Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model

Reviewer #1 (Public review):

Summary:

Wang, Po-Kai et al., utilized the de novo polarization of MDCK cells cultured in Matrigel to assess the interdependence between polarity protein localization, centrosome positioning and apical membrane formation. They show that the inhibition of Plk4 with Centrinone does not prevent apical membrane formation, but does result in its delay, a phenotype the authors attribute to the loss of centrosomes due to the inhibition of centriole duplication. However, the targeted mutagenesis of specific centrosome proteins implicated in the positioning of centrosomes in other cell types (CEP164, ODF2, PCNT and CEP120), as well as the use of dominant negative constructs to inhibit centrosomal microtubule nucleation did not affect centrosome positioning in 3D cultured MDCK cells. A screen of proteins previously implicated in MDCK polarization revealed that the polarity protein Par-3 was upstream of centrosome positioning, similar to other cell types.

Strengths:

The investigation into the temporal requirement and interdependence of previously proposed regulators of cell polarization and lumen formation is valuable. The authors have provided a detailed analysis of many of these components at defined stages of polarity establishment, and well demonstrate that centrosomes are not necessary for apical polarity formation, but are involved in the efficient establishment of the apical membrane.

Weaknesses:

Key questions remain regarding the structure of the intracellular cytoskeleton following depletion of centrosomes, centrosome proteins,or abrogation of centrosome microtubule nucleation. The authors strengthen their model that centrosomes are positioned independently of microtubule nucleation using dominant negative Cdk5RAP2 and NEDD-1 constructs, however, the structure of the intracellular microtubule network remains unresolved and will be an important avenue for future investigation.

Reviewer #3 (Public review):

Here the Wang et al resubmit their manuscript describing the events in the establishment of polarity in MDCK cells cultured in vitro. As with the original version, the description is throughout and is important to the field to report as it establishes a hierarchy of events in polarization, placing Par3 upstream of centrosome positioning and apical membrane component trafficking. Unfortunately, in the revised version, the authors addressed almost none of my points. They did a cursory job of responding in the rebuttal letter but made little attempt to actually address what was being asked or to incorporate any of my suggestions into the manuscript. The particularly egregious examples are cited below:

Comments on revisions:

(1) My original main experimental concern was not addressed: I had originally asked what role microtubules play in the process of polarization (either centrosomal or non-centrosomal). An obvious model is that Gp135, Rab11, etc. are delivered to the AMIS on centrosomal microtubules. Centrosomes might be also be pulled to the AMIS via cortically derived microtubules as is the case in the C. elegans intestine where the centrosome moves apically on apical microtubules via dynein directed transport to the cortically anchored minus ends. The authors do not explore the role of microtubules in the revision, citing that it was not possible to observe the microtubules directly or to perform nocodazole experiments during polarization. Instead, the authors use a relatively new genetic tool to disrupt centrosomal microtubules. They appear to succeed in displacing centrosomal g-tubulin using this tool, but without being able to observe microtubules, a remaining caveat of this experiment is that it is still unclear whether the authors have removed centrosomal microtubules. Compounding this issue is that this tool has never been used in MDCK cells. The authors conclude "we found that cells lacking centrosomal microtubules were still able to polarize and position the centrioles apically.", but they have not shown this, instead the data suggest this conclusion and the authors should acknowledge the caveat that they have no idea whether centrosomal microtubules are abolished. Similarly, the authors also state: "Additionally, although PCNT knockout cells show reduced microtubule nucleation ability, they still recruit a small amount of γ-tubulin". Where are the data that show that microtubule nucleation is reduced in these PCNT knock out cells?

(2) Many of my comments were addressed in the rebuttal, but not in the text.
The non-centrosomal GP135 in Figure 2 is not acknowledged or explained.

That the polarity index does not actually measure polarity, but nuclear-centrosome distance is not acknowledged or explained in the paper.

I still don't believe that the quantification in Figure 3D matches the images I am being shown in Figure 3A. In the centrinone treatment condition, there is certainly an enrichment of GP135 at the AMIS that is not detected in the quantification. The method described in the rebuttal might miss this enrichment if it is offset from line drawn between the centroid of the two nuclei.

