1. This review reflects comments and contributions by Elnaz Fazeli, Rachel Lau, Gregory Redpath, Mugdha Sathe and Wasim Sayyad.

    The preprint reports the use of two color-3D STORM imaging, quantitative live-cell TIRF microscopy, fluorescence lifetime, and atomic force microscopy to explore the load-adapted endocytic mechanism in actin networks of mammalian cells. The results show that the actin network forms at the base of the endocytic pit and grows around and over the tip of the clathrin coat. The branched actin network generator Arp2/3 is required for normal clathrin-mediated endocytosis (CME). CME is perturbed when the tension in the plasma membrane increases which also increases the actin coverage over the clathrin coat.

    This is a solid study; the manuscript reports appropriate control experiments and statistical analysis.

    Specific comments

    skin-melanoma cell line (SK-MEL-2)’ - Was this cell line chosen due to its availability in the lab? It would be interesting to see whether the results would be similar in normal human cell lines and not a cancer-specific mechanism. Perhaps this could be mentioned in the discussion as future work?

    ‘​​The standard deviations of positions of single fluorophores were 10 nm in-plane for XY and 19 nm in-depth for the Z dimension’ - This is obviously great resolution for STORM, but the manuscript reports using primary and secondary antibodies to detect clathrin etc. This will add a reasonable distance between the fluorophore and clathrin (or whatever protein) which will be relevant at this resolution. This should perhaps be mentioned as a potential caveat, or why it does not matter.

    Figures 1 and 4 - The schematics are great and make the interpretation of the images so much easier!

    Figure 1 ‘shape index’ - How was the shape index measured? Would it be possible to expand on how the two right columns of Figure 1E were separated into three categories (either in the Results or the Methods section).

    We detected actin associated with 74% of the clathrin coats, which is comparable to previous measurements for the same cell type that we made of endocytic traces in live cells of dynamin2-GFP events associated with actin-RFP, given that our super-resolved images are snapshots of a time-lapse event‘- What is the aspect ratio distribution of Clathrin coats that lacked actin associated with it?

    Coat with height similar to its width) (Figs. 1D’ - By looking at the fraction of shallow, u-shaped and omega-shaped clathrin coat shapes under steady state vs when membrane tension is altered, is it possible to see whether some stages are getting enriched or depleted?

    Figure 1F, ‘There was no significant correlation between actin/coat coverage and coat shape for any of the three geometries’ - What is the size/volume distributions of CCPs for a given geometry?

    Figure 1H ‘Indeed, as a function of actin/coat coverage, Dz increased from negative values to near zero’ - Conversely, should Dz decrease with regard to width of the clathrin pits?

    Consistent with our conclusions about where actin assembly occurs at CCPs, N-WASP localized to the base of both shallow and highly curved clathrin coats (Fig. S6A). More unexpectedly, at some CME sites, N-WASP covered the entire clathrin coat irrespective of coat geometry (Fig. S6B)’ - The N-WASP data is very interesting, recommend completing quantification similar to the actin data: if the N-WASP distribution come out the same as actin distribution, where it is clear it is "nucleated" at the bottom of a clathrin structure, that would be a very interesting result that supports the findings regarding actin.

    the CLTA-TagRFP-TEN and DNM2-eGFPEN lifetimes began to recover, most likely reflecting cellular adaptation to the hypotonic treatment’ - The recovery is noticeable for clathrin and dynamin2 lifetime, but is not reflected in endocytosis initiation and completion. Could some comments be provided about this?

    DNM2-eGFPEN-only events showed a moderate response to elevated membrane tension’ - Recommend referencing Fig S7 for this, and perhaps saying "moderate, statistically significant response to elevated membrane tension only with 75 mOsm hypotonic media" just to highlight the specificity of the response to clathrin-associated dynamin.

    Figure 3 Clathrin coat images for quantitative analysis were collected from at least 3 cells for each condition’ - Please report across how many independent experiments.

    2d-3D STORM’ - Should this read 2c-3D STORM?

    the average clathrin coat height decreased to 96 nm ± 24 nm’ - It seems that hypotonic shock in the background of arp2/3 inhibition actually rescues the Clathrin coat height back to DMSO levels. What is the height distribution with just hypotonic shock? Moreover, when arp2/3 is inhibited do all the CCPs lose actin around them? If yes, then is there any difference in shape distribution? If no, how does the actin distribution fare with regard to control?

    ‘​​actin grows higher in the z-dimension around clathrin coats’ - How will the actin distribution look under dynamin k44a (ideally temperature sensitive) mutant where the CCPs are stalled? Will the cell infer this as 'high tension'?

    We showed quantitatively that actin assembly and organization adapt to changes in membrane tension, which we measured by AFM membrane tether pulling’ - Will this actin recruitment change in hypertonic shock in a manner opposite to the hypotonic shock?

    such differences might reflect subcellular, local load variation resulting from variance in such factors as membrane tension and cell adhesion’ - Would it be possible to use actin coverage around CCPs to predict local tension? Computational modelling might be helpful in future studies.

    the U-shaped stage, consistent with the effects of actin assembly inhibition reported for other cell types’ - Does dynamic recruitment care if the CCPs have U vs omega shape? It might be interesting to see if dynamin localisation in altered in Z.

    for example by increasing contact of filaments associated with the coated pit with membrane-associated N-WASP-Arp2/3 complex’ - Through live TIRF imaging, Taylor & Merrifield et al. showed that 30-40% of CCPs showed N-WASP recruitment, so the question arises as to whether there are other regulators involved. Another question to consider is enrichment of BAR domain proteins. Would it be possible to comment on whether at high tension, more BAR domain proteins could also be recruited to help with membrane bending?


    • Data analysis, statistical analysis and data plotting’ - The following could come in this section: fluorescence lifetime, fluorophores used, shape index measurements, software used for image handling and image preprocessing, kymograph generation, STORM image reconstruction process and quality control, how was the data plotted and what parameters were used. This article discusses reporting for reproducible microscopy workflows: https://www.nature.com/articles/s41592-021-01156-w. Suggest a mention to whether the code and the original images are available at a repository (e.g. Github, Zenodo).
    • It may be worth adding some comments on why STORM was used in the study, the need for super-resolution imaging rather than confocal and why STORM in particular. Also some comments about the limitations of traction force microscopy over atomic force microscopy, owing to it being more suitable for measuring tension on the ventral surface where the endocytic events’ images were acquired.
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