of AXL. Because flotillin 2 is tagged with mCherry, this allowed its visualization revealing its localization both at the cell surface and intracellularly in the inset of the lower pane l of figure 6G.

After 6 hours of treatment using the opaganib inhibitor, we did not notice any major change in AXL-flotillin colocalization at the cell surface. Somehow, this is expected because blocking the generation of S1P is more likely to inhibit the invagination of flotillin-rich membrane microdomains rather than their formation.

3) I would remove the sentence on line 995-997 "to our knowledge this is the first report to describe ligand-independent AXL stabilization..." as the cells are not serum starved in all experiments and animal serum can contain variable amounts of the ligand GAS6.

We understand and agree with Reviewer #2, this sentence has been modified by “**To our knowledge this is the first report to describe AXL stabilization following its endocytosis”

Please note that the authors don't have to necessarily address comments 1-2, their paper is already very rich in convincing data.

Reviewer #2 (Significance (Required)):

AXL is a major oncogene that promotes EMT in a variety of tumour types. Understanding how its signalling can be triggered by endocytic pathways even in cells that are non-cancerous is very important and of high significance for the cancer field and the trafficking community.

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

This is an interesting and well written paper describing that upregulated flotillin promotes an endocytic pathway called upregulated flotillins-induced trafficking (UFIT) that mediates AXL endocytosis and allows its stabilization. Consequently, stabilized AXL in these flotillin-positive late endosomes enhances activation of oncogenic signaling pathways that promotes EMT. The authors suggest that Flotillin upregulation-induced AXL stabilization requires the activity of SphK2. However, this latter point is not supported by the data and further studies are needed to support this important conclusion.

**Major concerns:**

1. Most of the conclusions are based on effects of high concentrations (50 uM) of an ill-defined SphK2 inhibitor. The experiment described in Figure 6C-H need to be confirmed by downregulation of SphK2.

We understand that Reviewer #3 is concerned that in our experimental conditions, the effects we observed could be really explained by a specific inhibition of SphK2.

From the literature, among all the inhibitors described for SphK2, opaganib (ABC294640) is the most specific inhibitor available. It was shown to have no inhibitory effect on SphK1 up to 100 µM (French et al, J Pharmacol Experimental Exp Ther 2010; Neubauer HA and Pitson SM, The FEBS Journal 2013). In agreement, we found that PF543, the most specific SphK1 inhibitor, had no effect on AXL level (Figure S7F), unlike incubation with opaganib (Figure 6A and C), and that was confirmed in MCF10AF1F2 cells by the knock down of SphK2 with a specific siRNA (Figure 6B).

In the literature, depending on the cell lines, opaganib is used in vitro in the 10 to 60 µM range. Opaganib IC50 on recombinant SphK2 was established at 60 µM (French et al, J Pharmacol Experimental Exp Ther 2010). In our experiments, opaganib was used at a concentration of 50 µM, below the IC50 value, as previously done by Nichols’ group (Riento and al, PloS ONE, 2018). In most of our experiments (Figure 6, A, D, E-I, Figure S7D), opaganib was added for a maximum of 10 hours, which is shorter compared to what done in other studies (24-48 hours). Furthermore, it was shown that an opaganib concentration of 50 µM does not have any inhibitory effect in vitro on 20 protein kinases tested, including PKA, PKB, PKC, CDK, MAP-K, PDK1 and Src (French et al, J Pharmacol Experimental Exp Ther 2010).

In addition to inhibit SphK2, acting in a sphingosine-competitive manner, opaganib also was shown to act as an antagonist of estrogen receptor (ER), and inhibits ER-positive breast cancer tumor formation in vivo (Antoon JW et al, Endocrinology 2010). If Reviewer #3 is concerned about the possibility that the opaganib downstream effects we observed in our study might be explained by ER inhibition, we remind that we used cellular models that do not express ER. Indeed, the MDA-MB-231 cell line is a triple negative breast cancer cell line. MCF10A cells also do not express ER (Lane MA et al, Oncolgy Report, 1999,)** and our transcriptomic analysis (Table S1) did not reveal any increase in the expression of ER genes in MCF10AF1F2 cells in which flotillins are upregulated, thus eliminating a possible non-specific effect of opaganib in these cells.

In conclusion, we hope that these arguments help to convince Reviewer #3 that our experiments were performed in conditions where we carefully limited the possibility of opaganib off-target effects, on the basis of the currently available opaganib-related data from the literature.

We totally agree with Reviewer #3 that complementary experiments by downregulating SphK2 must be used. In agreement, we already downregulated SphK2 by siRNA in MCF10AF1F2 cells. This led to a significant decrease in AXL and ZEB1 expression. In the current revised version of the manuscript we have added data obtained with similar siRNA experiments performed in MDA-MB-231 cells (now Figure 6C). In agreement, we observed AXL and ZEB1 downregulation.

As shown below (Additional Figure 5) we recently obtained similar data in HS578T cells, showing that inhibiting SphK2 also affects AXL protein level in this triple negative breast cancer cell line (these data could be added in the manuscript).

ADDITIONAL FIGURE 5 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST

Additional figure 5: SphK2 inhibition decreases AXL level in HS578T cells. HS578T cells were incubated with opaganib (50µM, 10 hours) (A) or with siRNA Ctrl or siRNA SphK2 for 72 hours (B). Cell lysates were blotted with relevant antibodies against AXL, SphK2 and actin. The histograms show AXL level (normalized to actin) expressed as fold-increase compared with the control condition, and data are the mean ± SEM of 3 (A) and 4 (B) independent experiments.

Reviewer #3 also asks to use the siRNA approach on experiments shown in previous panels D-H (now panels E-I) of figure 6.

In complement to Figure 6D (now Figure 6E), experiments using a siRNA against SphK2 to show that “**AXL decrease upon SphK2 inhibition is not due to protein synthesis inhibition” are on-going and the obtained data could be added in the full revised version of our manuscript.

However, we are unfavorable to use a siRNA against SphK2, in addition to opaganib, in the experiments done to measure the effect of SphK2 inhibition on the rate of AXL internalization (previously in Figure 6E and F, now Figure 6F and G) and the level of AXL at the cell surface (previously in Figure 6G and H, now Figure 6H and I). Indeed, we carefully chose a short (4 hours) incubation with opaganib at the end of which the total cellular level of AXL was not yet decreased, allowing to measure unambiguously a defect in AXL endocytosis or a change in the level of AXL at the cell surface. We believe that it would be very difficult to achieve similar experiments using a siRNA against SphK2. It would require to determine the exact time after siRNA transfection leading to a sufficient SphK2 level reduction but in conditions where AXL level is still maintained. We think that due to the mosaic transfection efficiency, being able to precisely synchronize the effect of a siRNA at its beginning is impossible.

1. Does overexpression of SphK2 reverse the effects of the SphK2 inhibitor? In a similar manner, does overexpression of SphK2 enhance stabilization of AXL?

To answer the first question, it is not clear for us how to test whether SphK2 overexpression can reverse the effects of the SphK2 inhibitor because the ectopically expressed SphK2 would also be sensitive to the inhibitor. This would require to overexpress a SphK2 mutant that is catalytically active but insensitive to the inhibitor, and to our knowledge, such a mutant does not exist.

Regarding the second question, we are currently generating a retroviral DNA construct allowing to overexpress SphK2 homogeneously in the cell population. Then we will test whether it further increases AXL level through its stabilization. This will be tested in cells upregulated for flotillin. As we showed in Figure 6 A and D (previously Figure 6 A and C) that AXL level depends on SphK2 activity only in cells that overexpress flotillins, we anticipate that there will be no impact in a cell line with a moderate level of flotillin. Results could be added in the fully revised manuscript.

1. Although the authors suggest recruitment of SphK2 and formation of S1P in UFIT, there are no measurements of S1P. Also, there is no indication that SphK2 is activated despite the fact that ERK and AKT are activated in UFIT and are known to phosphorylate and activate SphK2. Is SphK2 that is recruited to flotillin phosphorylated?

To answer the first point raised by Reviewer#3, we recently performed, in collaboration with a lipidomic platform, a comparative analysis by quantitative mass-spectrometry of S1P levels between MCF10AmCh and MCF10AF1F2 cells. As we anticipated, the results show a 3,5-fold increase in S1P in MCF10AF1F2 cells compared with MCF10AmCh (Additional Figure 6). This data agrees with the fact that we found that the SphK2 catalytic activity is required for the UFIT pathway mediated AXL stabilization. This result is also in agreement with the study from the Nichols’ group which detect a decrease in S1P in cells in which flotillins were knocked out (Riento et al, PloS ONE, 2018). The results regarding the analysis of S1P level along with the complete methodology used will be added in the fully revised version of our manuscript.

ADDITIONAL FIGURE 6 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST

Additional figure 6: Upregulation of flotillins in MCF10A cells promotes an increase in the level of Sphingosine-1-phosphate. The level of sphingosine-1-phosphate was compared by quantitative mass-spectrometry analysis from three independent samples of MCF10AmCh and MCF10AF1F2 cells. The results are expressed in pmol equiv / 1 . 106 cells. The graph shows the value for each sample and the bar horizontal bars indicate the mean value for each condition.

Regarding the second point, we would like to clarify that we do not think that SphK2 interacts directly or indirectly with flotillins because SphK2 did not co-immunoprecipitate with flotillins (not shown). Thus, investigating by western blotting SphK2 phosphorylation status in flotillin immunoprecipitates is pointless. In theory, we could investigate the activity-related phosphorylation status of SphK2 associated with flotillin rich-membranes and endosomes. But this seems difficult to achieve because unfortunately, the only two commercially available antibodies against phosphorylated SphK2 are not described to work for immunofluorescence staining. One is against the Thr578 residue (https://www.abcam.com/sphk2-phospho-t578-antibody-ab215750.html), identified as phosphorylated downstream of ERK by Sarah Spiegel’s group (Hait et al, J Biol Chem, 2007). The second is designed to recognize specifically the phospho-Thr614 residue (https://www.abcam.com/sphk2-phospho-t614-antibody-ab111948.html), but this site has not been rigorously demonstrated to be phosphorylated downstream of AKT or ERK or to stimulate SphK2 activity. Thus, considering the lack of appropriate tools and considering that we already showed, using opaganib, that the catalytic activity of SphK2 is required for the UFIT pathway, we believe that investigating the phosphorylation status of SphK2 reflecting its activity in flotillin-positive vesicles will be complicated to achieve in a reasonable amount of time and we think that it will not bring a higher value to our present study.

To answer more broadly to the question “Is SphK2 recruited to flotillin phosphorylated?”, we anticipate that it could be the case at least on the Ser419 and Ser420 residues because Nakamura’s group demonstrated that the phosphorylation of these sites favors the nuclear export of SphK2 (Ding G et al, J Biol Chem, 2007). This group developed an antibody against these phospho-sites, potentially working by immunofluorescence. However, as it is unknown whether phosphorylation of these residues influences SphK2 activation status, we do not plan to perform immunofluorescence experiments with this tool (not available commercially) because the results would not address the Reviewer’s question.

1. It should be determined whether the optogenetic system used to induce flotillin oligomerization also induces recruitment and activation of SphK2.

As we already have all the available tools, optogenetic experiments will be performed to answer this point and the results could be added to the fully revised version of our manuscript.

As suggested, we plan to perform experiments in which exogenous S1P will be added to cells with a moderate flotillin expression level to check whether it could recapitulate the effect of flotillin upregulation on AXL expression. Results could be added to the fully revised version of the manuscript.

However, our current results on the localization and the involvement of SphK2 suggest that the generation of S1P involved in the UFIT pathway occurs at the plasma membrane and in late endosomes. Because the exogenous S1P that will be added in the culture medium will not go through the plasma membrane, we anticipate that it could be insufficient to mimic all the mechanisms of the UFIT pathway. Its effect will be limited to the plasma membrane. In addition, these mechanisms are very likely based on a local concentration of S1P in some microdomains (at the plasma membrane and in intracellular membranes) scaffolded by flotillins. It will be very difficult to mimic such local concentration of S1P just by adding S1P to the cells.

We agree that identifying the S1P receptors involved would be of valuable interest for a better characterization of the UFIT pathway. However, we think that this is beyond the scope of our present study. Among the five known S1P receptors, we do not know if any could be involved in membrane remodeling at the plasma membrane to promote endocytosis. To our knowledge, involvement of S1P receptors in endocytosis has never been reported. However, based on the work by Nakamura’s group (Kajimoto et al, Nat Comm, 2013 and Kajimoto et al, J Biol Chem, 2018), the S1P1 and S1P3 receptors are involved in membrane remodeling and cargo sorting from the outer membrane of late endosomes (where flotillins accumulate in our cell models). We could hypothesize that these receptors are influenced by flotillins and are involved in the UFIT pathway. But we think that testing this hypothesis would be the subject of a distinct study.

At the plasma membrane, we totally agree that the effect of S1P could be mediated, as suggested by De Camilli’s group (Shen et al, Nat Cell Biol 2014), by the formation of tubular endocytic structure rich in sphingosine after acute cholesterol extraction. Reciprocally, in our cell models, upregulated flotillins, thanks to their ability to bind to sphingosine (demonstrated by Nichols’ group (Riento et al, PloS ONE, 2018)) and to oligomerize, could create sphingosine-rich membrane regions.

1. There is a commercial antibody for endogenous SphK2 that can be used to validate and substantiate the data with GFP-SphK2. (F1000Res . 2016 Dec 6;5:2825. doi: 10.12688/f1000research.10336.2. eCollection 2016. Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence)

We thank Reviewer #3 for this suggestion and advice. Being able to detect the localization of endogenous SphK2 in late endosome would be valuable for our study. We already tried with no success with antibodies from Sigma and Cell Signaling Technology (not described to work in immunofluorescence experiments).

We will follow the advice from Reviewer #3 and test the anti-SphK2 antibody from ECM-Biosciences mentioned in the article by Neubauer and Pitson F1000 research, 2016. If we obtain interesting results, they will be included in the revised version of our manuscript.

However, in experiments using SphK2-GFP, we noticed that in live cells, the signal in late endosomes was completely lost after fixation using paraformaldehyde. Similarly, we also observed in live cells that NBD-Sphingosine, added in the culture medium, quickly accumulated in flotillin-positive late endosomes (Additional Figure 7, this data could be added in the fully revised version of the manuscript), but this accumulation was no longer detectable after fixation. Based on these observations, we believe that SphK2 recruitment to flotillin-positive late endosomes is highly labile probably because it mainly involves its interaction with sphingosine molecules that are enriched in these intracellular compartments. This is supported by our observation that addition of opaganib, characterized as a sphingosine competitive inhibitor, displaces SphK2-GFP from flotillin-positive late endosomes in live cells (Figure S7D). In addition, we showed that SphK2-Halo is more recruited in CD63-positive late endosomes in cells overexpressing flotillins (Figure 5E). This could be due to a higher concentration of sphingosine promoted by flotillins (that bind to sphingosine) accumulating in these compartments.

Thus, we will try the immunofluorescence staining of endogenous SphK2 using the recommended antibody, but it might be difficult to detect its presence in flotillin-rich late endosomes in fixed cells. The data could be added in the fully revised version of the manuscript.

ADDITIONAL FIGURE 7 CAN NOT BE ADDED BUT IS AVAILABLE UPON REQUEST

Additional figure 7: Visualization of NBD-sphingosine in flotillin-positive late endosomes. Live HS578T, MDA-MB-231 and MCF10AF1F2 cells expressing Flot1-mCherry were monitored by time lapse spinning disk confocal video-microscopy, 5 min after addition of fluorescent NBD-Sphingosine in the culture medium. On the right are shown still images corresponding to the boxed areas to illustrate the accumulation of NBD-sphingosine in virtually all flotillin-positive endosomes.

Reviewer #3 (Significance (Required)): This is an interesting paper. If the authors confirm the involvement of Sphk2 and mechanism of action of S1P, this would be an important contribution to the field.

Modifications done in the initial revised-version of our manuscript (at the time of the initial response). A full revised version will be provided after all the additional experiments asked by all the Reviewers will be achieved.

Revisions are highlighted in grey in the initial revised-version of the manuscript

1) Figure 1 has been modified and now includes results from a GSEA analysis as recommended by Reviewer #1. The texts of the corresponding legend and of the “Results” and “Methods” sections have been modified accordingly.

1) The Figure 2 version that was inserted in the manuscript was wrong because it was a copy of Figure S2. However, the correct Figure 2 was uploaded to the Review Commons website and accessible for the Reviewers. The correct Figure 2 is now inserted in the manuscript.

2) In the legend to panels C, E, F, J of Figure 2, the sentence: “The histograms show […] with control MCF10AmCh cells calculated from 4 independent experiments” was corrected to “The histograms show […] with control MCF10AmCh cells calculated from at least 4 independent experiments” because data shown in panel J are actually calculated from 8 independent experiments.

3) Figure 6 has been modified with the addition of panel C showing the effect of SphK2 downregulation by siRNA on AXL and ZEB1 level in MDA-MB-231 cells. The text has been modified accordingly.

4) In Figure 3 C, representative western blots have been added as asked by Reviewer #1.

5) In the Supplementary information section, the full clinicopathological characteristics of only 9 patients were indicated, whereas Figure S4 mentioned 37 patients. We corrected this mistake and now provide the characteristics of all patients.

6) In the sentence “Conversely, it induced ZEB 1 and 2 mRNA expression (Figures 1H and S1K) and ZEB1 protein expression (Figures 1I and S1L) (no anti-ZEB2 antibody is available)”, we removed “no anti-ZEB2 antibody is available”.

7) The sentence previously on line 995-997 "to our knowledge this is the first report to describe ligand-independent AXL stabilization..." has been modified to “**To our knowledge this is the first report to describe AXL stabilization following its endocytosis”

8) We are now referring to reference 18 (Planchon et al. J Cell Science, 2018) for the description of the cohort of 527 patients with breast cancer because this was missing.

.

Again not Lightning channels, and then one needs two transactions just to create the channel -- and then it only works on one side?