Cell height changes in the centrosome depleted cysts are still referenced in the text ("the cell heights of the centrosome-depleted cysts are less uniform"), but no specific data or image is called out. Currently, Figure 3G is referenced, but that is a graph of GP135 intensity

In my original review, I called on the authors to comment on the striking similarity of the mechanisms they documented in MDCK cells to what has been shown in in vivo systems. The authors did not do this, instead restating in the rebuttal some features of what they found. But, the mechanisms shown here are remarkably similar to the polarization of primordia that generate tubular organs in vivo. Perhaps most striking is the similarity to the C> elegans intestine where Par3 localizes to the cortex at the site of an apical MTOC that pulls the centrosome to the apical surface via dynein (Feldman and Priess, 2012). Instead of discussing this similarity, the authors state: "Par3 is likely to regulate centrosome positioning through some intermediate molecules or mechanisms, but its specific mechanism is still unclear and requires further investigation." Given the acetylated tubulin signal emanating from the Par3 positive patch in Figure 5E and F, I suspect similar mechanisms to the C. elegans intestine are at play here. Such a parallel should be noted in the Discussion.

I had originally commented that "I find the results in Figure 6G puzzling. Why is ECM signaling required for Gp135 recruitment to the centrosome. Could the authors discuss what this means?" The authors responded that "The data in Figure 6G do not indicate that ECM signaling is required for the recruitment of Gp135 to the centrosome". In Figure 6G, the localization of GP135 to the centrosome appears significantly delayed compared to its localization to the centrosome in images where cells were cultured in Matrigel. Indeed, the authors argue that the centrosomal localization precedes and contributes to its localization to the AMIS. In the absence of ECM, GP135 localizes to the membrane before it localizes to the centrosome and its localization to the centrosome appears significantly reduced. Thus, my original and current interpretation is that ECM signaling is somehow required for the centrosomal targeting of GP135. One could make a competition argument, i.e. that the cortex in the absence of ECM is somehow a more desirable place to localize than the centrosome, but this experiment also argues that the centrosome does not need to be a source of this material in order for it to end up on the cortex.

(3) There needs to be precision in the language used in many places:

I don't understand this line in the abstract: "When cultured in Matrigel, de novo polarization of a single epithelial cell is often coupled with mitosis." If a cell has divided, it is no longer a single cell.

The authors state in the Introduction "Because of its strong ability to nucleate microtubules, the centrosome functions as the primary microtubule organizing center", but then state ""In polarized epithelial cells, the centrosome is localized at the apical region during interphase, which contributes to the construction of an asymmetric microtubule network conducive to polarized vesicle trafficking". In the latter statement, I assume the authors are describing the well-characterized apical microtubule network in epithelial cells that is non-centrosomal. Thus, the latter sentence is at odds with the former.

The authors continually refer to Par3 as a tight junction protein. "Par3, which controls tight junction assembly to partition the apical surface from the basolateral surface". To my knowledge, PARD3 is an apical protein with similar localization to C. elegans PAR-3 and Drosophila Bazooka. PARD3B is a junctional protein. I assume that the antibody that the authors are using is to PARD3 and not PARD3B? Can the authors please clarify this in the text.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Wang, Po-Kai, et al., utilized the de novo polarization of MDCK cells cultured in Matrigel to assess the interdependence between polarity protein localization, centrosome positioning, and apical membrane formation. They show that the inhibition of Plk4 with Centrinone does not prevent apical membrane formation, but does result in its delay, a phenotype the authors attribute to the loss of centrosomes due to the inhibition of centriole duplication. However, the targeted mutagenesis of specific centrosome proteins implicated in the positioning of centrosomes in other cell types (CEP164, ODF2, PCNT, and CEP120) did not affect centrosome positioning in 3D cultured MDCK cells. A screen of proteins previously implicated in MDCK polarization revealed that the polarity protein Par-3 was upstream of centrosome positioning, similar to other cell types.

Strengths:

The investigation into the temporal requirement and interdependence of previously proposed regulators of cell polarization and lumen formation is valuable to the community. Wang et al., have provided a detailed analysis of many of these components at defined stages of polarity establishment. Furthermore, the generation of PCNT, p53, ODF2, Cep120, and Cep164 knockout MDCK cell lines is likely valuable to the community.

