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  1. Author Response:

    Reviewer #1 (Public Review):

    The manuscript is clearly written, the data are largely of sufficient quality, and the findings are certainly of interest to the endosomal research community. I also agree with the model the authors propose.

    We thank the reviewer for their appreciation of this study.

    One weak point of the study is the dependence on microscopic techniques to analyze integrin surface levels and endosomal recruitment of the retriever complex. The study would have benefited from additional methods to confirm the microscopy data.

    We agree. We have now added an experiment showing the changes in the surface levels of β1-integrins by flow-cytometry. This technique allowed us to easily sample 10,000 cells per experiment (New Figure 1G).

    It would also be good if the authors could confirm their inhibitor studies with genetic suppression/deletion of PIKfyve, ideally followed by rescues with a kinase deficient mutant.

    We agree. We now include experiments where we depleted PIKfyve with siRNA and rescued PIKfyve by exogenous expression of siRNA resistant PIKfyve. Depletion of PIKfyve caused a decrease in surface levels of β1- and α5-integrin by 21% and 17% respectively (Figure S3), which is very similar to what we observed by immunofluorescence.

    Later in the manuscript, we also tested changes in the endosomal localization of COMMD1 due to PIKfyve inhibition (Figure 7), which resulted in a 22% decrease. We now include new experiments showing PIKfyve depletion. For the depletion studies, we also tested rescue by PIKfyve expression. PIKfyve depletion resulted in a statistically significant but modest lowering of COMMD1 endosomal localization by 15%. This decrease was rescued back to basal levels on endosomes by re-expression of PIKfyve (Figure S8).

    Since PIKfyve has so many roles, and since the PIKfyve inhibitor has been shown to be specific by so many labs, we consider acute PIKfyve inhibition preferable. Long-term depletion studies are more likely to result in effects are indirect or provide cells with a chance to adapt.

    The authors rely on microscopy of integrin beta 1 for most of their data. However, SNX17 and the retriever complex are not required for the recycling of all beta 1 integrins (Steinberg et al., 2012). In HeLa cells, it is mainly integrin alpha 5/beta1 that is recycled by SNX17. Therefore, the other beta 1 integrins that recycle SNX17 independently tend to mask the recycling phenotypes caused by the loss of SNX17/retriever. I think that the authors could detect a much more pronounced recycling phenotype upon PIKfyve inhibition if they stained integrin alpha 5 instead of integrin beta 1. Does integrin alpha 5 "get stuck" in a LAMP1 positive compartment similar to what Steinberg et al., 2012 or McNally et al., 2017 describe in their studies? These two studies clearly show that almost all integrin alpha 5/beta 1 accumulates in a LAMP1 or LAMP2 positive compartment upon loss of retriever/SNX17 function. If the authors are correct in their assumptions, this should be happening upon loss of PIKfyve activity. One could use the Abcam antibody against integrin alpha 5 that was used in the McNally et al. study as it works very well.

    We did not see pronounced differences in surface levels of α5-integrin vs. β1-integrin upon inhibition or depletion of PIKfyve. For example, PIKfyve siRNA treatment lowered the surface levels of α5-integrin and β1-integrin in the similar range by 21% and 17 % respectively, as measured by immunofluorescence microscopy.

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  2. Evaluation Summary:

    The authors investigate the role of the PI3P 5-kinase protein (PIKfyve) in endosome to cell surface recycling. They report that PIKfyve function is necessary for cell migration and endsomal recycling of integrin proteins via the SNX17-Retriever pathway. The findings will be of interest to the endosomal research community.

    (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. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    In the present study Giridharan et al. identify a novel role for the phosphoinositide kinase PIKfyve in endosomal recruitment of the retriever complex and recycling of integrins as well as cell migration. Inhibition of PIKfyve with a specific inhibitor reduces endosomal levels of PI3,5P2 which in turns reduces the endosomal recruitment of VPS35L and other retriever components. The sorting nexin SNX17, which preferentially binds to PI3P, is not affected by PIKfyve inhibitors but cannot recycle integrins in the absence of endosomal retriever components. Based upon various inhibitor experiments, the authors propose a model of sequential recruitment of SNX17 and the WASH complex by PI3P followed by a shift to PI3,5P2 and recruitment of retriever, thus initiating actin mediated tubule formation and endosomal exit of SNX17 bound integrins.