Maybe this is a good idea if user A wants to send a bunch of payments to user B in that single channel that doesn't interoperate with anyone else, if it's really non-interactive. But I can't imagine concrete relevant use cases.

Generally requiring interactivity is an issue when the receiving end is a user that doesn't have an online wallet so Lightning doesn't work for them on that case -- it's hard to imagine many cases of services opening these wasteful channels to each user, or friends opening this kind of channel between themselves just to send a few bucks.

So the idea here is to actually increase blockchain space usage while simultaneously adding the requirement for the users who are withdrawing to have a wallet that understand OP_CTV and the way these transactions are batched? Sounds like a bad idea.

Even if I'm wrong on the specifics the general argument sounds weak: instead of making a single big transaction that will cost relatively less (or pay relatively more on fees for that) the idea is that exchanges will prefer to make a larger number of smaller transactions to reduce the immediate cost but increase the cost in the future? And then most users will have to wait anyway.

Why not make a single big transaction and just wait until it clears from the mempool?

Even if I'm wrong twice on the above and this is actually a good idea, this sounds like a super niche and debatable use case and it's hard to imagine anyone supporting it.

.

Dissonance of juxtaposed ideas, and particularly those just at the edge of chaos, can be some of the most fruitful places for learning.

Attempting to resolve these frictions can generate new knowledge.

This is what commonplace books are meant to do. Record this knowledge, allow one to juxtapose, and to think and write into new spaces.

It's also important to look more closely at things that don't cause dissonance. Is it general wisdom that makes them true or seem true? Question the assumptions underneath them. Where do they come from? Why do they seem comfortable? How could one make them uncomfortable. Questioning assumptions can lead to new pathways.

An example of this is the questioning the final assumption of Euclid (the "ugly" one) which led mathematicians into different geometry systems.

It's not really clear what the images for #2 and #2 in the list add to what is in the text, but they do make the page more visually appealing than just text alone. Try to select images that actually ADD TO the text, but at least select images that go with the text.

• about : secret (source|sauce) MindGraph Opidox
• benefits of using HTML as a media format ubiquity UI
• as a nice side effect magically becomes usable from the browser
• thing of the future
• Are we there yet?
• I've gone with HTML as Hypermedia before I encountered your talk.
• Built it into a Universal Lively Data format with MindGraph by 2018.
• Gone WebNative with Fission codes.
• That future is here:
• forging the
• Inter Planetary,
• Inter Personal IndyVerse of Web 3 HyperMedia as HTML
• with IndieHub https://social.coop/@indiehub
• https://social.coop/@indiehub

It's hard to pinpoint what ideas the author has the differentiates from other scholars that have looked into these writings. From what I am understanding, the author sees that Josephus is trying to use Abrahams progression as a parallel to Greek philosophers, making him a more relatable revered character to the Romans who would read about him. I am not sure why any other scholars would not make this same connection, I think the author is saying that other scholars are saying that Josephus is just generally describing Abraham in a Hellenistic approach.

It's amazing how they avoid interpreting the text accurately. They think these various numbers were just appended onto the text when the previous predictions failed. Their commentary is quite useless, because if they took the text seriously they would see that the numbers are referring to different events.

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

So, what's the conclusion from this review of this Wiki article? If you want to get a better understanding of something biblical, do not read Wikipedia. It is overtly anti-Christian and will deceive and mislead the reader to achieve its end. Go to one of the good, reputed bible commentaries. https://biblehub.com/ is a great place to start.

I haven't addressed damaging claims in other Wikipedia articles, such as:

• Darius the Mede was fictitious
• Belshazzar was fictitious

These probably come up in other Wikipedia articles, though they are easily answerable.

This statement is misleading and is worded as a deliberate attack on the authenticity of Daniel. It sounds like the Hebrew people EXCLUDED Daniel from their original canon of Scripture.

Note that the text mentions not 'the canon', but 'the canon OF THE PROPHETS'. That is just one section of the Hebrew canon. It's just like we divide the Old Testament into the Pentateuch, Major Prophets, Minor Prophets, etc. It's just categories, but it's all Scripture.

Daniel was not EXCLUDED from the Hebrew canon in any sense. Using that word 'excluded', as if there was an active renunciation of the book of Daniel by the Hebrews, is not only factually false, but it is designed to lead the reader against accepting Daniel as legitimate.

The truth is that Daniel was in the Hebrew canon, but just classified in the section called the Ketuvim ('Writings'), along with Ezra, Nehemiah and 10 other books of the Old Testament (see https://en.wikipedia.org/wiki/Ketuvim). Daniel was no doubt classified there because so much of his book is historical narrative (like Ezra and Nehemiah), not primarily prophetic like Isaiah, Ezekiel, etc. Daniel was in the canon, and was not excluded from it at all.

Again, it is clear this Wiki author has an agenda and will deceptively present his ideas accordingly.

Note that the author claims they're citing "evidence of the book's date", in a general sense. The unsuspecting reader would assume that what they are about to read is a good summary of all the data pertaining to the book's date. In reality, however, the author is only going to cite (loose) evidence of the date that THEY want to support, which is the 2nd century BCE. Why do they want this date, and what other dates might we consider?

They want (need) this date because then Daniel's amazingly accurate prophecies are not prophecies at all, and were actually just someone writing known history well after the events, and PRETENDING it was originally a prophecy. Thus this Wiki author avoids the notion of divine prophecy in Daniel and attempts to refute the inspiration of Scripture.

This would put the book in the inter-testimental period (that period of silence between the close of the Old Testament and Jesus' birth). But this is in contrast with the actual text of the book, which says it was penned in the 6th century BCE.

Normally, the actual text of the book would be considered primary evidence of when it was penned, but in this case it's ignored entirely because that would mean accepting that divine prophecy is real. Their materialistic worldview cannot tolerate a Divine essence. In the words of evolutionary Professor Richard Lewontin, "Materialism is absolute, for we cannot allow a Divine Foot in the door."

First—there are companies that specialize in negotiating ransomware attack responses. Secondly, it is entirely possible that a company may start negotiations to stave off harsher ransoms while figuring out if their internal processes can safely recover systems without paying the ransome.

.

It's sad that this seemed not shocking because people of colour deserve more recognition for their accomplishments and achievements. I just did a google search, and I'm glad to see that there's more music, news articals, and a magazine called "Black Girls Magazine" rather than pornographic media... but google can still do better.

We all know this smile. Foster Wallace has the talent of accurately pin pointing, and of questioning, universal experiences which, in my view, people never stop to think about. I have been on the receiving end of numerous professional smiles, and all I have ever done was to professionally smile in return, without thinking twice about what I was doing. It's just one of those things that are so engrained in human behavior that stopping to think about it may seem like a pointless task for most people. But it's not!

I can feel this on another level because this reminds me of the whole not judging a a book by a cover mainly because by personal experience because I do this a lot when I read the first pages and I don't like it I immediately think its bad you know then a lot of people tells me its good and that I judged too quick

@MMScotofGlasgow, Hopefully it's not too late...

Francis Yates discusses Petrus Ramus as an educational reformer in Chapter 10 and onward in The Art of Memory. There she outlines Ramus' crusade against images (based in part on the admonition from 4 Deuteronomy about graven images) and on their prurient use (sex, violence, etc.) which were meant to make things more memorable. Ramism caught on in the late 1500's and essentially removed memory by the root from the subject of rhetoric of which it had been an integral part. Ramus felt that structure and rote memorization would suffice in its stead. As a result the method of loci decreased in prominence in schools and disappeared from the scene based on educational reform which was primarily pushed by Huguenot/Protestants. I've not read anywhere that the practice was ever banned, it just fell out of fashion due to these reforms.

I'm sure it didn't help that printed books became ever cheaper during/after this time and so the prior need to memorize for those reasons wasn't helped either.

I'm sure another confounding factor was Erasmus' Copia: Foundations of the Abundant Style (1512) which dramatically popularized the keeping and use of commonplace books by the learned and literate. These became a regular place in which people collected and kept their thoughts and ideas rather than memorizing them as they may have done in the past.

Place names and songlines together reminds me of a great BBC segment "Disappearing Welsh Names" I saw recently: https://www.youtube.com/watch?v=yLQ6XlG0MQ4

It highlights by analogy the value of indigenous culture, knowledge, and creativity which the survival of songlines also provides us with. (It also saddens me because it starkly reminds me of all the knowledge and languages we've lost already.)

I've been learning Welsh since the pandemic started and just a few simple words of Welsh has given me a far greater appreciation of places in the UK and what they mean. It's helped not only to expand my vocabulary, but increased my creativity in creating local songlines. It's also made it much easier to learn to say and remember the town of Llanfairpwllgwyngyllgogerychwyrndrobwllllantysiliogogogoch.

It also uncovers quirks of place names like Breedon on the Hill which translates from Brythonic, Saxon, and Modern English to "Hill Hill on the Hill" and crystalizes, as if in amber, the fact that Brythonic, Saxon, and English speakers all conjoined for a time on a hill in England. Similarly there's also Barnack Hills in England which translates from old Celtic (barr), Scottish Gaelic (cnoc) and English as "flat topped hill hill hills". It's almost hillarious.

She keeps saying that it's just pretend so maybe she's accepting the fact that it might never happen

She wants him to still remember her even if it's not real. She wants him to hold on to those memories like she is. It seems that this might not have been genuine as she wants it to be.

This could mean that a lot of the things within her prior relationships have been pretend

she wants him to remember fondly with memories of them together, something that goes beyond physical love and attraction

Why does she care about him remembering her so bad? "Even if it's just pretend" implies that she only wants him to think about her even if it is the little things

She wants him to think about her and remember all of the good times that they had, even if it isn't in real life. But how is him seeing her in her dreams going to benefit her?

Saying that the relationship will be crazy and fun because she says it would be in his "wildest dreams"

This shows that it's not just African American people, but larger racial groups also being discriminated against.

The last line talks about how it's hard to see the whole picture of a thing when you're too close to it. It sounds like the spider catches the moth by camouflaging itself against the backdrop of the flower. Does the spider know that or does it just know that it can get some food if it hangs out in this spot? It reminds me of this trashy tv show called "Monster Bug Wars" I loved as a kid. The show was just edited video and commentary of some ants subduing and killing a wasp or something like that but the producers added these great monster noises. The show, like this poem, took something we would normally not notice and hyper focused on it. If there are designs governing this spider that it cannot see, there are probably designs governing us that we do not notice.

Her saying pretend again means that she knows that they will probably not see each other again even if its just in your...

She is saying that even if its not real she still wants him to at least pretend to see her again some day.

Even though he probably would have to pretend that they'll see each other again in order for him to say that he will, she still wants to hear him say that he'll see her again (she's searching and wants that false hope).

This to make me think that she is crazy for this guy and she would do anything to make him stay with her even if its in his dreams

Maybe how them breaking up is something that would only happen in a crazy dream. This sounds a little menacing, because it could mean they will never leave each other.

She wants him to tell her that he will come and see her, and if he can't see her in person than to jut think about her or maybe even dream about her. But as long as he remembers and see's her.

Even if seeing her again is some unrealistic fantasy that he has, she still wants him to maybe "give her false hope" that it can happen. The fact that she sighs and the end of this line can mean that she's already painfully aware that the two of them can never go back to the way they were when they were happy together.

No matter how impossible their relationship is she wants him to still hold on, despite the circumstances that try to keep them apart.

even if it is not in real life that he sees her, she is okay with it being pretend

Wildest dreams could be referring to like the least chance of something happening or a wish that will never come true in that sort of aspect.

The Web is not ...

Little confused by what this MORE is supposed to mean here--I assume it's about corporations failing to do more than they ought to, maybe there's a way to indicate that like "To do MORE." (just a suggestion though)

What is the best word/verb for this sort of pseudo-searching via word or idea association for generating new ideas?

I've used the related phrase combinatorial thought before, but he's also using the idea of artificial intelligence to search/find and juxtapose these ideas.

THIS IS SO GOOD! Women characters so often written to be either angels, demons, or the "quirky" relatable girl. It's so rare to find characters who get to be real and act like normal people. These characters are not completely revolutionary or even perfect, they're just enough and essentially catching up with their male counterparts who have been allowed to be so for so long.

I absolutely agree with this. It's very sad to me how the romance genre is so judged and shameful. It all goes back to how no literature is less valid than another. Just because it's not academic or some kind of classic, impressive content, doesn't mean it's a shameful story. And there absolutely is misogyny involved in the judgement of the romance genre. It's seen as a genre for silly preteens, or middle aged women and therefore seen as less valid. I saw a tiktok once of a girl talking to her mother, who has multiple degrees and I am pretty sure is a doctor. And this woman's favorite genre is romance. I think that just kind of speaks to the fact that the romance genre isn't just this uneducated, stupid genre.

I really do not like this statement. It is so degrading to Rooney's writing, which I do not believe deserves this level of criticism. I just think this feeling of superiority that people have in literature like this is baffling and really off putting. It's just like I've said before, just because one reviewer doesn't like it, doesn't make it bad. I think there's a sort of beauty in writing that isn't perfectly elegant, honestly.

I think this is an interesting comment because I feel that both Marianne and Connell sabotage their own happiness in a way. I don't think Connell is this inherently "good guy" who has just been pining away for Marianne the whole book, and I also feel that Marianne isn’t just this sad girl who has been waiting for Connell to sweep her off her feet. Both characters have good things and bad things about their personalities that occasionally hinder their ability to be fully committed to one another. I mentioned in my last comment that both Marianne and Connell make it known that they love each other but can't seem to really pull together. I think the tendencies they both have to sabotage their happiness and the lack of good communication is what keeps them from becoming a full-fledged couple throughout the whole book.

complete dismissal

This strikes me as that stereotype of "oh you're not white? what country were you born in then?" even though the character is literally born in America. It's just this insane thought that someone of color must obviously be born outside the country? I don't understand this logic, because it's not like America isn't a diverse country. Never mind that the author clearly said the character was born in America.

It's really interesting that these two genres are considered so similar in content but the classification is so different. Just small differences, probably hardly noticeable to the untrained eye, but they make or break which genre the book will be a part of. This has always been interesting to me, actually- how a book is classified in a genre vs how an author might want it classified.

I really really like this paragraph. I personally enjoy reading and learning about current world issues and other cultures. When people branch out and read things outside their comfort zone, there is so much room for growth. Like mentioned earlier in this essay, "It’s not entirely surprising that white critics gravitate toward writers in whom they see themselves, and who write about topics and lead the kinds of lives they are familiar with," by attempting to engage with material you aren't familiar with, you are actively fighting the status quo and stereotypes that surround different types of media. There are always more sides to a story, learning from those other perspectives doesn't just benefit you. Issues surrounding race are present in all areas of life like climate change and economic injustice, if you aren't educating yourself about those issues, then you could be a part of the problem.

This is a trend seen a lot in media nowadays. Often people of color are seen as outsiders and this can translate into any kind of work that they do. I mentioned previously in one of my comments that people of color and their work are a lot of times seen as outside the "mainstream" of things. Even though in the last few years society has made a lot of progress, we still have a long way to go before we can normalize pieces of work from people of color. There are many people of color that are born in the United States, so it's time to stop viewing their work as exotic and to simply just normalize their work. Even though this essay highlights the issue of race withing literature, these issues expand to every area of life like in the workplace, at school, or even in places of business.

crucial bit here — time is not as helpful a factor in qph as it is elsewhere

This is also roughly the way that zettelkasten and commonplace books have worked for centuries.

This passage is an excellent reason for "why" to keep one, which few sources I've seen bother to mention.

She is saying boys only wants girls that makes them go through a lot of bad mental states just to get with her. They probably don't like "easy" girls.

This to me is her saying that men only want to make woman lives hard by giving them a hard time but I think that is wrong just because you have gotten played doesn't mean that all men play.

The relationship might be a successful one, that will last through marriage. It's a possibility she thinks will happen even though she is just toying with the man.

Guys don't want a good relationship, she's trying to say that they all just cheat in a type of way or leave her and then they have to face her.

From the begining i feel like its a sort of survival place, the writer talks about streets and how its like- from the word streets i feel its talks about the difficulty of surviving the especially for those referred as negroes.

It would be good for you to explain this concept (perhaps through reference to some examples) - just so that it's clear that you're not simply telling me back what I told you. Here are a few examples I have (although you might find better): In-between Narratives: http://folksonomy.co/?keyword=28620 Eugène Atget 'Boulevard de Strasbourg 1912: http://folksonomy.co/?permalink=96 Some obvious film examples are: The Hangover (2009), Sunset Boulevard (1950)

This is not crucial to change now, but it just occurred to me that maybe the theme should go in the UI rather than server? Since it’s an aesthetic choice

Did blogging die off because the tools changed? Everyone had their own space on the internet and the internet itself was the medium which opened up the conversation. I could use WordPress while someone else might have been on Blogger, Moveable Type, Live Journal, TypePad, or something they made in HTML themselves.

Now it's all siloed off into tinier spaces where content is trapped for eyeballs and engagement and there's not nearly as much space for expression. Some of the conversation is broken up into 280 character expressions on Twitter, some on Instagram, and now people are aggregating content inside Substack. Substack at least has a feed I can subscribe to and a free form box to add a reply.

I appreciate Jeff's comment about the flywheel of social media. We're definitely going to need something like that to help power the resurgence of the blogosphere. I also like to think of it in the framing of "thought spaces" where the idea of a blog is to give yourself enough space to form a coherent idea and make an actual argument. Doing that is much harder to do on a microblog where the responses are also similarly limited. It just feels so rude to post 250 words in reply to a sentence or two that probably needed more space to express itself too.

I suspect that if we want a real resurgence of thought and discourse online, we're going to need some new tools to do it. As Friedrich Nietzsche famously conceded to his friend Heinrich Köselitz “You are right — our writing tools take part in the forming of our thoughts.”

It would help if we could get back to the bare metal of the internet in which to freely operate again. Substack at least feels close to that, though it could be much better.

