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  1. Evaluation Summary:

    This manuscript explores the relationship between stiffness of migrating cells and the stiffness of the substrate of which they are migrating. Specifically, the authors show that the actin-bundling protein Fascin limits the levels of activated myosin to alter stiffness of the migratory substrate. This is a well-controlled study, and with some additional clarifications of methods and extensions to some analyses that would strengthen the paper, it will be of broad interest to cell biologists, particularly those studying cell migration.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    The authors of this manuscript were trying to determine whether the stiffness of cells can influence the stiffness of the substrate the cells were migrating on and how this overall affect how cells migrate. Using the Drosophila border cell migration system the author found that by eliminating fascin expression in border cells, non-muscle myosin II activity increased, suggesting an antagonistic relationship between this actin bundler and non-muscle myosin II contractility. Further, increasing non-muscle myosin II activity in border cells increased non-muscle myosin II contractility in nurse cells (the substrate through which they migrated, suggesting a feedback mechanism between cells and their substrate.

    Some of the strengths of this manuscript capitalizing on Drosophila genetics of border cell migration allowing them to manipulate fascin expressing in different tissues (germline vs. somatic) and then determining how this affect border cell migration. Using atomic force microscopy the authors where also able to measure the biophysical properties of the egg chambers and thus were able to correlate these changes to the genetic/pharmacological manipulations. Increased non-muscle myosin II activity as assessed by increases in phosphomyosin staining inhibited border cell migration. Fascin-null border cells increase non-muscle myosin two activity in both border cells and the nurse cells through which they migrate. This could not be rescued by expression of phosphomimetic version of fascin that has been shown to inhibit fascin's bundling activity. This further suggests that it is the changes to actin architecture that is leading to changes in cellular contractility. The author's also demonstrate that this increased cellular contractility feedback loop is more generalizable as expression of constitutively active Rok (Rho kinase) also led to similar phenotype. A weakness of this manuscript is that the authors did not address any changes to the actin architecture, (actin-based structures) that likely correlated with the loss of fascin, nor did they explore exactly how increased contractility in border cells is communicated to the nurse cells. Overall, the data presented here support the authors claims.

    Increased fascin expression has been correlated with increased metastases as has increased cellular contractility, thus the results presented here begin to piece together this relationship. Furthermore, the feedback between cells and the role they play on augmented their substrates stiffness is also critical to number of migratory processes including metastasis.

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  3. Reviewer #2 (Public Review):

    In this manuscript, Lamb et al. investigated the role of Fascin in regulating myosin activity and cell stiffness during Drosophila border cell collective migration. Loss of fascin results in higher levels of activated myosin (p-MRLC), and altered myosin dynamics, in border cells and in the nurse cells, the cellular substrate upon which the border cells migrate. Further, loss of fascin increases the stiffness of the nurse cells as measured by atomic force microscopy (AFM). Reducing myosin activity, either pharmacologically or by RNAi of myosin, suppresses the delayed migration found in fascin mutants. Phosphorylation of Fascin is important for regulation of myosin, as a phosphorylation-mutant that disrupts actin-bundling activity (Fascin-S52E) fails to suppress the increased levels of activated myosin found in fascin mutants. The authors then perform cell type-specific RNAi knockdown of fascin. Knockdown of fascin in nurse cells results in elevated p-MRLC in nurse cells, though not in border cells. As expected, nurse cell fascin knockdown increased the stiffness of nurse cells. In contrast, knockdown of fascin in border cells elevated p-MRLC in both border cells and in nurse cells, and non-autonomously increased the stiffness of the nurse cells. Restoring Fascin in border cells (somatic cells) of fascin mutants reduced the stiffness of nurse cells to normal levels. The authors conclude that Fascin in border cells regulates the stiffness of their nurse cell migratory substrate by limiting the levels of activated myosin. This in turn promotes normal in vivo cell migration.

    Overall, this manuscript presents novel findings with broad interest to the fields of collective cell migration and actomyosin regulation. Many of the results are well-controlled and support the conclusions. The finding that Fascin limits the levels and dynamics of myosin in a migrating collective in vivo is generally convincing. Moreover, control of substrate stiffness by migratory cells has not been well explored.
    However, there are several key experiments that can be clarified with additional data or analyses to support the conclusions.