Weaknesses:

Additional quantifications would highly improve this manuscript, for example it is unclear whether the centrosome perturbation affects gamma tubulin levels and therefore microtubule nucleation, it is also not clear how they affect the localization of the trafficking machinery/polarity proteins. For example, in Figure 4, the authors measure the intensity of Gp134 at the apical membrane initiation site following cytokinesis, but there is no measure of Gp134 at the centrosome prior to this.

We thank the reviewer for this important suggestion. Previous studies have shown that genes encoding appendage proteins and CEP120 do not regulate γ-tubulin recruitment to centrosomes (Betleja, Nanjundappa, Cheng, & Mahjoub, 2018; Vasquez-Limeta & Loncarek, 2021). Although the loss of PCNT reduces γ-tubulin levels, this reduction is partially compensated by AKAP450. Even in the case of PCNT/AKAP450 double knockouts, low levels of γ-tubulin remain at the centrosome (Gavilan et al., 2018), suggesting that it is difficult to completely eliminate γ-tubulin by perturbing centrosomal genes alone.

To directly address this question, in the revised manuscript (Page 8, Paragraph 4; Figure 4—figure supplement 3), we employed a recently reported method to block γ-tubulin recruitment by co-expressing two constructs: the centrosome-targeting carboxy-terminal domain (C-CTD) of CDK5RAP2 and the γ-tubulin-binding domain of NEDD1 (N-gTBD). This approach effectively depleted γ-tubulin and abolished microtubule nucleation at the centrosome (Vinopal et al., 2023). Interestingly, despite the reduced efficiency of apical vesicle trafficking, these cells were still able to establish polarity, with centrioles positioned apically. These results suggest that microtubule nucleation at the centrosomes (centrosomal microtubules) facilitates—but is not essential for—polarity establishment.

Regarding Figure 4, we assume the reviewer was referring to Gp135 rather than Gp134. In the revised manuscript (Page 8, Paragraph 2; Figure 4I), we observed a slight decrease in Gp135 intensity near PCNT-KO centrosomes at the pre-Abs stage. However, its localization at the AMIS following cytokinesis remained unaffected. These results suggest that the loss of PCNT has a limited impact on Gp135 localization.

Reviewer #2 (Public review):

Summary:

The authors decoupled several players that are thought to contribute to the establishment of epithelial polarity and determined their causal relationship. This provides a new picture of the respective roles of junctional proteins (Par3), the centrosome, and endomembrane compartments (Cdc42, Rab11, Gp135) from upstream to downstream.

Their conclusions are based on live imaging of all players during the early steps of polarity establishment and on the knock-down of their expression in the simplest ever model of epithelial polarity: a cell doublet surrounded by ECM.

The position of the centrosome is often taken as a readout for the orientation of the cell polarity axis. There is a long-standing debate about the actual role of the centrosome in the establishment of this polarity axis. Here, using a minimal model of epithelial polarization, a doublet of daugthers MDCK cultured in Matrigel, the authors made several key observations that bring new light to our understanding of a mechanism that has been studied for many years without being fully explained:

(1) They showed that centriole can reach their polarized position without most of their microtubule-anchoring structures. These observations challenge the standard model according to which centrosomes are moved by the production and transmission of forces along microtubules.

(2) However) they showed that epithelial polarity can be established in the absence of a centriole.

(3) (Somehow more expectedly) they also showed that epithelial polarity can't be established in the absence of Par3.

(4) They found that most other polarity players that are transported through the cytoplasm in lipid vesicles, and finally fused to the basal or apical pole of epithelial cells, are moved along an axis which is defined by the position of centrosome and orientation of microtubules.

(5) Surprisingly, two non-daughter cells that were brought in contact (for 6h) could partially polarize by recruiting a few Par3 molecules but not the other polarity markers.

(6) Even more surprisingly, in the absence of ECM, Par 3 and centrosomes could move to their proper position close to the intercellular junction after cytokinesis but other polarity markers (at least GP135) localized to the opposite, non-adhesive, side. So the polarity of the centrosome-microtubule network could be dissociated from the localisation of GP135 (which was believed to be transported along this network).

Strengths:

(1) The simplicity and reproducibility of the system allow a very quantitative description of cell polarity and protein localisation.

(2) The experiments are quite straightforward, well-executed, and properly analyzed.

