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  1. Evaluation Summary:

    This paper will be of broad interest to scientists who study collective cell migration and cytoskeletal dynamics. The findings of this paper connect proteins involved in planar polarity to the actin protrusive machinery which establishes an axes for polarized collective cell migration. The data presented largely supports the claims of the authors who take advantage of quantitative imaging techniques and Drosophila genetics to establish this connection.

    (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 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Williams and colleagues explored the mechanisms that govern collective cell migration using Drosophila egg chambers as model system. The follicular epithelia of developing egg chambers undergoes a form of collective cell migration, migrating in a polarized manner on top of a basement resulting in the rotation of the entire egg chamber. As these cells rotate they also secrete more basement membrane protein that restricts the growth of the egg chamber into a single axis. This rotation is driven by lamellipodial protrusions of these epithelia cells which is planar polarized resulting in orchestrated collective cell migration. The atypical cadherin Fat2 was previously determined to be required for both planar polarity and cell migration. Through quantitative imaging techniques and the generation of mosaic mutants the authors of this study where able to demonstrate that Fat2 works in trans with WAVE complex, coupling planar with the actin protrusive machinery. More specifically, Fat2 establishes actin-bases protrusions at the leading-lagging axis of the migrating epithelia where the lagging edge with Fat2 enrichment stabilizes the WAVE complex in the leading edge of the proceeding cell. The use of fat2 null allele and mosaic clones of this allele helped to establish this cell-autonomous relationship. Loss of Fat2 led to a global destabilization of the WAVE complex which could now be found in protrusions on all sides of the cell instead of just the leading and lagging edges. The authors went on to demonstrate that the intracellular domain of Fat2 is dispensable and that the extracellular domain colocalizes with the WAVE complex. This finding does raise questions as to exactly how the extracellular domain is able to stabilize the WAVE complex which were not addressed in this study.

    The claims of the manuscript were largely supported by the data, however, the strength of this manuscript is also somewhat of a weakness. The combination of Drosophila genetics and quantitative images techniques they developed helped to define the role of Fat2 in this system show that it links planar polarity to the cellular protrusion machinery. However this same rigor of quantification was not carried out throughout the manuscript and some of the findings are more qualitative in nature. This manuscript, while convincing and compelling, could benefit from the inclusion of more quantitative approaches to better support their claims.

    This manuscript draws a clear connection between the planar polarity machinery and the actin protrusion machinery and how they both correlate with polarized collective cell migration. The impact of these findings will likely transcend this manuscript and will influence future studies seeking to further our understanding of collective cell migration. The quantitative methods used will also be of great use to the larger cell biology community.

  3. Reviewer #2 (Public Review):

    In this paper, Williams et al. seek to understand how Fat2 promotes formation and alignment of protrusions during Drosophila follicular epithelial cell migration in the egg chamber. The authors investigate how Fat2 regulates formation and localization of membrane protrusions by examining WAVE complex localization and activity in fat2 mutants. They find that the WAVE complex is is planar polarized to the leading edges of migrating follicle cells and is enriched at protrusions, which form at the leading edge. Furthermore, in fat2 mutant egg chambers, the WAVE complex is more uniformly distributed and protrusions are more transient and present around the cell perimeter. Using mosaic clones of fat2 mutant cells, the authors show Fat2 at the trailing edge acts non-autonomously to localize the WAVE complex to the leading edge of the cell behind. Fat2 puncta colocalize with the WAVE complex at positions of protrusion, suggesting that Fat2 acts very locally, at the level of punctate assemblies, to promote during protrusion formation. Furthermore, the authors show that the Fat2 intracellular domain is dispensable for WAVE-Fat2 puncta colocalization and protrusion formation. From these data, the authors propose the model that Fat2, at the trailing edge of a cell acts at the scale of puncta to localize the WAVE complex in puncta at the leading edge across the cell-cell interface.

    The major conclusions from this study are generally well supported by the data and the authors test alternative hypotheses where appropriate. Additionally, the authors developed new computational tools to segment and quantify protrusion dynamics. However, there are some aspects data that could be further quantified to better show the trends shown in the representative images. Specifically, Fig. 4D-E, S4, and S7 rely solely on representative images and it would greatly strengthen conclusions drawn from the data shown in these figures to include some quantification of these data.

  4. Reviewer #3 (Public Review):

    In this manuscript, the authors investigate the molecular mechanism of collective cell migration in the Drosophila egg chamber. They had previously identified planar cell polarity molecules (Fat2 and Lar) that coordinate the movement of leader and trailing cells, but how Fat2 regulates the protrusion and polarity of the trailing cell was not understood. Here they provide evidence that Fat acts in trans to restrict WAVE complex recruitment to a single surface of the trailing cell.


    The Drosophila epithelial follicular cells are a powerful system for probing the mechanism of collective cell migration given the exquisite coordination of movement and the lack of true leaders and followers in this edgeless topology. The authors had previously demonstrated that Fat2 at the back of a given cell facilitates protrusion of the cell behind it, but the basis of this facilitation was not known. By probing the spatial dynamics of a likely regulator of protrusion (WAVE complex), the current work represents an important extension of their previous findings. The authors find that Fat2 foci show a strong colocalization with WAVE foci in adjacent cells, suggesting sub-micron-scale coordination between Fat2 foci and WAVE-based protrusion in adjacent cells. In the absence of Fat2, WAVE-based protrusions are still observed, but they are shorter lived and lack polarity, leading to a lack of coordinated collective movement. The paper is very clearly written, and they employ nice quantitative image analysis schemes to understand the microscopy data. The authors make powerful use of clonal analysis to understand molecular function and protein distribution in the complex context of collective cell migration. The findings are likely to be of general interest to the cell migration and development communities.


    One of the most significant findings of this work is that Fat2 is not required for WAVE-based protrusions in the trailing cell, but it focuses protrusive activity to the Fat2-opposed membrane. The basis of this focusing is not known. The authors suggest this restriction is accomplished by Fat2-based sequestration of WAVE, but this seems unlikely given the amount of recruited versus free WAVE complex and the observation that significant WAVE depletion is needed to see a phenotype in other cells. This central point of how Fat2 restricts protrusions is not yet sufficiently bolstered.

    It is unfortunate that the story is not as simple as loss of Lar blocks Fat2-WAVE communication. This would have been the most direct molecular link from Fat2 to the WAVE complex, but of course it can be difficult to identify redundant molecular mechanisms. In the absence of this, it would be valuable to have a little more insight into the Fat2/WAVE coupling. Fat2 foci are characterized as being at the tips of filopodia, though show that filopodia are dispensable for follicular migration. Given that WAVE complex is not typically observed at filopodial tips, I wonder whether these are really filopodia.