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  1. Reviewer #3 (Public Review):

    In this revised manuscript (Oon and Prehoda), the authors performed additional live-imaging experiments and recorded aPKC and actin dynamics simultaneously in larval neuroblasts. They also provide evidence that aPKC polarization is lost upon F-actin disruption by Latrunculin A treatment. These are great improvements. The pulsatile dynamics of actin and myosin II showed in the manuscript are compelling. Images presented in this manuscript are of high-quality and impressive.

    However, the pulsatile apical myosin network in delaminating neuroblasts in Drosophila embryos was reported previously (An Y. et al., Development, 2017). This important and relevant paper should be cited in the introduction of the current manuscript. Therefore, the finding on the pulsatile actomyosin in larval brain neuroblasts reported in this manuscript is not a total novel discovery. Another major concern is that Lat-A did not specifically disrupt actomyosin pulsatile movements, as it generally disrupts the F-actin network. So these experiments only strengthened the link between the F-actin network and Par polarity (which was already demonstrated in Kono et al., 2019; Oon 22 and Prehoda, 2019). Low doses of Cytochalasin D are known to disrupt myosin pulses still allowing the assembly of the actomyosin network (Mason et al., Nature Cell Biology 2014). The author should treat neuroblasts with low doses of CytoD to only disrupt actomyosin pulses, not the entire F-actin network, and examine the effect on Par polarity. It is also worthwhile to knockdown sqh to disrupt apical pulsatile actin dynamics. Besides, most of the concerns previously raised by the reviewer were not addressed in the revised manuscript.

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  2. Reviewer #2 (Public Review):

    Previously, Oon and Prehoda showed apically directed movement of aPKC clusters during polarization of the neuroblast prior to asymmetric cell division. They found that these movements required F-actin, but the distribution of F-actin has only been reported for later stages of neuroblast polarization and division. Here, the authors report pulses of cortical F-actin during interphase, followed by an apically directed flow at the onset of mitosis, a strong apical accumulation of F-actin at metaphase and anaphase, followed by fragmentation and basally directed flow of the fragments. aPKC clusters are shown to colocalize with the F-actin networks as they flow apically. The F-actin networks are also shown have partial colocalization with non-muscle myosin II, suggesting a possible mechanism for their movement. Finally, the authors solidify the results of actin inhibitor studies from their 2019 study by showing that reported effects on aPKC localization are preceded by F-actin loss as would be expected but was not previously shown. Overall, the Research Advance extends the past study by more directly showing the involvement of F-actin and myosin in the apical localization mechanism of aPKC, and by describing F-actin and myosin dynamics prior to this transition. The following concerns should be addressed.

    1. The pulsatile nature of broad F-actin networks is evident during interphase, but these pulsations substantially subside upon entry into mitosis, and at this stage an apically directed flow of F-actin is the main behavior evident. This transition from pulses to flow is evident in both the movies and the kymographs of the F-actin probe. However, the authors state that the pulsations continue at the onset of mitosis and as the apical cap of aPKC matures. It is unclear whether the apical flow of aPKC and F-actin is associated with small-scale defined F-actin pulses, or small-scale random fluctuations of F-actin. The F-actin flow alone is an informative finding. The authors should consider revising their descriptions of these data (including in the manuscript title), or provide clearer examples of defined F-actin pulsations during the stage when aPKC polarizes.

    2. I checked the main text, methods, figures and figure legends, but could not find listings of sample sizes. Thus, the reproducibility of the findings has not been reported.

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  3. Reviewer #1 (Public Review):

    Oon and Prehoda report pulsatile contraction of apical membrane in the process of Par protein polarization in Drosophila neuroblasts. This explains how/why actin filament was required to localize/polarize Par complex. Specifically, using spinning disc confocal microscopy with high temporal resolution, they found the directed actin movement toward the apical pole, which nicely correlates with concentration of aPKC. They also show that myosin II is involved in this pulsatile movement of actin filament. This very much resembles the observation in C. elegans embryos, and nicely unifies observations across systems. Although descriptive in nature, I think this is an important observation and indicates a universal mechanism by which cells are polarized. I think this is a well executed study.

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  4. Evaluation Summary:

    Oon and Prehoda report pulsatile contraction of apical membrane in the process of Par protein polarization in Drosophila neuroblasts. This explains how/why actin filament was required to localize/polarize Par complex. This very much resembles the observation in C. elegans embryos, and nicely unifies observations across systems.

    (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.)

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