Can we have a conversational medium that isn't constrained by a handful of corporate silos that don't allow conversation across boundaries? Can we improve the problems of context collapse we're seeing in social media?

I'd like to think that some of the building blocks the IndieWeb movement has built might help guide the way. I love their idea of Webmention notifications that allow one site to mention another regardless of the platforms on which they're built. Their Micropub posting tools abstract away the writing and posting experience to allow you to pick and choose your favorite editor. They've got multiple social reader tools to let you follow the people and content you're interested in and reply to things directly in the reader. I presented a small proof of concept at a recent education conference, for those who'd like to see what that experience looks like today.

Perhaps if more platforms opened up to these ideas and tools, we might be able to return, but with a lot more freedom and flexibility than we had in the nostalgic blogosphere?

Yet, we'll still be facing the human work of interacting and working together. There are now several magnitudes of order more people online than there were in the privileged days of the blogosphere. We're still going to need to solve for that. Perhaps if everyone reads and writes from their own home on the web, they're less likely to desecrate their neighbor's blog because it sticks to their own identity?

There's lots of work to be done certainly, but perhaps we'll get there by expanding things, opening them up, and giving ourselves some more space to communicate?

he feels bad for eating them but sounds like that he doesn't regret it, it's a difference between feeling bad and regret

In the original paragraph I merely state how I looked for distractions to prevent me from starting my work, while in the revised one, I discuss what I am feeling as I procrastinate. Rather than just saying I am procrastinating, I show how this moment allows me to connect with the most present version of myself. By paying more attention to myself, I am not focusing on other distractions. I also used vivid language when detailing my surroundings, as I compare my attention of the birds to that of an ornithologist.

I added more vivid details to this paragraph as before the details did not further the main idea of my essay. Before, I just described what I was seeing around me, without responding to it. I tried making the stimuli more meaningful as I linked the yellow caution tape to Covid-19 and how it changed my college experience, ironically as I write in a college library. The yellow tape stimulated ideas about what I was missing out on, in terms of the college experience, which I later address in the essay. By mentioning this, my final paragraph is more effective as it is linked to more paragraphs in the essay.

Rather than just stating that I feel alone, which is what I did in the original paragraph, here I explore this feeling. At the time of writing this, I did not truly know what I was going to say. However, taking another look at it after freewriting about what I was feeling, I gained insight on what I was going to write. Instead of saying I am learning to be comfortable with being alone, I discuss how I am able to connect with an authentic version of myself, as I use being alone as a tool to access this part of me. These additions make the paragraph far more powerful because I completely explore the idea which makes it more effective.

In the old paragraph, I just state I am happy with this reflection, but don’t go on to explain why. In this version, I present two possible explanations as to why. I show how I have gained respect for myself by being alone and explore the feeling of solitude. This addition makes the paragraph much more powerful as earlier I mention how I struggled with being alone and would distract my brain from being present with myself. Additionally, towards the end of the paragraph I write towards the complexity, by exploring why I hated being alone. These two additions, which are polar opposites, help show where I started in the journey of solitude to where I end up now.

it's crazy to think about just how much everything effects one another

It's hard to suggest that scientific publishing move under the ultimate control of an individual.

In a similar way we started using the internet (and HTML) to mimic the user interfaces of CDs and DVDs. We still use HTML to create faux book interfaces for magazines, complete with page flip animations (although thankfully these are less common than they used to be).

Maybe she is getting back with an old boyfriend? Or maybe he just stopped talking to her and suddenly wants back in. But the way she says "since I have even heard from you" makes me think that she is questioning herself

Did he ghost her and suddenly see that she's doing good now and just popped back into her life.

This suggests that the Nabataeans's religion was heavily based on the sky. It's interesting then that the statues were just stone blocks. Perhaps it is because they thought it was futile to accurately portray the Divine, similar to modern religions today?

I love a good asyndeton that turns into a polysyndeton. Good stuff, Ginsburg. These devices make the people he is describing sound so incredibly active, like they are just constantly going, doing, and taking up space in this world. Even making their mark in a way, whether it’s in silly, drunken stupor or intentional, meaningful purpose.

I really think it's something deeper than just wanting attention because you shouldn't want to keep going back to that...

I think that the city is getting more polluted and the tree just can't take it anymore so it's getting weaker. It can't keep helping the city with it's pollution.

I used to get frustrated at trying to come up with a good topic sentence. I always felt like it wasn’t strong enough or blatant enough. I found that just writing my entire paragraph without focusing on what the topic sentence will be made it so much simpler for me because I either ended up writing one within that paragraph without even realizing it or, re reading the paragraph to myself that I just wrote made me think of a strong one.

It's so important to keep in mind that the tools are to be used to drive the learning and not just used for the tool's sake.

I like this idea since would eliminate some of the cost that cause many Americans to not seek medical health until it's life threatening and give people warning signs just in case something happens so they can get any problems early. Jessica F. -B

"Just pretend, Just pretend" -----> She's telling the boy that she could care less if it was not serious, she wants to be forever in the boy's memories.

She wants to be in his mind for eternity even if it was just for kicks and giggles. She still wants some kind of sign that he loves her as much as he loves him.

The girl wants to see him in his dreams but everyone knows that dreams are your imagination

She wants him to see her in reality or even if his dreams that he would remember her.

So Nas recognizes the circular motion that not just he but also his peers and neighbors are in.

Though it's a cycle, it's rooted in a complex system meant to sabotaged Black Americans. It's hard to find a way out.

It very difficult to figure out whether an individual is in genuine need of help or not.

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

We would like to thank our reviewers for their critical reading and constructive comments. We have addressed all of their points and have included below a detailed reference to the changes we made accordingly. We have also added an additional supplemental figure.

Reviewer #1 :

**Major comments:**

1. The authors highlight in their conclusion that the new Python library has the potential to accelerate and expand microscopy development. I agree with this statement since classes and methods do not need to be written in Python from scratch anymore. However, I would recommend that the authors include in their conclusion the value of the library for reproducibility if the final python acquisition code is shared along with publications. Nowadays, scientists frequently write in their publications that LabView or a specific commercial scope's acquisition software was used without any acquisition code. Python-Microscope would have the potential to change this trend, and the authors need to stress this aspect and its value for reproducibility in science accordingly. This is a good point. We have added the following to the discussion section.

“A further advantage of the approach provided by Microscope is in increasing reproducibility in science. Scientists frequently write in their publications that LabView or a specific commercial scope's acquisition software was used without any specific acquisition settings, code or macros to assist with reproduction. This is especially critical in complex experimental setups where specifics of acquisition are particularly important. Microscope has the potential to change this trend, allowing authors to freely publish simple code demonstrating exactly how their control and acquisition operates. Additionally, the defined device interfaces allow such code to be ported to other specific hardware with minimal changes.”

The authors need to provide a more comprehensive overview of the currently used data acquisition strategies in their introduction. Currently, they highlight the acquisition software provided by vendors for data acquisition (mainly used by life scientists and not necessary scope builders/developers), Micro-Manager (mainly used by life scientists; currently also restricted to wide-field systems), and LabView (for advanced microscope systems; used by advanced developers).

However, most advanced microscope builders use MatLab (Chmyrov et al. Nature Methods (2013) - https://doi.org/10.1038/nmeth.2556, Ta et al. Nature Communications (2015) - https://doi.org/10.1038/ncomms8977 , etc.), Python (York et al. Nature Methods (2013) - https://doi.org/10.1038/nmeth.2687, etc.), and LabView to write their acquisition software. Since the manuscript focused on advanced microscopes, the authors need to position their library with respect to Matlab and Python's current use as well.

We thank the reviewer for pointing out the omission of Matlab control solutions and extending the references to other Python based approaches. We have also added a reference to the Pycro-Manager framework for Micro-Manager which has been published since our original submission.

We have added Matlab to the LabVIEW generalised control software section which now reads:

“custom control software often in LabVIEW or Matlab, both proprietary software. LabVIEW offers a visual programming environment that is commonly used for building instruments in the physical sciences, whereas Matlab is a programming platform with a focus on numeric computing.”

And extended the description sections in the introduction with the following paragraphs and references:

“Matlab is a numerical focused programming environment that scientists are often familiar with for data processing. It has frequently been used for microscopy, leveraging a number of available Matlab sub packages to provide GUI’s and easy access to complex data processing steps. The use of Matlab for microscope control is common in the field but the actual code is rarely shared and often custom to a single microscope setup and associated to image reconstruction (Chmyrov et al., 2013, Ta et al., 2015). Exceptions are ScanImage for the control of laser scanning microscopes (Pologruto et al., 2003), and Matlab Instrument Control (MIC) for the control of individual microscope components (Pallikkuth et al., 2018). Matlab provides a textual programming language simplifying code sharing and version control, however, Matlab is proprietary closed source software and the general requirement of many extensions significantly adds to the cost of implementing many systems.”

“There is currently an increasing number of software options for microscope control in Python, many of which are in the form of custom scripts specific to a microscope (Alvelid and Testa, 2019, York et al., 2013) but some provide a fully integrated microscope control environments, namely PYME https://www.python-microscopy.org/ for SMLM and ACQ4 (Campagnola et al., 2014) for electrophysiology. While this code is freely available and can be modified, their design around a specific setup, technique, or environment reduces its potential for code reuse in other projects.”

The authors need to give (a) software provided by vendors, (b) LabView, and (c) Micro-Manager, more credit.

(a) Several microscope vendors (e.g., Abberior Instruments - https://imspectordocs.readthedocs.io/en/latest/specpy.html ) allow their scopes can be externally controlled to enable the execution of customer-driven acquisition strategies which the vendor's acquisition software itself might not have implemented with. The authors might want to include that scope vendors aim for more customer modifiable acquisition software.

The reviewer makes a good point, especially in the fact that a number of microscope vendors provide Python interfaces for their systems. We have added the following text:

Several microscope vendors, such as Abberior Instruments and Zeiss, provide Python interfaces to enable instrument control from Python. These are all very useful additions to proprietary systems, however they have a fundamental draw back that each manufacture produces their own abstractions meaning code from one system is not compatible with another. Although these interfaces leverage the substantial Python infrastructure they are not generalisable and hence fail to enhance portability or reproducibility.

The fact that these companies are providing Python interfaces to their instruments indicates the general interest of the community in Python as a programming language to extend hardware capabilities. This demonstrates the potential benefit of an entirely Python based interface to a wide range of hardware.

(b) The authors criticize that LabView code can be hard to understand, reproduce and maintain. However, similar to writing good code in general, there are best practice strategies for writing good LabView code to ensure scalability, readability, and maintainability available as well (https://learn.ni.com/learning-paths/labview-core-3-2016-english ). The primary problem might lie more on the side of lousy coding practice than on LabView's side to perform appropriately.

This is a fair point and we have revised the manuscript as indicated below. However, it remains true that it is much harder for a non-expert to write high quality code in LabView than in Python. This is particularly evident in complex systems.

We have changed the section about LabView to read:

“The visual nature of the programming environment makes simple projects easy but systems with a large number of hardware components or complicated control architecture can become hard to understand, reproduce, and maintain. Although this complication can be reduced with good programming practices, it is not uncommon to outsource such work to a commercial company \citep{chhetri2020software} because good code writing in LabView is significantly more challenging than in popular general purpose languages such as Python. Additionally, the LabView work flow does not integrate well into modern distributed source control infrastructure such as mercurial or git, a necessity for modern open source development.”

(c) The authors should include the current effort by Pinkard et al. (Pinkard et al. Nature Methods (2021) - https://doi.org/10.1038/s41592-021-01087-6 ) in their discussion.

A pre-print version of this paper was available on arXiv and cited in our original submission. Now this paper is published we have included the published reference and the following text has been added to our discussion section.

“As mentioned in the introduction, micromanager has a recently introduced Python interface, Pycro-Manager (Pinkard et al. 2021). This simplifies connections between micromanager based hardware interfaces and Python based analysis and control. Although this reduces the effort in using Python for control and online analysis compared to other approaches it does not provide direct access to the hardware via Python. This interface keeps the existing micromanager infrastructure. Particularly new hardware interfaces still need code in both C/C++ and Java before they are accessible via the Python interface.”

The authors might want to explain how they plan to facilitate the library's adoption and the long-term maintenance within the microscopy community. Do they plan to create a new category on Image.sc, which would allow the community to interact with the developers? etc. Furthermore, who will keep writing wrappers to the libraries provided by the vendors? etc

This is a critical point, as the reviewer states, community involvement is essential to continuation of the project and provide a useful tool going forward. We have already published several systems utilising this software platform and are working hard to expand its user base. We have asked for people to post question on the image.sc forums (https://image.sc/) and we also interact with developers and users on the github issue pages (https://github.com/python-microscope/microscope/issues). We have recently implemented a fully automated microscope on a simple motorized stand from Zaber. This provides a fully automated microscopy solution for a very low cost.

We have edited the end of the discussion to read

Microscope is a free and open source project currently being used in several labs with an open development approach. Our aim is that the microscope development community will find it a useful tool and engage in this development to increase its general usefulness. With that aim in mind, we perform our development conversations and user support in the open as github issues and the project is an image.sc community partner. In particular, expanding the number of devices supported by Microscope would be extremely beneficial. However, adding support for a device requires physical access to the device and the current list of supported devices echoes the devices we and our collaborators have access to. This is a chicken and egg problem. Python-Microscope needs broad device support to be widely adopted by the community but it needs contributions from the community to support those devices. We believe that, Microscope currently provides enough devices and infrastructure to support adoption by more developers. There are contribution guidelines within the ``Get Involved' section of the documentation, available online at https://www.python-microscope.org/doc/get-involved.

The authors stress using their library for complex scopes but do not provide an example of complex implementation (they only provide paper references). Only a code for a simple time-series is provided. It would be very beneficial to provide the code for implementing a complex microscope and its GUI with the author's library as separate figures or in the paper's supplement. This would also support point 1 in the review.

The GUI elements provided by Python-Microscope are deliberately minimal implementations to allow basic connectivity and functionality of specific hardware to be tested. Python-Microscope is specifically designed to provide a hardware interface layer separate from the user interface. We provide a very simple examples to demonstrate how easily devices can be controlled. For more complete examples we have developed two associated packages providing GUIs, both are referenced in the text, BeamDelta is an optical alignment tool, while Microscope-Cockpit provides a full user interface to complex microscope systems. We have added a supplemental figure demonstrating the full GUI provided by Cockpit.

**Minor comments:**

It would help the paper if several phrases would be changed: Title: 'Python-Microscope: High-performance control of arbitrarily complex and scalable bespoke microscopes." To: e.g., Python-Microscope: A new open-sources Python library for the control of microscopes

Why? The authors use the word "high-performance" to address their Python library's trigger feature within the text. Unfortunately, that is not how most people would use the term for. Therefore, it should be avoided not only in the title but throughout the text. Furthermore, the word "complex" combined with microscopes should be avoided. A complex microscope is, for most microscope builders, a microscope that needs precise times and synchronization, includes several feedback active feedback loops, incorporates several devices, is very stable, etc. The context in which the term "complex microscopes" is used here is when the authors talk about the library's features to connect devices to servers either locally or remotely. I agree that the library can connect devices over arbitrary complex networks, but using the term "arbitrary complex microscopes" would be misleading considering the library's current speed limitations, the limited number of currently integrated devices, etc.

We have changed the title to:

Python-Microscope: A new open-source library for the control of microscopes

1. Various section titles: "Library features" would be more suitable than "Use Cases" since the individual new features at the new library are described in this section. Also, the description of the individual features should be mentioned more accurately. The following list might be a better, more accurate fit: (1) "Device modularity" instead of "Device independence."

Also, the current title "Write once, run with any device" is inaccurate since the wrapper for multiple devices has not been implemented. (2) "Experiment- and scope-specific layout" instead of "Experiments as programs." (3) "Complex network integration" instead of "Easy implementation of complex systems and scalability" (see reasoning under point a). (4) "Hardware and software trigger integration" instead of "High performance, " (5) "Developer-friendly programming features" instead of "Simple development tool."

We have renamed the specified sections and subsections title and expanded the description in the list of use cases to be more accurate.

1. The authors should avoid using the term "Microscope" when talking about "Python-Microscope." It facilitates the manuscript's readability since it is occasionally not evident in the paper if they refer to the library or a microscope. We have changed “Microscope” to “Python-Microscope” in multiple places of the manuscript where it was unclear whether we were referring to the software or to a physical microscope.

2. The authors should avoid the phrase "pythonic software platform" in the abstract since Python-Microscope is a library / Python package and not a software platform. Furthermore, the term "pythonic" describes the desired way to write Python code. It means code that does not just get the syntax right but follows the Python community conventions and uses the language in the way it is intended to be used. Instead, it might be advisable to write, "Python-Microscope offers elegant Python-based tools to control microscopes...". We have changed the abstract as suggested.

Figure 1 should be supported by comments, e.g., #Load packages, #Parameter Initialization, #Create Devices, # Set camera parameters, etc.

Comments have been added the sample code.

The paragraph under the section "Experiments as programs" about the advantages of using Python (starting from "We have developed the software in Python, ...") should be moved into the Introduction section.

We have moved this segment to the end of the introduction.

Reviewer #2:

1)The introduction does a good job describing the current situation (using multiple software from multiple vendors simultaneously, Micro-Manager, Labview), although it could be highlighted a bit more that several groups have created custom Python code for microscope control (such as https://github.com/ZhuangLab/storm-control, https://github.com/Ulm-IQO/qudi, https://github.com/fedebarabas/tormenta, https://github.com/AndrewGYork/tools), some with at least the hope that their code will be generally usable. It also could be noted that the Micro-Manager device abstraction layer has been accessible from Python for more than a decade (currently the Python 3 interface is at https://github.com/micro-manager/pymmcore).

We have significantly expanded the references to previous Python code and made other changes to the relevant sections as detailed in the response to reviewer #1 and quoted below. We have made reference to the recently published Pycro-Manager package (the previous version referenced the arXiv preprint of this paper. It should be noted that although the Python bindings for mmcore have been available for more than a decade, they have been rarely used, the only published paper referencing them appears to be the whitepaper from a workshop on microscope control software published on arXiv in 2020 (https://arxiv.org/abs/2005.00082).