    The manuscript is clearly written, the data are largely of sufficient quality, and the findings are certainly of interest to the endosomal research community. I also agree with the model the authors propose. One weak point of the study is the dependence on microscopic techniques to analyze integrin surface levels and endosomal recruitment of the retriever complex. The study would have benefited from additional methods to confirm the microscopy data. It would also be good if the authors could confirm their inhibitor studies with genetic suppression/deletion of PIKfyve, ideally followed by rescues with a kinase deficient mutant.

    The authors rely on microscopy of integrin beta 1 for most of their data. However, SNX17 and the retriever complex are not required for the recycling of all beta 1 integrins (Steinberg et al., 2012). In HeLa cells, it is mainly integrin alpha 5/beta1 that is recycled by SNX17. Therefore, the other beta 1 integrins that recycle SNX17 independently tend to mask the recycling phenotypes caused by the loss of SNX17/retriever. I think that the authors could detect a much more pronounced recycling phenotype upon PIKfyve inhibition if they stained integrin alpha 5 instead of integrin beta 1. Does integrin alpha 5 "get stuck" in a LAMP1 positive compartment similar to what Steinberg et al., 2012 or McNally et al., 2017 describe in their studies? These two studies clearly show that almost all integrin alpha 5/beta 1 accumulates in a LAMP1 or LAMP2 positive compartment upon loss of retriever/SNX17 function. If the authors are correct in their assumptions, this should be happening upon loss of PIKfyve activity. One could use the Abcam antibody against integrin alpha 5 that was used in the McNally et al. study as it works very well.

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  4. Reviewer #2 (Public Review):

    Giridharan and colleagues have sought to examine the role of phosphoinositide switching on the function of endosomes in the sorting and transport of integral proteins, in particular integrins. Their focus has been on the enzyme PIKfyve which catalyses the conversion of PI(3)P, an identity feature of early endosomes, to PI(3,5)P2, an identity cue for late endosomes. Through experiments that inhibit PIKfyve function, they have examined the resulting effects on the localization and function of components of the SNX17-retriever-CCC-WASH pathway in the sorting of internalized integrins and the impact on cell migration. They conclude that the activity of PIKfyve, along with its upstream kinase VPS34 (this generates PI(3)P), are coordinated to regulate integral protein sorting through this pathway. Strengths of the study include: the research topic, analysis of the functional significance of efficient and correct endosomal sorting is an expanding area of interest; its breath, from analysis of cell migration down to molecular analysis of integrin sorting; the combination of quantitative biochemical and imaging based analysis; and, the use of genetic tools and acute chemical inhibition. However, the data supporting the conclusions could be strengthened by additional controls and the integration of published data into the final model that argues for phosphoinositide switching in the ordered assembly of the SNX17-retriever-CCC-WASH pathway could be improved.

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  5. Reviewer #3 (Public Review):

    This study extends what is known about the importance of phosophoinositides in endosomal protein sorting and will be of interest to groups studying endosomal protein sorting. It is perhaps to be expected that PIKfyve, the only PI3P 5-kinase in could have a role in this process as it is important for sorting/trafficking events at other points in the endocytic pathway.
    It remains to be determined whether the SNX17-Retriever machinery is solely responsible for integrin recycling and it is noteworthy that some of the effects of the loss of PIKfyve function are somewhat marginal. Indeed, there is evidence that SNX27, along with the WASH complex is necessary for integrin recycling and cell migration. There is a possibility that PIKfyve has rather wide-ranging roles in endosomal protein sorting and therefore many cargo proteins will be affected to a greater or lesser degree if PIKfyve function is impaired. A question left open is what machinery is involved in forming the tubular carriers (or vesicles) that transport integrins to the cell surface as SNX17 lacks the membrane-bending BAR domains in many other sorting nexins that can drive membrane tubulation.

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