Physical association between a novel plasma-membrane structure and centrosome orients cell division

Thank you for submitting your article "Physical association between a novel plasma-membrane structure and centrosome orients cell division" for consideration by eLife. Your article has been favorably evaluated by K VijayRaghavan as the Senior editor and three reviewers, one of whom, Yukiko M Yamashita (Reviewer #1), is a member of our Board of Reviewing Editors.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this report, Negishi et al. describe a novel cell invagination during the early development of ascidian embryo that captures the centrosomes to orient the spindle. They describe this novel structure that is formed during oriented divisions of ascidian embryos (11th cell cycle). Long range centrosome migrations are known to be a feature of many large invertebrate embryonic cells, most notably ascidian embryos, but the origins of the directed force that drive this migration have been a mystery for over 100 years. Wilson for example talks about this type of behavior in his famous book from the last century. The apparent "action at a distance" of some cortical cue on the centrosome has always struck me as a very interesting problem. Here the authors suggest a novel solution whereby the membrane forms deep invaginations that reach out and attach to the centrosome, after which they exert elastic forces to pull the centrosome to the appropriate direction.

The morphological observations in this paper are truly striking and have been carried out with great thoroughness. The authors use a wide range of techniques from simple membrane dyes, to GFP tagged membrane proteins, to serial block face SEM, all of which reveal a highly novel structure composed of nested membranes from two adjacent cells diving deep into the interior of a cell to contact its centrosome. This invagination is associated with microtubules, which are apparently required for its formation or at least maintenance. This is thus a very interesting structure that many cell biologists, especially those interested in cytoskeleton-membrane interactions, will find very interesting. The images are of extremely high quality and all of the observations are rigorously quantified.

The reviewers agreed that this is a very interesting story describing a novel cellular structure that likely plays a critical role in spindle orientation during early development of ascidian embryos. The major concern was the lack of direct proof of a causal role of this invagination in spindle orientation. However, there is tremendous circumstantial evidence that this structure may play a direct mechanical role in centrosome migration. Given the novelty of the discovery, we believe that this paper will spark further research into the possible role of elastic membrane tethers in moving centrosomes, not only in ascidians but other systems as well. Given the complete mystery surrounding the long-range action at a distance underlying centrosome migration in invertebrate embryos, the demonstration of at least a potential mechanism is extremely welcome to the field. We therefore support the publication of this paper in eLife. But the critical lack of direct evidence must be appropriately acknowledged in the text.

Essential revisions:

Please go through the text to make it clear that the causal relationship (i.e. invagination orients spindle) is not proven. (Particularly because the laser ablation of invagination did not work in a way to provide insights).

The authors utilize disruption of planar cell polarity to test the idea of invagination being required for spindle orientation. Indeed, they observed that disruption of PCP causes invagination with randomized orientation, which nicely correlated with perturbed spindle orientation. But this does not necessarily prove that the invaginations play a causal role in moving the centrosomes, because it leaves open the possibility that there is some other down-stream process regulated by the planar cell polarity pathway that regulates centrosome movement independently of the invaginations. Thus, please make it clear that this is still correlational.

In addition, there have been a few suggestions that can potentially add mechanistic insights to the paper. However, after discussion, we felt that these are likely beyond the scope of the present manuscript. Therefore, the following suggestions are not required for acceptance of the manuscript. If the authors have such data already, they can be added. Or such possibilities may be discussed.

1) The actomyosin cortex appears to restrict the formation of those plasma membrane structures. It would be interesting to determine whether the cortex is weaker at the place where this structure forms, and how such potential anisotropy of the actomyosin cortex is established in the first place.

2) Microtubules appear to be required for the formation of those plasma membrane structures, but it remains unclear how microtubules generate the observed tensile forces. Presumably microtubule motors are involved, and it would thus be very interesting to identify any of such motors and their possible function in this process.

3) The orientation of the observed plasma membrane structure seems to determine the position of the primary cilium and the mitotic spindle, thus overriding other potential mechanisms, such as cell shape. It would be quite interesting to see whether e.g. cell division orientation is truly randomized in cases where the plasma membrane structure is cut, or whether other factors take over and bias division orientation.