“There is currently an increasing number of software options for microscope control in Python, many of which are in the form of custom scripts specific to a microscope (Alvelid and Testa, 2019, York et al., 2013) but some provide a fully integrated microscope control environments, namely PYME https://www.python-microscopy.org/ for SMLM and ACQ4 (Campagnola et al., 2014) for electrophysiology. While this code is freely available and can be modified, their design around a specific setup, technique, or environment reduces its potential for code reuse in other projects”

2) Manuscripts describing software tools have to balance the goal to "announce" and advertise the software package with the goal to objectively explain the design principles and choices made. In my opinion, this manuscript finds a nice balance, and leaves the reader with a decent understanding of the capabilities, advantages, limitations and high level architecture of the Python-Microscope package. Possible exceptions are the use of the word "elegant" in the abstract, and extensive use of the word "bespoke" that I mainly know from real estate agent language and that likely is confusing to many readers for whom English is a second language.

We have reworded the abstract to say

“Python-Microscope offers simple to use Python-based tools to control microscopes…”

We use the term “bespoke” to refer to the construction of novel optical microscopes, as opposed to controlling existing integrated systems from commercial vendors. We have reworded paper to refer to custom built microscopes and optical systems to clarify this point.

As far as I am aware, "Microscope" is the most developed microscope abstraction layer written in pure Python. Remarkably, its design (device classes that inherit from a device-base class and have their own function calls, supplemented with "Settings" that can be declared by each device), is extremely similar to that of the Micro-Manager device abstraction layer (where "Settings" are called "Properties"), with the main difference being that one is written in Python and the other in C++ with C bindings. Writing these device classes in Python hopefully brings the advantage that more people can write them, however, the Micro-Manager C interface has the advantage that it can be used from any programming language on any platform, hence is more future proof than pure Python code. The downside of having multiple microscope device abstraction layers is that resources will be diluted and confuse partners in industry (which toolkit should they support with their limited resources?). The number of devices supported is currently much, much greater in the Micro-Manager platform than in Microscope, and a translation layer to make Micro-Manager device adapters in Microscope does not seem out of the question, and could possibly benefit many.

We are aware of the similarity between our approach and that in micromanager. There is therefore significant overlap and possible duplication of effort, however when we started this project we reviewed the Python bindings of micromanager core and felt that using this approach would add significantly, not only to our development effort, but also to end user effort as they would also have to install Micro Manager and its Python bindings. In addition, we believe that there is significant value in having a pure Python implementation. As the reviewer suggests "Python is at the moment probably the most widely used computer programming language by scientists". Having Python-Microscope in a language that the end user can code, invites them to look into the “box” and eases the process for these, possible casual, Python users to contribute with fixes and support for new devices.

Reviewer #3:

• I miss more information regarding the latency of the device-server and software triggering, how fast can it be? How much delay would you have between computers/devices? For example, could we have the devices sincronized at the microsecond range? I think this is super important so that the reader knows if it's worth using a software triggering approach with Python-Microscope or they should buy a DAQ instead. We generally expect high performance hardware to require hardware triggering, software triggers are very unlikely to be performant, or reliable enough to achieve ms, yet alone, µs timing accuracy and reproducibility. Software triggering is implemented as a basic approach to allow simple low speed hardware control, such as basic image snapping. Our systems all utilise external timing devices to provide digital triggering and, in some cases, analogue voltage control. This is becoming increasingly easy with high performance microprocessors such as the ardiuno or higher spec solutions such as National Instruments DAQ boards. We are currently investigating the recently released Raspberry Pi Pico boards, which provide very high performance digital triggering at very low cost (~£4). We are passionately promoting open source, low cost solutions, so requiring a NI DAQ board and LabView licenses goes against the spirit of this project.

1b) It's good though that they don't want to limit themselves to software triggering but also mention hardware triggering, but it's important to better explain where are the limitations.

This is a significant issue but we feel it is beyond the scope of this paper. We utilise microscope as a low level interface to hardware for our systems. The hardware control software has no internal knowledge of device connectivity eg which filter wheel might be in front of what camera, so any integrated control, such as synchronising light sources and cameras is beyond the scope of this package. We use the cockpit package as a GUI and to provide this higher level control integration. We then utilise hardware timing devices interfaced to cockpit to run experiments. We feel that this is a relatively cheap and approachable solution while allowing high performance from even complex systems.

1c) Needs info adding to the text, but in general python-microscope doesn’t concern itself with this, just allows setting of trigger types and you are then responsible for triggering.

As suggested by the reviewer, Python-Microscope does not generally concern itself with triggering. It allows setting of trigger types in a consistent manner, and on relevant devices can initiate a software trigger event. The end of the section “Fast and furious” now reads:

“The microscope interface was designed with the concept of triggers that activate the individual devices and software triggers are handled as simply another trigger type. This approach provides an interface that supports software triggers but is easily upgraded to hardware triggers. The source of such hardware triggers can be other devices --- typically a camera --- or a dedicated triggering device. The recommended procedure is to prepare an experiment template that is then loaded on a dedicated timing device which triggers all other devices, as described in Carlton 2010.

The existence of fast and cheap microprocessors and single board computers mean providing a dedicated hardware timing to sequence and synchronise a number of devices is relatively easy and extremely cost effective. We would recommend systems are designed around using an external device to provide hardware triggers to devices. This provides reliable timing and much more flexible sequencing than directly connecting outputs from one device to trigger inputs for another.”

1d) I also miss information about the triggering, do the software offer a platform that can synchronize devices, or that's more left to the developer to do? They say they can generalize to arbitrarily complex devices so therefore I think it needs to be specified how. Same with the server feature, how fast is that link?

The software triggering depends very much upon the individual devices and delays such as context switching within the OS. We offer no solution to synchronise devices. Our claim to generalise to arbitrarily complex systems is based on the fact that you can trivially run devices on different computers to allow horizontal scaling. If you wish to have 25 cameras, simply run them on different computers, then none will be speed limited by computational resources. Synchronisation can be achieved by an external hardware timing device as described above.

The server link is passed over standard ethernet, likely now 1GB/s, however data packets must be serialised before transmission and deserialised on receipt by Python, as well as standard network overhead and latency. We have only seen network limitations on image transfer from cameras to remote server computers. This has not been a significant issue as the cameras drivers typically have memory buffers, which can be enlarged to cope with backlogs, as well as the Python-Microscope image transmission processes acting on a FIFO memory queue. Possibly long experiments utilising fast, high pixel count cameras could saturate these buffers, but such a specialised application could use specialised solutions such as multi-path networking or a computer with a very large amount of RAM for temporary buffering.

2a) Some critical comments are that, first of all there are not so many drivers yet available (for example Hamamatsu camera).

The reviewer is correct, device support is critical. There are two components to this, a) the resources to implement new devices, and b) the physical hardware to enable testing and debugging of these devices. We have focused on the hardware that we own and use but hardware support is expanding. As described in our reply to reviewer #1, we hope that a community of experienced hardware and software developers will evolve and help support new devices. We have instructions on how to support new hardware devices and are happy to help interested parties. We also plan to apply for continuing funding to enable us to further develop Python-Microscope, especially to expand its range of supported hardware,

The well defined interface with the abstract base class in Python enforces what is required for a minimal implementation of a specific device type. Most devices are relatively easily supported by reference to existing devices of the same type. For instance, a stage is likely to be communicated to by serial over USB, taking simple text commands and returning easy to interpret responses. Adding a new device simply involves defining what commands to send and how to deal with the replies from the hardware. With a suitable manual this can typically be done with a few hours of programming and testing.

2b) I guess this paper is also to show proof of concept and then upon interest they will include more devices, but in that case it should be more documented how one can contribute to the project and generate new drivers. For example, if we want to try it tomorrow in our setups, and we have a specific device such as an Hamamatsu camera, What should we do? Should we contact the authors, write an issue in the github page or write the driver ourself?

We have added the following paragraph on contributing to the project at the end of discussion section of the paper:

Microscope is a free and open source project currently being used in several labs with an open development approach. Our aim is that the microscope development community will find it a useful tool and engage in this development to increase its general usefulness. With that aim in mind, we perform our development conversations and user support in the open as github issues and the project is an image.sc community partner. In particular, expanding the number of devices supported by Microscope would be extremely beneficial. However, adding support for a device requires physical access to the device and the current list of supported devices echoes the devices we and our collaborators have access to. This is a chicken and egg problem. Python-Microscope needs broad device support to be widely adopted by the community but it needs contributions from the community to support those devices. We believe that, Microscope currently provides enough devices and infrastructure to support adoption by more developers. There are contribution guidelines within the ``Get Involved' section of the documentation, available online at https://www.python-microscope.org/doc/get-involved.

• Second, the graphical interface is maybe good enough for developers and builders but in order to have a solid microscope that biologists are going to use it needs a bit more work in that direction. The GUI in microscope is extremely basic and designed for quick testing. For a microscope system aimed at biological users we would recommend using Microscope-Cockpit, our paper is now referenced and a supplemental figure shows an example of its interface, or implementing an alternative more specialised GUI. We have released Python-Microscope as a separate package to separate low level hardware control from a GUI front end, enable relatively easy automated control of microscope systems directly from Python, or allow others to create GUI base interfaces without having to deal with interfacing to specific hardware.

This part where the mother is murdered in the bathroom is scary, because it shows that no one is safe

当然，我们知道「氛围」这个词的含义。它是一种你无法确定的抽象品质的占位符——一种气氛（「一种悠闲的氛围」）。它是你喜欢或不喜欢某事或某人的原因（好的氛围与坏的氛围）。它是一种没有明显解释的直觉（「只是我感受到的一种氛围」）。许多氛围没有具体的名字，但有些氛围有。

Reviewer #2 (Public Review):

In cognitive neuroscience, a large number of studies proposed that neural entrainment, i.e., synchronization of neural activity and low-frequency external rhythms, is a key mechanism for temporal attention. In psychology and especially in vision, attentional blink is the most established paradigm to study temporal attention. Nevertheless, as far as I know, few studies try to link neural entrainment in the cognitive neuroscience literature with attentional blink in the psychology literature. The current study, however, bridges this gap.

The study provides new evidence for the dynamic attending theory using the attentional blink paradigm. Furthermore, it is shown that neural entrainment to the sensory rhythm, measured by EEG, is related to the attentional blink effect. The authors also show that event/chunk boundaries are not enough to modulate the attentional blink effect, and suggest that strict rhythmicity is required to modulate attention in time.

In general, I enjoyed reading the manuscript and only have a few relatively minor concerns.

1) Details about EEG analysis.

First, each epoch is from -600 ms before the stimulus onset to 1600 ms after the stimulus onset. Therefore, the epoch is 2200 s in duration. However, zero-padding is needed to make the epoch duration 2000 s (for 0.5-Hz resolution). This is confusing. Furthermore, for a more conservative analysis, I recommend to also analyze the response between 400 ms and 1600 ms, to avoid the onset response, and show the results in a supplementary figure. The short duration reduces the frequency resolution but still allows seeing a 2.5-Hz response.

Second, "The preprocessed EEG signals were first corrected by subtracting the average activity of the entire stream for each epoch, and then averaged across trials for each condition, each participant, and each electrode." I have several concerns about this procedure.

(A) What is the entire stream? It's the average over time?

(B) I suggest to do the Fourier transform first and average the spectrum over participants and electrodes. Averaging the EEG waveforms require the assumption that all electrodes/participants have the same response phase, which is not necessarily true.

2) The sequences are short, only containing 16 items and 4 cycles. Furthermore, the targets are presented in the 2nd or 3rd cycle. I suspect that a stronger effect may be observed if the sequence are longer, since attention may not well entrain to the external stimulus until a few cycles. In the first trial of the experiment, they participant may not have a chance to realize that the task-irrelevant auditory/visual stimulus has a cyclic nature and it is not likely that their attention will entrain to such cycles. As the experiment precedes, they learns that the stimulus is cyclic and may allocate their attention rhythmically. Therefore, I feel that the participants do not just rely on the rhythmic information within a trial but also rely on the stimulus history. Please discuss why short sequences are used and whether it is possible to see buildup of the effect over trials or over cycles within a trial.

3) The term "cycle" is used without definition in Results. Please define and mention that it's an abstract term and does not require the stimulus to have "cycles".

4) Entrainment of attention is not necessarily related to neural entrainment to sensory stimulus, and there is considerable debate about whether neural entrainment to sensory stimulus should be called entrainment. Too much emphasis on terminology is of course counterproductive but a short discussion on these issues is probably necessary.

.

Skip all remaining sections.

The rest of this article is N/A, so you should stop here! :)

In summation, your spec sheets should include the following sections: features, requirements, problem management strategy.

Chorus Repetition: You know how the chorus is soft at first but then gets more and more evil by the second? Yeah, this just became 100% psychopathic now. And remember how I said that "Love is either heaven or hell?" Well, this wasn't really heaven at all!

I'm starting to think that the nasty scar was the girl giving the boy a cut from trying to kill him. The blank space is probably in the list of exes that she wants to kill, the flames is that she probably burned everything that had to do with the relationship, and the "take things way too far" is probably that the boy was being optimistic but the girl was being super jealous and manipulative.

not trying to set a stereotype because not all boys are the same but there are some that just are the worst

This tends to be a bit stereotypical towards men because not all of them are like that. Some men actually treat their women in the right way.

But this also is true, some men are only in it for the money or for "the thing." Some just tend to lie by saying nice things but they turn out to be abusive.

We absolutely have. Just like with online video platforms, the ability to browse and wonder while picking up, reading, and putting down to move on to the next title is all but gone. It's replaced with suggestions based on prior history, what other people have liked, and a preprogrammed auto-playing queue over which there is little control, and over which most people exert no control.

After uncomfortably long times perusing stuff online, I often settle for something that is familiar and (often) stale. This is never the case when I go to a bookstore in person or peruse the shelves of a comic book shop or thumb through the LPs at a local used music store.

Online course catalogs are the worst offenders, often poorly programmed to only show the next open section of a pre-programmed list of options.

You can't just leave things and expect it to be recycled.

once information is in the open commons transfer over to the knowledge repository

The sign/symbol I'm choosing is the stage direction when Ophelia lifts up her hands and says "How Shiftless". Ophelia is repulsed by seeing how Eva and Tom kiss. She believes it to be inappropriate and just wrong, possibly even a waste of time to be giving such affection to Uncle Tom. It is such an emotional moment for Ophelia because she is clearly not used to seeing any sort of decency towards a slave and you can tell it boils her blood. I also think it's interesting that when she says "How Shiftless" this time, she lifts her hands. The other times she said it there was no stage direction of any sort of gesture. This gesture I think is Ophelia trying to show that she wants nothing to do with this "behavior" the family is indulging in with Uncle Tom and to show that it's below her. If this were being performed on stage I would laugh for sure. I think it's funny how dramatic she is, and especially because she does it over and over. It's almost like a broken record.

His father's ideas of pride and preserving were all coming down, with having to now be PITIED by the system that they were crushed by. It's like not only has his father died, but what he represented has also died.

hypocritical because they are doing the same thing, just one blames the other and refuses to take accountability it's also sad that Malcom was okay with this (maybe not okay, but he had experienced this treatment his whole life, so feels like it is normal and won't fight it)

I think it's interesting that the concern for ethics in computing has been a cause for concern since the 1990s. Although it wasn't as expansive as it is today, it was still something people were careful about. I think that's fascinating, but not as fascinating as the line "including for organist guilds." THERE'S A CODE OF ETHICS FOR ORGANISTS. I don't know what I'm going to do with this information but it is fascinating nonetheless.

It's incredible how much technology has evolved even in just the past 20 years, and it has done a lot of good for society. However, it's sad to see it being used in malicious ways that can be very damaging to people. It's good to see how they are updating the ethics code to mitigate these problems, and it was something I wasn't aware of. As technology advances so should our regulations. Lots of research and collaboration has gone into preventing the harmful uses of AI as well as increasing testing to ensure programs are working properly.

I'm using it to do both of these things and definitely find it more useful for the knowledge base work. I've never used Simplenote heavily, but it's definitely more focused on your use case Colin.

For the quick notes scratchpad idea, I've been relying on Markor and syncing the results from my phone to my Obsidian data store to get those notes into my notebook more easily. Often when I'm at my desktop I may move those notes to other more appropriate places to keep track of them. Hopefully Obsidian's mobile version (in beta) will make this portion easier.

Someone else is steering your life. What this snippet says is that there is no you. Just experience

Everything is either pre-determined or random. Either way, you are a puppet

This is much the way I feel and write. I keep my site more as a personal commonplace book and write primarily for myself. Others read it from time to time and comment, but in the end, it's really all just for me.

I love this idea about heirlooms and utility.

Author Response:

Reviewer #1:

Guo et al. describes interesting experiments recording from various sites along a cortico-cerebellar loop involved in limb control. Using neuropixels recordings in motor cortex, pontine nuclei, cerebellar cortex and nuclei, the authors amass a large physiological dataset during a cued reach-to-grasp task in mice. In addition to these data, the authors 'ping' the system with optogenetic activation of pontocerebellar neurons, asking how activity introduced at this node of the loop propagates through the cerebellum to cortex and influences reaching. From these experiments they conclude the following: the cerebellum transforms activity originating in the pontine nuclei, this activity is not sufficient to initiate reaches, and supports the long standing view that the cerebellum 'fine tunes' movement, since reaches are dysmetric in response to pontine stimulation. Overall these data are novel, of high quality, and will be of interest to a variety of neuroscientists. As detailed below however, I think these data could provide much more insight than they currently do. Thus below I provide some suggestions on improving the manuscript.