    First, because border cells are surrounded by nurse cells, the authors would need to more explicitly indicate how they measured p-MRLC levels in border cells versus nurse cells. How p-MRLC "puncta" are measured, and in particular what the authors mean by "length" of puncta, would need to be clarified. More notably, the p-MRLC staining looks quite different from the MRLC-GFP images shown. MRLC-GFP at the membrane should represent the phosphorylated and active pool of myosin, but somehow looks more disperse both in control and fascin mutants compared to p-MRLC staining.

    Second, the authors would need to clarify how many stage 9 follicles (egg chambers) they measured in each AFM experiment and for each genotype. In the materials and methods, it says that 2-3 follicles were measured for each experiment. This seems like a low number, although it is a technically challenging method. A recent study from the Bilder lab (Chen et al., Nature Communications 2019) appeared to measure at least 8 follicles per genotype. This is particularly important, since the data points for the stiffness measurements are generally quite broad and overlap between controls and mutants, e.g., with ~5-15 kPa in control nurse cells and ~15-45 kPa in fascin null nurse cells (e.g., Figure 2D; but also Figure 5G). There may be technical reasons why this number of follicles was measured, but it would be helpful to describe the reasoning in more detail.

    Third, the non-autonomous control of nurse cell substrate stiffness, and levels of activated myosin in nurse cells, by loss of fascin in border cells (and by overexpression of activated Rho-kinase in border cells) is interesting and novel. The authors propose that the border cells regulate the stiffness of nurse cells to facilitate border cell migration. Further clarification of this phenotype would strengthen the manuscript. Specifically, it is unclear whether the authors find elevated p-MRLC in nurse cells that are in front of the border cells, or a more general elevation of p-MRLC levels (and presumably nurse cell stiffness).

    Finally, the authors use pharmacological inhibition of myosin and/or activation of myosin to rescue border cell migration (Figure 3 and Figure 3, figure supplement 1). The Y-27632 drug and MRLC-RNAi should be fine. However, Drosophila myosin has been reported to be insensitive to blebbistatin (Straight et al., Science 2003; Heissler et al., FASEB J. 2015). Therefore, caution should be taken in assessing the results with blebbistatin in Drosophila.

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  4. Reviewer #3 (Public Review):

    The authors had previously demonstrated that fascin was critical for border cell migration in Drosophila oogenesis, but were not able to fully identify the definitive molecular underpinnings.

    Here, the authors use genetic tools enabled by Drosophila system to selectively remove fascin from specific cell types, and then measure myosin 2 RLC phosphorylation, as a readout for contractility, in both the border and nurse cells. This primary method is complemented with migration analysis, rescue experiments, and what appears to be a very challenging AFM experiment to measure cell stiffness in the follicle! By doing this, they are able to modify fascin in the border cells and determine the impact on their substrate (the nurse cells).

    While taking advantage of the wonderful toolset enabled by Drosophila, this manuscript, in its current form, would benefit from a better explanation of which cells are being manipulated in each experiment. Non-Drosophila biologists might struggle with some of the terminology and could use a more "guided tour" of the work. In addition, it would be very interesting to know more about where actin is in the different cell types upon manipulation of fascin.

    Despite these limitations, the authors are able to demonstrate that fascin is somehow regulating myosin activation in multiple cell types. This is almost certainly happening in many other cells. A future challenge lies in understanding how direct this link is. It is feasible that altering the bundling of actin could be altering many myosin-modulating proteins.

    While previous works have demonstrated that migrating cells can alter the stiffness of ECM at their anterior (van Helvert and Friedl, 2016; Doyle et al. 2021), this work demonstrates this concept in cells migrating on other cells, requiring an added level of complexity, and demonstrates it in a living organism. While many studies have looked at cell migration in 2D and 3D ECM environments, semi-recapitulating physiological settings, fewer studies have carefully investigated cells migrating on other cells, as must happen with high frequency throughout multicellular life. Collectively then, this is an exciting addition to our understanding of cell migration.

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