1) Since the loop is the focus of this study, it would be nice if the authors better characterized latencies of responsivity to pontine stimulation through the loop, to address how cortically derived information routed to the cerebellum may loop back to influence cortical function. In the data provided, we know that pontine stimulation modulates Purkinje and deep nuclear firing (but latency to responses are not transparently provided in the main text, if anywhere), while motor cortical responses peak at 120 ms (after stimulus onset?, unclear), and that this responsivity is preferentially observed in neurons engaged early in the reaching movement. Is the idea, then, that cortical activity early in the reach is further modulated by cerebellar processing to (Re) influence that same cortical population? Does this interpretation align with the duration of reaches, the duration of early responsive activity during reach, and the latency of responsivity; or is the idea that independent information from other modalities entering the pontine nuclei modulates early cells? Latency to respond at the different nodes, might aid in thinking through what these data mean for the function of the loop.

We thank the reviewer for this important suggestion, and we have now added measurements of the latency from the onset of sinusoidal PN stimulation to neural responses in Purkinje cells, DCN neurons, and motor cortex (Supplemental Fig. 7), and observe a progressive recruitment of laser-evoked spiking along this pathway. There is a tradeoff between temporal resolution (which increases with decreasing bin width) and statistical power (which decreases with decreasing bin width), and we have opted to use 10 ms bins in a sliding window, which provides a reasonable compromise between these criteria. Although we potentially detect fewer tagged neurons at shorter latencies than we would with larger bins, this approach enables us to detect the timing of the earliest responses (defined as the earliest time point at which 5% of the neurons eventually recruited are responsive). Note that the sinusoidal stimulation used in these experiments is not ideal for latency measurements, as it takes 6.25 ms for the laser to reach peak power. We have also added a similar analysis for the response latency of PN neurons to pulse train stimulation of motor cortex (Supplemental Fig. 1). Based on these analysis, our estimate of the delay for signals to propagate across the entire loop is 26 ms: PN to motor cortex (21 ms) + motor cortex to PN (5 ms). Given that the movement duration (lift-to-grab) is approximately 110 ms on average, this would allow ~4 full feedback cycles throughout the reach. Thus, these delays are consistent with the possibility that cortical activity during planning or early in the reach is further modulated by cerebellar processing to influence that same cortical population later in the reach. Regarding the earliest motor cortical responses that we observe in PN-tagged units, it's possible that they may result from ponto-cerebellar input driven by other cortical regions. Alternatively, the responses of motor cortical neurons early in the movement may be driven more directly by other cortical areas or the basal ganglia, but these early-responding neurons may also receive strong ponto-cerebellar input due to plasticity during development or learning.

2) Many of the figures need work to aid interpretation. Axis labels are often missing (eg 2F); color keys are often unlabeled (2F); color gradients often used but significance thresholds are hard to evaluate (using same colors for z scores and control / laser is confusing 6, 8); and within-figure keys would be useful (5D-h). These issues occur throughout the manuscript.

We have added the axis and color labels in Fig. 2F, and have added additional annotation throughout the main and supplemental figures. For firing rate z-score heatmaps, we have kept the gray color scale for control and laser to facilitate direct comparison between the panels, but have added orange and blue boxes around the heatmaps in Fig. 6, 7, S8, and S9 to emphasize that they reflect different experimental conditions.

3) Relatedly, but also conceptually, Figure 3B has particular issues, such as identifying where the neuropixel multiunit activity is coming from. I assume that in the gray boxes illustrating the spatio-temporal profile of spiking band activity that the lower part of the box is the ventral direction, upper, dorsal. This is not spelled out. From the two examples it would seem that the spiking band is in different places in the cerebellum, undermining, I think, the objective of the figure. It would be sensible to revisit this entire figure to identify the key takeaways and design figures around those ideas. As it stands, these examples appear anecdotal. Consider moving this to a supplement. Powerband density strength is missing an axis. More importantly, it would be nice to corroborate the interpretation of the MUA with the single unit recordings, since the idea is that many neurons are entraining to the PN activity. Yet, the examples don't seem particularly entrained. Is the activity being picked up on just axonal firing of the PN axons? Fourier analysis of spiking of isolated neurons in cerebellum should be used to corroborate the idea that cerebellar neurons are entraining, rather than the neuropixel picking up entrained PN axons.

To examine spike entrainment to the 40 Hz PN stimulation for Purkinje cells and DCN neurons, we computed the phase of sinusoidal stimulation coinciding with each individual spike. If a neuron is entrained to the stimulation, the phase distribution for its spikes will differ from the uniform distribution on the circle; this can be assessed for each cell using a Rayleigh test. Furthermore, we can calculate the strength of entrainment and preferred phase by calculating the magnitude and angle of the mean resultant for each cell. If a neuron’s spikes are completely unrelated to the stimulation phase, the mean resultant length will tend to 0 as the number of spikes observed goes to infinity. If, on the other hand, a neuron is completely entrained (with every spike occurring at exactly the same phase), the mean resultant length will be 1. This approach is illustrated schematically in Supplemental Fig. 6A.

This new analysis revealed two key features of the data we had not previously appreciated. First, it revealed PN-stimulation-induced changes in neural activity that were not apparent from the mean firing rate profiles: most Purkinje cells and DCN neurons were significantly entrained to the 40 Hz stimulation. Second, the entrainment strength was higher in the DCN than Purkinje cells (Supplemental Fig. 6B-D), suggesting the corticonuclear pathway amplifies the rhythmic input. This result is strikingly similar to published observations obtained from slice electrophysiology and anesthetized mice (Person & Raman, 2012), which we now discuss in the text. It is also possible that direct excitation from PN collaterals contributes to the DCN entrainment.

We agree that the original analysis of multiunit activity is difficult to interpret, for two reasons: (1) the signal likely reflects the combined contribution of multiple cell types, including pontine mossy fiber terminals, and (2) the depth profile will differ for different electrode penetrations, due to the geometry of the cerebellar cortex. Furthermore, this analysis is largely redundant, since we have recorded from individual Purkinje cells and added new analyses demonstrating their entrainment to the 40 Hz stimulation (Supplemental Fig. 6). We have now moved this figure to the supplement and added labels to all axes (Supplemental Fig. 3).

4) The use of the GLM is puzzling. In addressing the question of how cerebellum and motor cortex interact (from the Abstract, "how and why" do these regions interact) it is unclear why these regions are treated separately. I would have expected some kind of joint GLM where DCN activity is used to predict M1 variance (5 co-recordings are reported but nothing to analyze?); or where DCN + M1 activity is used to decode kinematics to see if it is better than one or the other alone. As it stands, we learn that there is more kinematic information in the motor cortex than in DCN. This is not necessarily surprising given previous literature on cerebellar contributions to reaching movements. In principle the idea that 'PN stimulation might perturb reaching kinematics through descending projections to the spinal cord, or by altering activity in motor cortex' is treated as mutually exclusive outcomes, though it is highly unlike to be so.' Analyzing M1+DCN together could address whether DCN activity adds nothing to decoding kinematics that isn't there in M1 or adds something that M1 does not have access to. The main point here is that the physiological datasets could be better leveraged with these fits to derive insight into the interactions of the loop. R2 should be provided in the GLMs (Fig 8) to assess statistically how well they perform relative to one another, not just correlations between the two.

We have added two additional analyses to address these questions. First, in addition to motor cortex-based and DCN-based decoders for all sessions (Fig.8 and Supp. Fig.12A-D, G-H; all the R2 values are reported in Supp. Fig. 12C-D, G-H) we now also train a decoder using both motor cortical and DCN multiunit activity in sessions with simultaneous recordings (Supp. Fig.12E-F, I-J). When we train only on control trials, the decoder performs about equally well with or without the DCN multi-units for control trials (Supplemental Fig. 12E), but performs slightly worse on laser trials in comparison to using only cortical data (Supplemental Fig. 12F). When we train on both control and laser trials, adding DCN multi-units slightly degrades decoding performance on both control and laser trials in 3 out of 5 sessions (Supplemental Fig. 12I-J). Based on this comparison, it does not appear that DCN contributes kinematic information that is not already present in cortex. However, there are several cautionary notes to consider in interpreting these results. (1) This dataset consist of only 5 sessions, in all of which the recording yield in DCN was not as high as in cortex, so it is possible that dimensions of activity unique to DCN may not have been sampled enough in these experiments. (2) Our task involves only a single reaching target (in comparison to, e.g., center-out reaching tasks with eight targets which are possible in primates) so we cannot assess whether DCN contains directional-specific kinematic information not present in cortex. Thus, in light of these factors, it is difficult to draw strong conclusions from our experiments about differences in kinematic information between motor cortex or DCN. A more rigorous comparison requires carefully controlled experiments with many reaching targets, as in Fortier, Smith, & Kalaska (1993).

Second, we have added an additional analysis to determine how predictive cortical activity is of DCN activity at the single-trial level, and vice versa. We considered several possible statistical approaches to this issue. Computing pairwise correlations of neurons in the cortex and DCN would be one possible method, but the outcome of this analysis would be difficult to interpret, as the sign and timing of firing rate peaks will vary across neurons. Another approach would be to regress principal component scores in one region - or their derivatives, as in Sauerbrei et al., 2020 - on the scores in another region. However, because cortex and DCN are bidirectionally connected, the choice of which region’s scores should be considered as the dependent variables is ambiguous, and this approach will merely “align” activity in one region (as a projection onto regression coefficients) with activity in the other. Ideally, we would like to find simultaneous linear transformations of both cortical and DCN activity that would maximally “align” them with one another, and to compute the correlations of the aligned neural trajectories. This is precisely what canonical correlation analysis (CCA) does, and CCA has been used increasingly in recent years to align population activity from different brain regions or samples - e.g., Lara et al., Nat. Comm. (2018), Perich et al., Neuron (2020), and Gallego et al., Nat. Neuro (2020). We took this approach with our simultaneous recordings of multiunit activity in the motor cortex and DCN, and found that:

(a) In each of the 5 sessions, CCA found two pairs of canonical variates that were strongly correlated (Supplemental Fig. 11A, first two columns; Supplemental Fig. 11B, correlations in the range 0.58-0.88 for the first two canonical variates), and two pairs of canonical variates weakly correlated (Supplemental Fig. 11B, correlations <0.27 for the last two canonical variates)

(b) The first two canonical variates accounted for half or more of the variance in each region (49%-64% in cortex, 51%-70% in DCN; Supplemental Fig. 11C, left column)

(c) Between a quarter and a half of the variance in each region was accounted for by canonical variates in the other region (25%-50% of variance in DCN explained by cortex, 26%-47% in cortex explained by DCN; Supplemental Fig. 11C, right column)

From these results we conclude that, within the constraints of our behavioral task, some but not all of the dominant dimensions of cortical and cerebellar activity are strongly correlated. We also performed additional CCA analyses using only laser trials or only control trials, to assess whether PN perturbation strongly affected the similarity in population activity between the two regions, but found limited differences between the results of the two analyses (Supplemental Fig. 11D).

Reviewer #2:

Guo et al examine the cortico-cerebellar loop during skilled forelimb movements in mice. The authors use optogenetic stimulation of the pontine nuclei (PN) and recordings in PN, cerebellar cortex, cerebellar nuclei (DCN), and motor cortex to show that PN output is transformed into a variety of activity patterns at different stages of the cortico-cerebellar loop. Stimulation only slightly alters movement-related activity in these structures and degrades movement accuracy. The authors conclude that the cortico-cerebellar loop fine tunes dexterous movement. The study is technically impressive, employing recordings in 4 brain regions, and recordings during optogenetic manipulations and behavior. The experiments are well done and the analyses are appropriate. The comparison across brain regions is comprehensive. The results that PN perturbation alters skilled movement and the perturbed activity could predict perturbed movement are important. The study adds to a long line of work supporting the view that the cortico-cerebellar pathway is required for fine motor control. I have a few comments on the interpretation and analysis which I believe could be addressed with changes to the text and additional analysis.

1) The authors conclude that the cortico-cerebellar loop "does not drive movement" but "fine tunes" the movement. While I generally agree with this interpretation, I wonder if the authors could flush out the concepts of "driving movement execution" vs. "fine-tuning movement" more clearly. Do authors consider them separate processes? How can they be disentangled? I also feel the data on its own has some limitations that should be considered or discussed. First, the data shows that PN stimulation degrades movement accuracy. However, this does not yet reveal the function of the cerebellar loop in fine motor control. Certain places in the text makes stronger assertions (for example, "cortico-cerebellar loop fine-tunes movement parameters") that I feel the data does not support. It is not clear from the data how the loop tunes movement parameters. Second, Fig. 5F shows that stimulating PN blocked movement initiation in some sessions (this is also mentioned in the Methods). Could the authors consider the possibility that stimulating PN at a higher intensity might block movement? This is related to the distinction between "driving" vs. "fine-tuning" movement. At the very least, the authors should discuss these limitations and possibilities.

In our view, the claim that a brain area drives reaching means that it is necessary for generating the large changes in muscle activity that set the limb in motion towards the target. The claim that a brain area fine-tunes reaching means that it is necessary for generating smaller changes in muscle activity that subtly adjust the limb trajectory and enable precise and accurate behavior. Previous work has demonstrated that motor cortex drives reaching: if it is transiently silenced, the initiation of reaching is robustly blocked (see Guo et al. 2015, Sauerbrei et al. 2020, and Galinanes et al. 2018). In the present manuscript, we show that perturbation of the PN has a very different effect: mice are usually able to initiate reaching, but they are less skillful (the success rate drops), slower (movement duration increases), and less precise (endpoint standard deviation increases). Our interpretation of these results is that while the total output of cortex drives movement (likely through corticospinal and cortico-reticulospinal routes), the cortico-cerebellar loop makes more subtle adjustments to the ongoing movement; that is, it fine- tunes. We have updated the text (in particular, the Abstract, Introduction par. 1, and Discussion par. 1-2) to clarify the distinction between driving and fine-tuning.

We agree that several interpretive statements in the previous version (especially concluding sentences at the end of some Results paragraphs) were not clearly connected with the data, and we have removed or modified these statements. We now lay out our interpretation of the data as evidence for a cortico-cerebellar contribution to fine-tuning, rather than driving, in the first two paragraphs of the Discussion, but emphasis that this is an interpretation, rather than a direct description of the data. We have also changed the title to more directly state our experimental observations.

We now mention the possibility that stronger stimulation or inactivation of PN neurons might have robustly blocked movement, and also mention several experimental variables which might have contributed to animal-to-animal variability in behavioral effects: “It is possible that the variability of behavioral effects ...” (Discussion).

2) Related to point 1, in Fig. 5F, for stimulation trials in which mice failed to initiate movement, did mice fail to move altogether, or did they move in an abnormal fashion?

We have added a new video documenting the behavior of the animal with the largest blocking effect from PN stimulation (supplemental video 2). This animal does not struggle through a partial reach, but fails to initiate movement. Small movements of the arm occurred (this also occurred in control trials), but these were not tightly synchronized with the onset of the laser across trials.

3) In the abstract, the authors state that PN stimulation is "reduced to transient excitation in motor cortex". Also in the results (page 5) and discussion (page 8), "pontine stimulation only led to increases in cortical firing rates". These statements are based on the comparison between Fig 3D, 3F, and 4B. But I think the current presentation is somewhat misleading. First, Fig 3D, 3F, and 4B use different neuron selections that make direct comparison difficult. Fig 3 shows all neuron from Purkinje cell and DCN recordings. Fig 4B shows only PN-tagged motor cortex neurons. Furthermore, based on the methods description, it appears that PN-tagged neurons were defined using one-sided sign-rank test. Since the test is one tailed, does that mean neurons shown in Fig 4B are, by definition, neurons significantly excited by photostimulation? Looking at Fig 4B and 4C closely, there appear to be neurons suppressed by PN stimulation. Could the authors organize the rows in Fig 4 in the same way as Fig 3, where neurons that show suppression are grouped together?

We now display the PN stimulation-aligned firing rates in the same format for Purkinje cells (Fig. 3B), DCN neurons (Fig. 3D), and motor cortical cells (Fig. 4A, lower), with all neurons in a single panel, sorted by response magnitude, for each area. The dominant response pattern in the cortical population is a transient firing rate increase, and this is more readily apparent with the new panel in Fig. 4A (lower). We also use a two-tailed test (which has slightly less statistical power, but allows us to test for both firing rate increases and decreases) for the identification of PN-tagged cortical neurons, and display neurons with stimulation-locked increases (n = 94) and decreases (n = 13) separately (Fig. 4B). In Fig. 4B-C, we still sort the neurons by their reach- related responses, as this reveals a difference in lift-aligned patterns between tagged and non- tagged neurons, which would be masked if we ordered according to stimulation-aligned responses. In Fig. 4D-E, we pool neurons with PN-stimulation-aligned increases and decreases into a “PN-tagged” group, as the small number of stimulation-aligned decreasing neurons (n = 13) does not allow adequate statistical power for a 3x3 contingency table test or for within-group averaging of lift-aligned firing rates.

4) Fig 7 shows that PN stimulation has only subtle effects on movement-related activity in motor cortex. However, only a small portion (1/8) of the motor cortex neurons show modulation to PN stimulation. Fig 7 shows all neurons. Would the results look similar for PN-tagged neurons?

We have added a new analysis to address this question, shown in Supplemental Fig. 10. The laser - control difference in lift-aligned activity are indeed larger for PN-tagged neurons; however, the largest peak in this difference occurs before lift, when the laser has been turned on, but the animal hasn’t started to move (Supplemental Fig. 10C).

5) Page 3 "Our observation that the activity of some motor cortex-recipient PN neurons is aligned both to the cue and movement suggests that these neurons might integrate signals of multiple modalities." Presumably, motor cortex neurons also have cue and movement-related activity and PN simply inherits this activity from the motor cortex.

As described in our response to the first reviewer’s seventh comment, we cannot conclude that the cue-related responses in the PN are inherited entirely from motor cortex. Briefly, (1) it has been difficult for us to reliably disassociate cue and movement responses for individual motor cortical cells (for instance, the GLM approach we took with PN neurons resulted in very poor model fits when applied to cortical cells), though our previous work has suggested that at the population level, the dominant signal in motor cortex is aligned to movement onset. To reliably disentangle cue and movement responses in cortex, we would need to train mice to wait for a relatively long and variable delay period before reaching. (2) The PN receive convergent input from many cortical areas, and there is likely a convergence of multiple inputs onto the motor- cortex-tagged PN units (c.f. the convergence of inputs from visual and somatosensory cortex onto individual PN neurons in rats reported in Potter, Ruegg, & Wiesendanger,1978). Hence it is possible (if not likely) that the multi-modal activity we observe in PN neurons results from the integration of inputs from different cortical areas, rather than being entirely inherited from motor cortex.

6) Do Purkinje cells follow the 40 Hz PN stimulation like in the multi-unit recordings. The PSTHs in Fig 3 are too smoothed out to see this.

As described in the response to reviewer 1.3 above, we have added a new analysis to the manuscript to address this question (Supplemental Fig. 6). Most Purkinje cells and DCN neurons are entrained to the 40 Hz stimulation, and the entrainment is much stronger in the DCN, consistent with previous work (Person & Raman, 2012).

7) For the correlation analysis in Fig 6C top and 7C top, is the correlation computed from z-scored firing rates rather than on raw firing rates? This is not clear from the text. If computed on raw firing rates, one would expect the correlation to be above 0 even before photostimulation, since different neurons exhibit different baseline firing rates that presumably will be the same across control and stim trials.

The correlations were indeed computed on z-scores, rather than raw firing rates, for this reason. We have clarified this in the Methods section. This analysis was designed to capture correlations in movement-related modulation between control and laser trials, and we z-scored the firing rates to avoid the confound that would have been introduced by baseline differences.

Reviewer #3:

It is generally thought that the cerebellum is primarily involved in the short-timescale control of movements, while motor cortex is involved in motor planning. The present paper follows classic studies in primates and a recent study in mouse that investigated the role of cortico-cerebellar loops in motor control. To date, studies in both species applied perturbations to the cerebellum to then study changes in cortical activity. For example, it has been long known that cooling deep cerebellar nucleus produces changes in the responses of motor cortex neurons in primate (e.g., Meyer-Lohmann et al., 1975). Further, Gao and colleagues' recent paper (Nature 2018) used optogenetics to perturb responses in the deep cerebellar nucleus before licking movements. The authors of this 2018 nature paper conclude that persistent neural dynamics are maintained during voluntary movements by connectivity in within this cortico-cerebellar loop.

The experiments are well performed, and the results are logically organized and presented. However, a main concern is that the authors have not well justified that these experiments prove a conceptual advance. The conclusions appear to be largely consistent with those of prior work, both regarding changes in the responses of motor cortex neurons, and resultant (subtle) changes in behavior (i.e., altered arm kinematics). The impact of the paper would be improved if the authors adapted a more precise style of reporting the novelty of their results throughout.

Major concerns:

1) The experiments are well performed, and the results are logically organized and presented. However, a main concern is that the authors have not well justified that these experiments prove a conceptual advance. As noted above, prior studies have probed the role of cortico-cerebellar loops by applying perturbations to cerebellar activity (cerebellar cortex and/or deep cerebellar nuclei) and quantifying changes in cortical activity prior to and during movement. The main novelty of the present study is that the authors perturbed the loop at a different locus, namely in the pontine nuclei (PN) which send projections to the cerebellum rather than directly to the cerebellum. The rationale for why this specific perturbation provides a conceptual advance to the field was not adequately motivated.

The authors do clearly review prior literature showing that perturbation of cortico-cerebellar projections impacts the rest of the loop and behavior, they also well explain the application of their exciting new tool to specifically target PN neurons with their optogenetic stimulation. Yet, the authors do not motivate why it is important to specifically perturb the pontine nuclei (PN) to gain new insights into the role of "cortico-cerebellar loops" nor do they provide any reason to expect a difference in changes in loop dynamics for perturbations applied versus to the DCN. Indeed, the conclusions appear to be largely consistent with those of prior work, both regarding changes in the responses of motor cortex neurons, and resultant (subtle) changes in behavior (i.e., altered arm kinematics). Generally, these results are similar to those previously reported in primate DCN cooling experiments characterizing changes in hand movement in in a voluntary tracking task (e.g., Brooks et al., 1973; Conrad and Brooks 1974).

We agree that the rationale and conceptual advance require clarification. Previous work has established that silencing motor cortex blocks reaching (Guo et al. 2015, Sauerbrei et al. 2020, Galinanes et al. 2018), but the perturbations used in these studies were not selective to specific output channels (e.g., corticospinal, corticoreticulospinal, or corticocerebellar), and simultaneously influenced many projection targets of motor cortex. Other work from the Brooks, Prut, Person, and Svoboda groups has shown that altering cerebellar output impairs movement planning or execution, but their methodology did not test the effects of disrupting specific cerebellar inputs (e.g., from cortex). Thus, we would argue that previous studies have not provided direct evidence of the behavioral and neural effects of disrupting cortico-cerebellar signals. The central goal of the present manuscript is to test how selective impairment of cortico-cerebellar communication - not the simultaneous impairment of corticospinal, corticoreticulospinal, and cortico-cerebellar communication, and not a nonselective disruption of cerebellar output - disrupts behavior and neural dynamics across the cortico-cerebellar loop. Our conceptual advance, then, is to show that impairment of cortico-cerebellar communication does not typically block movement execution (as simultaneous perturbation of all motor cortical outputs does), but disrupts the fine kinematic details, similar to a direct manipulation downstream in the cerebellum. We have updated the text, particularly the Abstract, Introduction par. 1, and Discussion par. 1-2, to clarify this rationale and conclusion.

2) The description of the connectivity of the loop illustrated in Figure 1 is straightforward. Motor cortex recipient PN neurons project to PN neurons, which then project directly to the cerebellar cortex and deep cerebellar nuclei, etc. Thus, the effect of any perturbation to PN neurons should be realized rapidly within neurons in the cerebellar cortex and deep cerebellar nuclei if they are part of this direct loop. However, onset latencies for the effect of the perturbations are not documented for these experiments (Figs 3&6 in the test/reaching conditions, and associated text). Similarly, latencies are not reported for the onset of changes in motor cortex neuron responses to PN perturbations in either condition (Figs 4&7 in the test/reaching conditions, and associated text). The only reference I could find to latencies specified the that required to reach the peak firing rate - not latency of the change. Specifically: "these were stereotypical, mostly consisting of transient excitation (Fig. 4B, left; median time of firing rate peak 120 ms)" - 120ms seems very long for the loop in Fig 1. It would be useful to know the latency between optogenetic stimulation in PN and changes in PN firing rate. And then the question is at what latency are the neurons in subsequent nodes altered? Quantification of latencies of the effects that are observes in the different nodes of the cortico-cerebellar loops would strengthen the authors' conclusion that they are actually studying the direct loop in Figure 1 which would then make the study's conclusions more compelling.

We agree that it is important to characterize the latencies of neural responses to PN stimulation, and now provide these numbers for Purkinje cells, DCN neurons, and motor cortical neurons in the text and Supplemental Fig. 7. On stimulation of the PN, activity propagates first to Purkinje cells, then the DCN, and finally to motor cortex. We also quantify the latency of PN responses to motor cortical stimulation in Supplemental Fig. 1. (For a discussion of the rationale and limitations of our method, see also our response above to reviewer 1’s first comment.) Unfortunately, we have not been able to measure the delay from stimulation onset to the earliest spikes induced by ChR2 currents in PN neurons, as this would require simultaneous insertion of a stimulation fiber and recording probe to a deep target in the PN. Furthermore, we note that the earliest measurable response in Purkinje cells occurs 10 ms after stimulation onset, and this is likely an overestimate of the minimum latency, as it takes 6.25 ms for the laser to reach peak power under sinusoidal stimulation.

3) Overall, there was often a sharp incongruity between the complexity of many of the findings described in results and accompanying figures and the short summary conclusion provided for the Results. Here is one of many examples (bottom of page 5), where the authors conclude "These results demonstrate that the cortico-cerebellar loop does not drive reaching, but fine-tunes the behavior to enable precise and accurate movement." Yet, what the results above describe is considerable heterogeneity and variability across animals and cases. These conclusion should be more aligned with/ justified by the author's description of their actual results.

Throughout the Results section, we have now tied the interpretations more closely to the data. For example, in the instance the reviewer mentions, we now state: “These results demonstrate that PN stimulation impairs reaching performance, typically by disrupting precision, accuracy, duration or success rate of the movement.” In the first two paragraphs of the Discussion, we lay out our interpretation of the data as evidence that the cortico-cerebellar loop contributes to fine- tuning the movement, rather than driving it, but emphasize that this is an interpretation rather than a description of experimental results. Furthermore, we now address possible factors that could underlie the diversity of behavioral effects in the fourth paragraph of the Discussion (“It is possible that the variability of behavioral effects ...”).

4) A related issue is the disconnection between description and summary, in the description of Figure 6- 8. The emphasis on correlation, yet the authors' main point here seems to be that there are changes in the activity in cortex and DCN induced by the PN stimulation during movement explain the changes in hand trajectory. For example, Figure 6D and its implications are not effectively described in the text.

The main conclusion of figures 6 and 7 is that PN stimulation during movement alters movement-aligned cortical and DCN activity, but this modulation is typically subtle; that is, activity on control and laser trials is highly correlated for most neurons and time points. This is in contrast with more dramatic effects observed for perturbations delivered to other nodes in the loop; for instance, thalamic perturbations can robustly prevent the generation of the cortical pattern that drives movement (Sauerbrei et al. 2020). Supplemental Fig. 8D-E and Supplemental Fig. 9D-E suggest that these subtle stimulation-induced changes during movement are largely consistent with the changes that would be expected based on neural responses to laser alone, outside engagement with the task. Finally, the decoding analysis in Fig. 8 allows us to interpret these subtle neural changes: they do not appear to be random, but are consistent with the effects of stimulation on the hand. That is, the difference in hand velocity between laser and control trials decoded from neural activity is correlated with the observed hand velocity difference. We have added a video (supplemental video 3) to better visualize this result in all three spatial dimensions simultaneously, and have edited the text in the Results section to clarify these findings.

5) Finally, the authors conclude that changes in the activity in cortex and DCN induced by the PN stimulation during movement explain the subtle deviations in hand trajectory and conclude that the cortico-cerebellar loop is responsible for fine-tuning movement parameters (bottom pf page 5 and top of page 8). However, i) the statement that this pathway fine-tunes motion is not justified by the analysis, and ii) the novelty is not made clear relative to prior work that has investigated cortico-cerebellar loop (beyond the experimental difference in perturbation site).

Regarding (i), we agree that the fine-tuning is an interpretation rather than a direct reflection of the data presented in the paragraph, and have altered the statement accordingly: “Overall, these results show that the subtle changes in the activity in cortex and DCN induced by the PN stimulation during movement are consistent with the changes in hand trajectory for individual mice.” We now explain our interpretation of the data as supporting a fine-tuning role in the Discussion, rather than the Results. Regarding (ii), we have now clarified in the Abstract, Introduction, and Discussion that perturbation of the PN enables us to test the effects of a selective disruption of cortico-cerebellar communication, in contrast with direct manipulations of motor cortex or cerebellum (see also our response to comment 3.1 above).

Overall, the text that follows in the discussion presented the findings in a far more clear and compelling way than much of the text in the Abstract, Introduction and Results "perturbing cortico-cerebellar communication did not block movement execution: animals were typically able to generate the basic motor pattern during optogenetic stimulation of the PN, and neural activity in cortex and cerebellum largely recapitulated the firing patterns observed during normal movement. Instead, PN perturbation altered arm kinematics, decreasing the precision and accuracy of the reach, and perturbation-induced shifts in neural activity explained these behavioral effects." The paper would be improved if the authors adapted this more precise style of reporting throughout.

We have edited the main text throughout to improve clarity and precision.

Here’s an example: “Signal is our annual customer event and afterwards the whole company is exhausted. But the next day, when we all really want to sleep in, we do one thing — a recap where we capture what we call ‘worked, not worked.’ It would be easy for everyone to say, ‘All right, I'm taking the next week off. We just worked our butts off.’ But two weeks later, if we came back and did the post-mortem, they would have forgotten a lot of stuff,” he says. With “worked, not worked,” documented for posterity, it’s a valuable resource to return to again and again. “When we start working on the next year’s conference, we can pull out the ‘worked, not worked’ and we've already documented the outcomes we liked, the outcomes we didn't like, and some of the decisions that went into that. By doing that every year, you just get a little bit better,” says Lawson. This approach is perhaps best summarized by one of Twilio’s favorite mottos, which comes from Chief Product Officer Chee Chew: Every day, our goal is to suck a little bit less. "It expresses this idea that it’s okay that everything’s not perfect and we suck at certain things, but if we just suck a little bit less every day, then we’re building a stronger company as a result."

If I understand correctly, 'witness' trees represent trees who were singled out for their significance on a plot of land As said, these are often obscure and scattered, but put in a larger database could contribute to broader studies. While the primary objective in this study was to find records by new landowners, the concept of witness trees also applies to those who stood durring significant events, not just ownership. Im captured by this concept: I find importance in all species, but 'witness' trees proposes that trees become significant when something significant happens around them. It also gives a sort of anthropomorphic personality as a being who 'sees' and event or situation but will live decades after its human witnessers have passed on. It's almost humbling in a way. In a smaller sense, every creature you interact with is witness to your life, and depending on the longevity of that organism that moment could be held onto for a long time.

It's probably still frustrating though to hear white girls saying the n-word, even if they're just singing along, because that word is still so divisible and painful for African-Americans to hear, especially from non-black people. As long as that word still carries pain and oppression with it, it's still going to cause arguments based on whether it should be said by certain people or not.

Education on this issue shouldn't primarily fall onto the African-American community, I think it's silly to believe that Black people are responsible for educating others on the history and hurt behind the n-word. People need to educate themselves at some point and not rely on others to explain why something hurts them-- sometimes it's just common sense why you shouldn't say certain things.

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

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

Review of "Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence." by Diehl et al for Review Commons.

**Major Comments.** __

1. In this paper the function of Plasmodium falciparum exported protein PFA66, is investigated by replacing its functionally important dnaJ region with GFP. These modified parasites grew fine but produced elongated knob-like structures, called mentulae, at the surface of the parasites infected RBCs. Knobs are elevated platforms formed by exported parasite proteins at the surface of the infected RBC that are used to display PfEMP1 cytoadherance proteins which help the parasites avoid host immunity. The mentulae still display some PfEMP1 and contain exported proteins such as KAHRP but can no longer facilitate cytoadherence. Complementation of the truncated PFA66 with full length protein restored normal knob morphology however complementation with a non-functional HPD to QPD mutant did not restore normal morphology implying interaction of the PFA66 with a HSP70 possibly of host origin is important for function. While a circumstantial case is made for PFA66 interacting with human HSP70 rather than parasite HSP70-x, is there any direct evidence for this eg, protein binding evidence? I feel that without some additional evidence for a direct interaction between PFA66 and human HSP70 then the paper's title is a little misleading.

   We thank the reviewer for their kind words. They are correct that we do not show direct evidence of such an interaction, but would like to note that we, and others, despite concerted efforts to produce direct evidence, have always been hindered by the nature of the experimental system. As noted also in our reply to Reviewer 3, the inability to genetically modify the host cell leads us to suggest that indirect evidence is the best that can conceivably be provided at this time. Our evidence, although indirect, is the first experimental evidence for the importance of such an interaction, all other suggestions having been based on “guilt by association” i.e. protein localisation or co-IP analyses.

Was CSA binding restored upon complementation of ∆PFA with the full-size copy of PFA66?

As this project grew organically and was driven by the results already obtained, we decided to use knob morphology via SEM as a “proof-of-principle” to show that we could reverse the phenotype. Thus, while we cannot comment on whether ALL functions of PFA66 are complemented, we suspect that if the knobs revert to their WT morphology, this is likely to be true for the other tested phenotypes. We do not feel that revisiting all of our assays (which would basically entail repeating almost every experiment so far carried out) would really be much more informative. We have added a note in the discussion stating “We wish to note that we cannot unequivocally state that our complementation construct allows reversion of all the aberrant phenotypes herein investigated, however we feel it likely that all abnormal phenotypes are linked and thus our “proof-of-principle” investigation of knob/eKnob phenotypes is likely to be reflected in other facets of host cell modification and can thus be seen as a proxy for such.”.

**Minor Comments**

Line 36, NPP should be NPPs if referring to the plural.

Changed

Line 37, MC should be MCs if referring to the plural. By the way this acronym is never used in the text, it's always written 'Maurer's clefts'.

Changed

Abstract, Line 52-53, could be changed to "uncover a new KAHRP-independent..." as it currently implies (albeit weakly) that that this is the first observation of a KAHRP-independent mechanism for correct knob biogenesis. Maier et al 2008, have previously shown that knock out of PF3D7_1039100 (J-domain exported protein), greatly reduced knob size and knock out of PHISTb protein PF3D7_0424600, resulted in knobless parasites.

Correct. In line with the suggestions of another reviewer, this section has been changed.

In the Abstract it is mentioned that "Our observations open up exciting new avenues for the development of new anti-malarials." This is never really expanded upon in the rest of the paper and so seems like a bit of a throwaway line and could be left out.

Good point, changed

Line 59, WHO world malaria report should be cited here since these numbers are from the report not a paper from 2002.

Done

Line 67, Marti et al 2004 should be cited here as its published at the same time as Hiller et al 2004.

Our mistake. Done

Line 76, I suggest using either 'erythrocyte' or 'red blood cell' throughout the text not both.

We now use erythrocyte throughout

Line 80, Maier et al 2008 should be referenced here.

Done

Line 87, the authors should cite Birnbaum et al 2017 for the technique used. This is cited immediately after (line 98) in the results section but could be addressed at both points in the text.

Done

Line 123, IFAs and live cell imaging failed to detect the PFA-GFP protein and the author proposes this is due to low expression levels. However, PFA66 is expressed at ~350 FPKM in the ring stage and previous studies from your own group have visualised it using GFP before. Is there another explanation for this such as disruption of the locus here has served to greatly reduce the expression level of the fusion protein?

The truncated protein is now distributed throughout the whole erythrocyte cytosol, not concentrated into J-dots, likely making detection difficult. We wish to note that our original GFP tagged PFA66 lines (Külzer et al, 2010) did not really show a strong signal in comparison to other lines we are used to analysing. We further believe that the sub-cellular fractionation (Figure S1) demonstrates the erythrocyte cytosolic localization of the truncated PFA66. We have no evidence that truncation causes lower expression, but any future revision will include a comparison of expression levels of endogenously GFP tagged dPFA and PFA66.

Line 147, for consistency it would be best to introduce infected red blood cell (iRBC) at the beginning of the main text and use throughout the text instead of switching between 'infected human erythrocyte' and iRBC.

We agree, and have changed accordingly

Line 153, Fig S2A does not exist.

We apologise, this has been changed

Lines 156-158: Different knob morphologies are described with repeated reference to Fig2 and FigS2. Since multiple whole-cell SEM images are displayed in these figures it would be worth adding lettering and/or zoomed-in regions of interest highlighting examples of each aberrant knob type.

This has now been added to Figure S2.

Line 178-179, "Although not highly abundant in either sample, the morphology of Maurer's clefts appeared comparable in both samples (data not shown)." Why is the data not shown? Representative images of Maurer's clefts from each line should be included in the supplementary figures or this in-text statement should more clearly justified.

Figure S3 has been adjusted to also show Maurer´s clefts in more detail. An Excel table of Data can be provided if necessary.

Line 196, indirect immunofluorescence assay (IFA).

Changed

Line 201, how was the 'non-significant difference' measured? PHISTc looks quite different by eye. Rephrase the term "significant difference" as localisation of these exported proteins was compared visually rather than quantified. Otherwise, a measure of mean fluorescence intensity could be taken for each protein as a basic comparison between the two lines. In the Figure legend of S4, the term "no drastic difference", is used suggesting this was not quantified. By the way, PHISTc appears different by the represented figure.

We apologise for our use of a specific term for non-statistically verified observations. The PHISTc image the reviewer comments on, was presented incorrectly (too much brightness introduced during processing) and is now correct. We mean to say that we could not (in a blinded check), tell the difference between WT and KO IFA images. Only KAHRP (in our opinion) demonstrated a different fluorescence pattern. As KAHRP has previously been implicated in knob formation, we then analysed this phenotype in more detail. A detailed analysis of the fluorescence pattern in the other IFAs does, in our eyes, not add to the story or add any real value to our observations.

Line 213, you now have 3 versions for the word wild type, 'wild type', 'wild-type' and 'WT', best to choose one for consistency.

Changed

Line 232, 'tubelike' to 'tube-like'.

Changed

Line 279, just use 'IFA', the acronym has already been explained earlier in the text.

Changed

Line 319, 'permeation' should be 'permeability'.

Changed

Line 353, 'The action of host actin is known' to 'Host actin is known'.

Changed

Line 373, 'through their role as regulators'.

Changed

Line 402, either use 'HSP70-x' or 'HSP70-X' throughout the text.

Changed

Line 540, the speed used to pellet the samples for sorbitol lysis assay, 1600g is quite high and could reflect RBC fragility rather than direct sorbitol induced lysis. The parasitemia is also very low, and previous published methods have used ~90% parasitemia rather than the 2% used here. We are not saying the method is wrong but please check it is accurate.

We used the method of our former colleague Stefan Baumeister (University of Marburg), who is an expert in analysis of NPP, thus we are sure the method is correct. We are in fact tempted to remove the NPP data as they deflect from the main narrative of the manuscript, this being the reason we include them only as supplementary data

Line 479, 10µm should be 10 µM.

Changed

In Fig 1A, the primers A, B, C etc are not explained anywhere that I can see.

This information has now been included in the 1A Figure legend and table 2A.

Figure 1B, I do not see any clear band for the 3' integration indicated with the *. Can a better image be shown?

We apologise. Integration PCRs are notoriously challenging. Any revised manuscript will include better quality images

It seems from Fig 3G,H,I that the KAHRP puncta are bigger in ∆PFA but are as abundant as CS2. Given that KAHRP is associated with knobs how do you reconcile this with there being fewer knobs per unit area in ∆PFA compared to CS2 as in Fig 2B? The numbers of knobs/KAHRP spots/Objects per um2 seems to vary between Fig 2 and 3. Please provide some commentary about this.

We are not sure if all KAHRP spots actually label eKnobs, and it is possible that there are KAHRP “foci” that are not associated with eKnobs. We also wish to note that the data in figure 2 and 3 were produced using very different techniques. Sample preparation may lead to membrane shrinkage or stretching, and the different microscopy techniques have very different levels of resolution. For this reason we do not believe that the data from these very different independent experiments can be compared, however a comparison within a data set is possible and good practice.

In the bottom panels of Fig 4, KAHRP::mCherry appears to extend beyond the glycocalyx beyond the cell. Is this an artifact?

We checked assembly of the figure and are sure that this was not introduced during production of the figure. Our only explanation is that WGA does not directly stain the erythrocyte membrane, but the glycocalyx. A closer examination of the WGA signal reveals that it is weaker at this point (and also in the eKnobs i, ii) so potentially the KAHRP signal is beneath the erythrocyte plasma membrane, but the membrane cannot be visualised at this point.

Line 837, does this refer to 10 technical replicates or was the experiment repeated on 10 independent occasions? This should at least be done in 2 biological replicates given the range in technical replicates on the graph. Was CS2 considered as '100% lysis' or the water control described in the method? Please provide more detail.

This figure is the result of 10 biological and 4 technical replicates. A number of data points were removed as lying outside normal distribution (Gubbs test). The highest value within a biological replicate was set to 100% to allow comparison of results. This has now been corrected in the text.

Reviewer #1 (Significance (Required)):

This is a reasonably significant publication as it describes knob defects that to my knowledge have never been observed before. Importantly, the deletion of the J domain from PFA66 is genetically complemented to restore function really confirming a role for this protein in knob development. Amino acids critical for the function of the J-domain are also resolved. Apart from some minor technical and wording issues the paper is really nice work apart from one area which is the proposed partnership of PFA66 with human HSP70 for which there is not much direct evidence. If this evidence can be provided, we think this work could be published in a high impact journal. Without the evidence, it could find a home in a mid-level journal with some tempering of the claims of PFA66's interaction with human HSP70.

**Referee Cross-commenting**

There seems to be a high degree of similarity in the reviewers' comments and I think as many issues as possible should be addressed. I definitely agree that the term mentula should be not be used.

We have now adopted the suggestion of Reviewer 3, and use the term eKnobs.

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

Plasmodium falciparum exports several proteins that contain J-domains and are hypothesized to act as co-chaperones to support partner HSP70s chaperones in the host erythrocyte, but the function of these co-chaperones is largely unknown. Here the authors provide a functional analysis of one of these exported HSP40 proteins known as PFA66 by using the selection-linked integration approach to generate a truncation mutant lacking the C-terminal substrate binding domain. While there is no fitness cost during in vitro culture, light and electron microscopy analysis of this mutant reveals defects in knob formation that produces a novel, extended knob morphology and ablates Var2CSA-mediated cytoadherence. These knob formation defects are distinct from previous mutants and this unique phenotype is exploited by the authors to show that the HSP70-stimulating "HPD" motif of PFA66 impacts rescue of the altered knob phenotype. In other HSP40 co-chaperones, this motif is critical to stimulate partner HSP70 activity, suggesting that PFA66 acts as a bona fide co-chaperone. Importantly, previous work by the Przyborski lab and others has shown that deletion PfHSP70x, the only HSP70 exported by the parasite, does not phenocopy the PFA66 mutant, implying that the partner HSP70 is of host origin. The results are exciting but I have some concerns about controls needed to properly interpret the functional complementation experiments. My specific comments are below.

We agree that some control experiments are missing, and these will be included in any future revision.

**Major comments**

__

• The failure of the HPD mutant PFA66 to rescue the knob-defect is very interesting. However, the authors need to determine that the HPA mutant is expressed at the same level as the WT (by quantification against the loading controls in the western blots in Fig 1D and Fig S6H) and is properly exported (by IFA and/or WB on fractionated iRBCs, as done for the GFP-fused truncation in Fig S1A). Otherwise, the failure to rescue is hard to interpret. If these controls were in place, the conclusion that a host HSP70 is likely being hijacked by PFA66 is appropriate. This genetic data would be greatly strengthened by in vitro experiments with recombinant protein showing activation of a host HSP70 by PFA66, but I realize this may be out of the scope of the present study. Along these lines, it might be worth discussing the finding by Daniyan et al 2016 that recombinant PFA66 was found to bind human HSPA1A with similar affinity to PfHSP70x but did not substantially stimulate its ATPase activity, suggesting this is not the relevant host HSP70. This study is cited but the details are not discussed. __

As in our answer to Reviewer 1, we will examine the expression and localisation of both WT and mutant PFA66.

We are currently expressing and purifying a number of HSP40/70 combinations for exactly the kind of analysis suggested and hope to include such data in future revisions, but as the reviewer fairly notes, this is really beyond the scope of the current study.

Regarding Daniyan et al (and other) papers: The fact that PFA66 can stimulate PfHSP70x does not preclude that it also interacts with human HSP/HSC70, and indeed there is some stimulation of human HSP70. Daniyan and colleagues did steady-state assays in the absence of nucleotide exchange factors. Therefore, the stimulation of human HSP/HSC70 is not very prominent. One should either do single-turnover experiments or add a nucleotide exchange factor to make sure that nucleotide exchange does not become rate-limiting for ATP hydrolysis. This is completely independent of the results for PfHSP70-X the intrinsic nucleotide exchange rates of the studied HSP70s could be very different. Also, it is important to understand that J-domain proteins generally do not stimulate ATPase activity much by themselves but in synergism with substrates, allowing the possibility that such an in vitro assay may not reflect the situation in cellula. dditionally the resonance units in the SPR analysis for PFA66-HsHSP70 are lower than those for PFA66-PfHSP70-X. This could mean that PFA66 is a good substrate for PfHSP70-X but not for HsHSP70, but this does not mean that PFA66 does not cooperate with HsHSP70.

- The authors claim that truncation of PFA66 alters the localization of KAHRP but not the other exported proteins they evaluated by IFA (Fig S4). This seems baseless as they don't apply the same imageJ evaluation to these other proteins. Similarly, the statement that KAHRP structures "appear by eye to have a lower circularity, although we were not able to substantiate this with image analysis" is subjective/qualitative and should probably be removed.

We mean to say that we could not (in a blinded check), tell the difference between WT and KO IFA images. Only KAHRP (in our opinion) demonstrated a different fluorescence pattern. As KAHRP has previously been implicated in knob formation, we then analysed this phenotype in more detail. A detailed analysis of the fluorescence pattern in the other IFAs does, in our eyes, not add to the story or add any real value to our observations.

The statement on the circularity has been removed according to the reviewers wishes.

-The section title "Chelation of membrane cholesterol...causes reversion of the mutant phenotype in ∆PFA" seems an overstatement given the MBCD effect on the knob morphology is fairly weak and remains significantly abnormal.

The title of this section was misleading, we agree. We have retitled it “Chelation of membrane cholesterol but not actin depolymerisation or glycocalyx degradation causes partial reversion of the mutant phenotype in ∆PFA” to clarify that the reversion was only partial (as explained by the following text in the manuscript).

**Minor comments**

- The DNA agarose gel image in Fig 1B is not very convincing. Most of the bands are faint and there is a lot of background/smear signal in the lanes. Also, it would help for clarity if the primer pairs used for each reaction were stated as shown in the diagram (rather than simply "WT", "5' Int" and "3' Int").

We apologise. Integration PCRs are notoriously challenging. Any revised manuscript will feature clearer images.

- Given the vulgar connotation of "mentula", the authors might consider an alternative term.

We have now adopted the term “eKnobs” suggested by Reviewer 3.

- lines 67-69: The authors may wish to cite a more recent review that takes into account updated Plasmepsin 5 substrate predication from Boddey et al 2013 (PMID: 23387285). For example, Boddey and Cowman 2013 (PMID: 23808341) or de Koning-Ward et al 2016 (PMID: 27374802).

A fair point, we have now added Koning-Ward.

- lines 77-79: "deleted" is repetitive in this sentence.

Changed

- line 115: It might be clearer to state "endogenous PFA66 promoter"

Changed

- lines 131-132: "...these data suggests that deletion of the SBD of PFA66 leads to a non-functional protein." Behl et al 2019 (PMID: 30804381) showed the recombinant C-terminal region of PFA66 (residues 219-386, including the SBD truncated in the present study) binds cholesterol. The authors may wish to mention this along with their reference to Kulzer et al 2010 showing PFA66 segregates with the membrane fraction, suggesting cholesterol is involved in J-dot targeting.

We should have noted this connection and thank the reviewer for bringing it to our attention. This section has been revised to include this important information.

- line 198: It's not clear what is meant by "+ve" here and afterward. Please define.

We have now changed this to “structures labelled by anti-KAHRP antibodies”, or merely “KAHRP”.

- lines 749-750: "Production of PFA and NEO as separate proteins is ensured with a SKIP peptide". Translation of the 2A peptide does not always cause a skip (see PMID: 24160265) and often yields only about 50% skipped product (for example, PMID: 31164473). Because of the close cropping in the western blots in Fig 1C or S1A this is difficult to assess. Is a larger unskipped product also visible? Beyond this one point, it is general preferable that the blots not be cropped so close.

A very valid point, and in other parasite lines we have indeed detected non-skipped protein. In our case, we visualise a band at the predicted molecular mass for the skipped dPFAGFP and the commonly observed circa. 26kDa GFP degradation product. The full-length blots have now been included as supplementary data (Figure S7).

- lines 867-868: Explain more clearly what "Cy3-caused fluorescence" is measuring.

The Cy3 channel refers to anti-var2CSA staining, and we have now included this information.

- Several figure legends would benefit from a title sentence describing what the figure is about (ie, Fig legends 1, 3, 5, S1, S5 & S6)

This has been added.

Reviewer #2 (Significance (Required)):

This manuscript by Diehl et al reports on the function of the exported P. falciparum J-domain protein PFA66 in remodeling the infected RBC. Obligate intracellular malaria parasites export effector proteins to subvert the host erythrocyte for their survival. This process results in major renovations to the erythrocyte, including alteration of the host cell cytoskeleton and formation of raised protuberances on the host membrane known as knobs. Knobs serve as platforms for presentation of the variant surface antigen PfEMP1, enabling cytoadherence of the infected RBC to the host vascular endothelium. This process is of great interest as it is critical for parasite survival and severe disease during in vivo infection. The basis for trafficking of exported effectors within the erythrocyte after they are translocated across the vacuolar membrane is not well understood but is known to involve chaperones. This is a particularly interesting study in that it provides evidence in support of the hypothesis, initially proposed nearly 20 years ago, that the parasite hijacks host chaperones to remodel the erythrocyte. This is biologically intriguing and also suggests new therapeutic strategies targeting host factors that would not be subjected to escape mutations in the parasite genome. The work will be of interest to the those studying exported protein trafficking and/or virulence in Plasmodium (such as this reviewer) as well as the broader chaperone and host-pathogen interaction fields.

**Referee Cross-commenting**

I also agree with similarity in comments. Some additional discussion on the failure to localize the PFA66 truncation by live FL is warranted, as noted by reviewer #1. Seems likely that either the level of PFA66 protein is reduced by the truncation or the truncated PFA66 is dispersed from J-dots and harder to visual when diffuse instead of punctate. In either case, the complementing copy (WT or QPD) should be visualized by IFA.

As noted above, we believe our inability to visualize the truncated protein is likely due to its dispersal throughout the whole erythrocyte cytosol as opposed to lower expression levels, but we will be checking this, and also the localisation of WT and mutant PFA66 complementation chimera and expect to have this result for the next revision.

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

The data are for the most part well controlled and reveal a potential function for PFA66 in knob formation. The assays are state of the art and the data provides insight into knob formation.

However, some conclusions are not fully supported by the data. For example, 'uncover a KAHRP-independent mechanism for correct knob biogenesis' (line 52-53) is not supported by the data because PFA66 truncation could result in misfolding of KAHRP and thus lead to knob biogenesis defects.

We meant to imply that not only perturbations/absence of KAHRP lead to aberrant knobs. This is now changed to “…uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.”.

The other major issue is that despite having a complemented parasite line in hand, the parental parasite line is used as a control for almost all assays. This is a critical issue because an alternative explanation for their data would be that expression of truncated PFA66 leads to expression of a misfolded protein that aggregates in the host RBC OR it clogs up the export pathway and indirectly leads to knob biogenesis defects. It is surprising that the authors do not test the localization of dPFA using microscopy especially since it is tagged with GFP. While the complemented parasite line does revert back, this could also be due to the fact that the complement overexpresses the chaperone helping mitigate issues caused by the truncated protein.

As all virulence characteristics we monitor in this study have been verified many times in the parental CS2 parasites in the literature, we think that the best comparative control is indeed the truncated cell line. The large part of our study aimed to characterize differences in various characteristics upon inactivation of PFA66 function, and for this reason we used the parental WT line as a control. Using the complementation line would not truly reflect the effect of PFA66 truncation, as PFA66::HA was not expressed from an endogenous locus, but rather from an episomal plasmid. This itself may result in expression levels which differ from WT, and thus this parasite line cannot be seen as the gold-standard control for assaying PFA66 function.

We did indeed try to localize dPFA (lines 122-123 in the original manuscript), but were unsuccessful, likely due to diffusion of dPFA throughout the entire erythrocyte cytosol (as opposed to concentration into J-dots as the WT). For this reason we carried out fractionation instead, and could show that dPFA is soluble within the erythrocyte cytosol. This experiment additionally excludes any blockage of the export pathway as no dPFA was associated with the pellet/PV fraction. Other proteins were still exported as normal (Figure S4), further supporting a functional export pathway. Indeed, as reported by ourselves and our colleagues (particularly from the Spielmann laboratory, Mesen-Ramirez et al 2016, Grüring et al 2012), blockage of the export pathway is likely to lead to non-viable parasites as the PTEX translocon seems to be the bottleneck for export of a number of proteins, many of which are essential for parasite survival.

Reviewer #3 (Significance (Required)):

The malaria-causing parasite extensively modifies the host red blood cell to convert the host into a suitable habitat for growth as well as to evade the immune response. It does so by exporting several hundred proteins into the host cell. The functions of these proteins remain mostly unknown. One parasite-driven modification, essential for immune evasion, is the assembly of 'knob' like structures on the RBC surface that display the variant antigen PfEMP1. How these knobs are assembled and regulated is unknown.

In the current manuscript, Diehl et al target an exported parasite chaperone from the Hsp40 family, termed PFA66. The phenotypic observations described in the manuscript are quite spectacular and well characterized. The truncation of PFA66 results in some abnormal knob formation where the knobs are no longer well-spaced and uniform but instead sometimes form tubular structures termed mentulae. The mechanistic underpinnings driving the formation of mentulae remain to be understood but that will probably several more manuscripts to be deciphered.

We thank the reviewer for their kind comments, and also for the recognition that this current manuscript is merely the exciting beginning of a story!

**Major Comments:**

General comment on the use of controls: The large part of our study aimed to characterize differences in various characteristics upon inactivation of PFA66 function, and for this reason we used the parental WT line as a control. Using the complementation line as a control in this context would not truly reflect the effect of PFA66 truncation, as PFA66::HA was not expressed from an endogenous locus, but rather from an episomal plasmid. This itself may result in expression levels which differ from WT, and thus this parasite line cannot be seen as the gold-standard control for assaying PFA66 function. Our complementation experiments were initially designed to verify that phenotypic changes ONLY related to inactivation of PFA66 function and were (as unlikely as this is) not due to second site changes during the genetic manipulation process. To avoid lengthy and not really very informative analysis of the complementation line, we used knob morphology via SEM as a “proof-of-principle”. However, as the reviewer is formally correct, we have added a passage to the discussion stating that “We wish to note that we cannot unequivocally state that our complementation construct caused reversion of all the aberrant phenotypes herein investigated, however we feel it likely that all abnormal phenotypes are linked and thus our “proof of principle” investigation of knob/eKnob phenotypes is likely to be reflected in other facets of host cell modification and can thus be seen as a proxy for such.“.

Fig 3: The control used here is the parental line. Was there a reason why the complemented parasite line was not used as the control? Showing that the KAHRP localization and distribution is restored upon complementation would greatly increase the confidence in the phenotype.

Please see our general comments above.

Fig 5: The data showing a defect in CSA binding are convincing but again only the parental control is used and not the complemented parasite line. The complemented parasite line should be used as a control for the PFA binding mutant.

Please see our general comments above, and also our reponse to reviewer 1.

In 5D, the defect in dPFA seems to be occur to a lesser degree than Fig. 2C. How many biological replicates are shown in each of these figures? The figure legend says 20 cells were quantified via IFA but were these cells from one experiment? The expression of mentulae seems quite variable, while the authors mention '22%' (line 164), it seems in most other experiments, its more ~10% (5D and S6B, D-E). Were these experiments blinded?

As the reviewer is likely aware, subtle differences in parasite culture conditions, stage, fixation, SEM conditions and length of time in culture between time experimental time points can lead to variations in results. Due to the time required to generate the data for figure 5, these experiments took place months after the original (i.e. Figure 2C) analysis. It is not possible to directly compare the results of these two independent experiments, however it is possible to compare the results of the parasite lines included within each set of experimental data. Due to the time and cost involved, each of these experiments represents only one biological replicate. If required, we can include more replicates, although this is more likely to further complicate the situation due to the reasons mentioned above.

Fig S6G: The staining suggests that most PfEMP1 in is not exported, in any parasite line. Staining for PfEMP1 is technically challenging and these data are not enough to show that expression level is 'similar' (Line 279-280). It may be more feasible to use the anti-ATS antibody and stain for the non-variant part of PfEMP1 (Maier et al 2008, Cell).

It is well known that a large portion of PfEMP1 remains intracellular. This figure does not aim to differentiate between surface exposed and internal PfEMP1, but merely to show that similar TOTAL PfEMP1 is expressed in the deletion line, and also that the parasites have not undergone a switching event which would lead to loss of CSA binding ability. We will endeavour to address this in future revisions by Western Blot but wish to note that WB analysis of PfEMP1 is notoriously difficult.

Lines 320-322: The logic of why increased robustness of the RBC membrane would lead to faster parasite growth is confusing. It is likely that the loss of PfEMP1 expression leads to faster growth. The loss of NPP is minimal and may not cause growth defects in rich media.

As far as we can detect, there is no loss of total PfEMP1 expression (as verified by figure S6G), but rather a drop in surface exposure and functionality, which is unlikely to affect parasite growth rates. What we intended to say was that the NPP assay is influenced by fragility of the erythrocyte, and therefore a stiffer erythrocyte may be more resistant to sorbitol-induced lysis. As the NPP result does not really add much to the main narrative of this manuscript, we would prefer not to invest unnecessary effort for a minimal potential readout. Indeed, we are tempted to remove the NPP data as they deflect from the main findings of the manuscript, this being the reason we include them only as supplementary data

Lines 433-434: These data do support a function for HsHsp70 but these data are among many others that have previously provided circumstantial evidence for its role in host RBC modification. May be a co-IP would help support these conclusions better.

Despite all our best efforts and publications, we have been unable to detect this interaction in co-IP or crosslink experiments, although we were successful in detecting interactions between another HSP40 (PFE55) and HsHSP70 (Zhang et al, 2017). Although this is disappointing, it may be explained due to the transient nature of HSP40/HSP70 interactions. We agree that our suggestion (that parasite HSP40s functionally interact with human HSP70) is not novel (we and others have noted this possibility for over 10 years), however the challenging nature of the experimental system makes it very difficult to show direct evidence of the importance of this interaction in cellula. Over the past decade we have use numerous experimental approaches to try to address this but have always been confounded by technical challenges. In 2017 the corresponding author took a sabbatical to attempt manipulation of hemopoietic stem cells to reduce HSP70 levels in erythrocytes, however it appears (unsurprisingly) that HsHSP70 is required for stem cell differentiation, and thus this tactic was not followed further. The authors believe that, due to the lack of the necessary technology, indirect evidence for this important interaction is all that can realistically be achieved at this time, and this current study is the first to provide such evidence.

We would further like to note that a successful co-IP would not directly verify a functional interaction between PFA66 and HsHSP70, but could also reflect a chaperone:substrate interaction between these proteins, and is therefore not necessarily informative.

**Minor Comments:**

Fig1: The bands are hard to see in WT and 3’Int. May be a better resolution figure would help? Also, the schematic shows primers A-D but the figure legend does not refer to them. It would be useful to the reader to have the primers indicated above the PCR gel along with the expected sizes.

We apologise. Integration PCRs are notoriously challenging. Any revised manuscript will contain clearer images.

Fig S1: The NPP data could be improved if tested in minimal media. It has been shown that NPP defects do not show up in rich media (Pillai et al 2012, Mol. Pharm. PMID: 22949525). Does complementation restore NPP and growth rate?

As the NPP result does not really add much to the main narrative of this manuscript, we would prefer not to invest unnecessary effort for a minimal potential readout. Indeed, we are tempted to remove the NPP data as they deflect from the main findings of the manuscript, this being the reason we include them only as supplementary data. Likewise the complementation experiments are, we feel, unnecessary.

Fig 4: It is not clear what the line scan analysis are supposed to show. What does ‘value’ on the y-axis mean?

These are line scans of fluorescence intensity (arbitrary units) along the yellow arrows shown on the fluorescent panels. This is now indicated in the figure legend.

Fig S5D: Maybe it was a problem with the file but no actin staining is visible.

The actin stain was visible on the screen, but unfortunately not in the PDF. We have applied (suitable) enhancement to produce the images in the new version.

Fig 6: A model for mentulae formation is not really proposed. Only what the authors expect the mentulae to look like.

We have changed the legend to reflect this “Figure 6. Proposed model for eKnob formation and structure.”. We do propose that runaway extension of an underlying spiral protein may lead to eKnobs, thus would like to keep the word “formation”.

Lines 312-313: It is not clear what 'highly viable' means, parasites are either viable or not.

This has been changed.

Lines 400-405: The authors forgot to cite a complementary paper that showed no virulence defect upon 70x knockout or knockdown (Cobb et al mSphere 2017). Those data also support a role for HsHsp70.

We apologise for the omission. This is now included.

**Referee Cross-commenting**

I agree, the comments are pretty similar. The authors could tone down their conclusions or add more data to support their conclusions. May be call them elongated knobs or eKnobs, instead of mentula? __

We have now removed the offending term and use eKnobs.

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Review of "Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence." by Diehl et al for Review Commons.

Major Comments.

1. In this paper the function of Plasmodium falciparum exported protein PFA66, is investigated by replacing its functionally important dnaJ region with GFP. These modified parasites grew fine but produced elongated knob-like structures, called mentulae, at the surface of the parasites infected RBCs. Knobs are elevated platforms formed by exported parasite proteins at the surface of the infected RBC that are used to display PfEMP1 cytoadherance proteins which help the parasites avoid host immunity. The mentulae still display some PfEMP1 and contain exported proteins such as KAHRP but can no longer facilitate cytoadherence. Complementation of the truncated PFA66 with full length protein restored normal knob morphology however complementation with a non-functional HPD to QPD mutant did not restore normal morphology implying interaction of the PFA66 with a HSP70 possibly of host origin is important for function. While a circumstantial case is made for PFA66 interacting with human HSP70 rather than parasite HSP70-x, is there any direct evidence for this eg, protein binding evidence? I feel that without some additional evidence for a direct interaction between PFA66 and human HSP70 then the paper's title is a little misleading.
2. Was CSA binding restored upon complementation of ∆PFA with the full-size copy of PFA66?

Minor Comments

1. Line 36, NPP should be NPPs if referring to the plural.
2. Line 37, MC should be MCs if referring to the plural. By the way this acronym is never used in the text, it's always written 'Maurer's clefts'.
3. Abstract, Line 52-53, could be changed to "uncover a new KAHRP-independent..." as it currently implies (albeit weakly) that that this is the first observation of a KAHRP-independent mechanism for correct knob biogenesis. Maier et al 2008, have previously shown that knock out of PF3D7_1039100 (J-domain exported protein), greatly reduced knob size and knock out of PHISTb protein PF3D7_0424600, resulted in knobless parasites.
4. In the Abstract it is mentioned that "Our observations open up exciting new avenues for the development of new anti-malarials." This is never really expanded upon in the rest of the paper and so seems like a bit of a throwaway line and could be left out.
5. Line 59, WHO world malaria report should be cited here since these numbers are from the report not a paper from 2002.
6. Line 67, Marti et al 2004 should be cited here as its published at the same time as Hiller et al 2004.
7. Line 76, I suggest using either 'erythrocyte' or 'red blood cell' throughout the text not both.
8. Line 80, Maier et al 2008 should be referenced here.
9. Line 87, the authors should cite Birnbaum et al 2017 for the technique used. This is cited immediately after (line 98) in the results section but could be addressed at both points in the text.
10. Line 123, IFAs and live cell imaging failed to detect the PFA-GFP protein and the author proposes this is due to low expression levels. However, PFA66 is expressed at ~350 FPKM in the ring stage and previous studies from your own group have visualised it using GFP before. Is there another explanation for this such as disruption of the locus here has served to greatly reduce the expression level of the fusion protein?
11. Line 147, for consistency it would be best to introduce infected red blood cell (iRBC) at the beginning of the main text and use throughout the text instead of switching between 'infected human erythrocyte' and iRBC.
12. Line 153, Fig S2A does not exist.
13. Lines 156-158: Different knob morphologies are described with repeated reference to Fig2 and FigS2. Since multiple whole-cell SEM images are displayed in these figures it would be worth adding lettering and/or zoomed-in regions of interest highlighting examples of each aberrant knob type.
14. Line 178-179, "Although not highly abundant in either sample, the morphology of Maurer's clefts appeared comparable in both samples (data not shown)." Why is the data not shown? Representative images of Maurer's clefts from each line should be included in the supplementary figures or this in-text statement should more clearly justified.
15. Line 196, indirect immunofluorescence assay (IFA).
16. Line 201, how was the 'non-significant difference' measured? PHISTc looks quite different by eye. Rephrase the term "significant difference" as localisation of these exported proteins was compared visually rather than quantified. Otherwise, a measure of mean fluorescence intensity could be taken for each protein as a basic comparison between the two lines. In the Figure legend of S4, the term "no drastic difference", is used suggesting this was not quantified. By the way, PHISTc appears different by the represented figure.
17. Line 213, you now have 3 versions for the word wild type, 'wild type', 'wild-type' and 'WT', best to choose one for consistency.
18. Line 232, 'tubelike' to 'tube-like'.
19. Line 279, just use 'IFA', the acronym has already been explained earlier in the text.
20. Line 319, 'permeation' should be 'permeability'.
21. Line 353, 'The action of host actin is known' to 'Host actin is known'.
22. Line 373, 'through their role as regulators'.
23. Line 402, either use 'HSP70-x' or 'HSP70-X' throughout the text.
24. Line 540, the speed used to pellet the samples for sorbitol lysis assay, 1600g is quite high and could reflect RBC fragility rather than direct sorbitol induced lysis. The parasitemia is also very low, and previous published methods have used ~90% parasitemia rather than the 2% used here. We are not saying the method is wrong but please check it is accurate.
25. Line 479, 10µm should be 10 µM.
26. In Fig 1A, the primers A, B, C etc are not explained anywhere that I can see.
27. Figure 1B, I do not see any clear band for the 3' integration indicated with the *. Can a better image be shown?
28. It seems from Fig 3G,H,I that the KAHRP puncta are bigger in ∆PFA but are as abundant as CS2. Given that KAHRP is associated with knobs how do you reconcile this with there being fewer knobs per unit area in ∆PFA compared to CS2 as in Fig 2B? The numbers of knobs/KAHRP spots/Objects per um2 seems to vary between Fig 2 and 3. Please provide some commentary about this.
29. In the bottom panels of Fig 4, KAHRP::mCherry appears to extend beyond the glycocalyx beyond the cell. Is this an artifact?
30. Line 837, does this refer to 10 technical replicates or was the experiment repeated on 10 independent occasions? This should at least be done in 2 biological replicates given the range in technical replicates on the graph. Was CS2 considered as '100% lysis' or the water control described in the method? Please provide more detail.

Significance

This is a reasonably significant publication as it describes knob defects that to my knowledge have never been observed before. Importantly, the deletion of the J domain from PFA66 is genetically complemented to restore function really confirming a role for this protein in knob development. Amino acids critical for the function of the J-domain are also resolved. Apart from some minor technical and wording issues the paper is really nice work apart from one area which is the proposed partnership of PFA66 with human HSP70 for which there is not much direct evidence. If this evidence can be provided, we think this work could be published in a high impact journal. Without the evidence, it could find a home in a mid-level journal with some tempering of the claims of PFA66's interaction with human HSP70.

Referee Cross-commenting

There seems to be a high degree of similarity in the reviewers' comments and I think as many issues as possible should be addressed. I definitely agree that the term mentula should be not be used.

The work she does is incredibly important. It's super inspiring to see that harmful traditions can indeed be changed, and changing them will transform the whole community into a better version of itself. I love how Ntaiya's work includes and involves the whole community so that they can instantly see the benefits of change. The world needs to see this and I see her as a hero. This continued narrative that women need helping and empowerment because they for some reason need this more than men is just continuing to make women feel less than men. It doesn't empower them at all. Empower a PERSON and empower a community.

It is terrible that this is accurate. It's not even just on the way to school. Sadly it can happen anywhere like the mall, gym, or really anywhere. We need to be more aware of this so we can fix it and so girls don't feel this way. It's not ok that that girls are nervous about walking to school or the gym or even when they want to leave the house. They should be focused in what they need to get done and not how to be safe. This is the biggest thing we need to fix.

Sometimes we may think "it's just an idea or a thought", but that idea can change so many lives and have a positive impact. There are many people out there who go through rough paths and don't have the right guidance or support team, we can be their voice and show them the way.

It's very impressive that she had the motivation to not only build a school, but also to have the vision to completely rebuild her community. Like she said, the school is a very good foundation because it brings together like-minded kids who will be able to transform their community. I'm also interested to know if she had any other ideas other than building a school. Maybe a group of volunteer workers similar to boy/girl scouts could work to help the community as well.

I agree 100%. Girls (and boys) are what the future depends on. In order to become someone, hold a degree, or make a change there needs to be education behind it all. A community is stronger if school is in the mix. If you think about it, we wouldn't want to live in a community where there were no doctors, where would we take our children if they get sick? School is definitely just the start, but it's the start to a brighter future for ourselves and families. It opens up life changing opportunities.

It is incredible to hear that change is possible and it is happening through education. Just one person can make a huge impact, like we just witnessed through this Ted talk. Not only did the speaker inspire her community, she inspired me! Sometimes I think that my voice or opinions don’t matter because there are so many people who are thinking like me or simply won’t listen. To think that this woman had a goal that was “taboo” in her community but turned out that many other females in her community had the same goal. And now change is occurring in her community. This Ted talk has made me realize that maybe my voice does matter and maybe change can happen if I speak up on my thoughts instead of belittling them.

its interesting to see the new perspective that the speaker has opened up to men in this country. by introducing education to people's daughters it triggers an emotional response of pride and shows them what they can create and achieve. By opening this idea up to fathers first, it's only natural that it becomes a common understanding for men all over the community, by giving something for them to relate and open up to