(3) The writing is clear and conclusions are convincing.

Weaknesses:

(1) The simplicity of the system may not capture some of the mechanisms involved in the establishment of cell polarity in more physiological conditions (fluid flow, electrical potential, ion gradients,...).

We agree that certain mechanisms may not be captured by this simplified system. However, the model enables us to observe intrinsic cellular responses, minimize external environmental variables, and gain new insights into how epithelial cells position their centrosomes and establish polarity.

(2) The absence of centriole in centrinone-treated cells might not prevent the coalescence of centrosomal protein in a kind of MTOC which might still orient microtubules and intracellular traffic. How are microtubules organized in the absence of centriole? If they still form a radial array, the absence of a centriole at the center of it somehow does not conflict with classical views in the field.

Previous studies have shown that in the absence of centrioles, centrosomal proteins can relocate to alternative microtubule-organizing centers (MTOCs), such as the Golgi apparatus (Gavilan et al., 2018). Furthermore, centriole loss leads to increased nucleation of non-centrosomal microtubules (Martin, Veloso, Wu, Katrukha, & Akhmanova, 2018). However, these microtubules typically do not form the classical radial array or a distinct star-like organization.

While this non-centrosomal microtubule network can still support polarity establishment, it does so less efficiently—similar to what is observed in p53-deficient cells undergoing centriole-independent mitosis (Meitinger et al., 2016). Thus, although the absence of centrioles does not completely prevent microtubule-based organization or polarity establishment, it impairs their spatial coordination and reduces overall efficiency compared to a centriole-centered microtubule-organizing center (MTOC).

(3) The mechanism is still far from clear and this study shines some light on our lack of understanding. Basic and key questions remain:

(a) How is the centrosome moved toward the Par3-rich pole? This is particularly difficult to answer if the mechanism does not imply the anchoring of MTs to the centriole or PCM.

Previous studies have shown that Par3 interacts with dynein, potentially anchoring it at the cell cortex (Schmoranzer et al., 2009). This interaction enables dynein, a minus-enddirected motor, to exert pulling forces on microtubules, thereby promoting centrosome movement toward the Par3-enriched pole.

In our experiments (Figure 4), we attempted to disrupt centrosomal microtubule nucleation by knocking out multiple genes involved in centrosome structure and function, including ODF2 and PCNT. Under these perturbations, γ-tubulin still remained detectable at the centrosome, and we were unable to completely eliminate centrosomal microtubules.

To address this question more directly, we employed a strategy to deplete γ-tubulin from centrosomes by co-expressing the centrosome-targeting C-terminal domain (C-CTD) of CDK5RAP2 and the γ-tubulin-binding domain of NEDD1 (N-gTBD). As shown in the new data of the revised manuscript (Page 8, Paragraph 4; Figure 4—figure supplement 3), this approach effectively depleted γ-tubulin from centrosomes, thereby abolishing microtubule nucleation at the centrosome.

Surprisingly, even under these conditions, centrioles remained apically positioned (Page 8, Paragraph 4; Figure 4—figure supplement 3), indicating that centrosomal microtubules are not essential for centrosome movement during polarization.

Given these findings, we agree that the precise mechanism by which the Par3-enriched cortex attracts or guides centrosome movement remains unclear. Although dynein–Par3 interactions may contribute, further studies are needed to elucidate how centrosome repositioning occurs in the absence of microtubule-based pulling forces from the centrosome itself.

(b) What happens during cytokinesis that organises Par3 and intercellular junction in a way that can't be achieved by simply bringing two cells together? In larger epithelia cells have neighbours that are not daughters, still, they can form tight junctions with Par3 which participates in the establishment of cell polarity as much as those that are closer to the cytokinetic bridge (as judged by the overall cell symmetry). Is the protocol of cell aggregation fully capturing the interaction mechanism of non-daughter cells?

We speculate that a key difference between cytokinesis and simple cell-cell contact lies in the presence or absence of actomyosin contractility during the process of cell division. Specifically, contraction of the cytokinetic ring generates mechanical forces between the two daughter cells, which are absent when two non-daughter cells are simply brought together. While adjacent epithelial cells can indeed form tight junctions and recruit Par3, the lack of shared cortical tension and contractile actin networks between non-daughter cells may lead to differences in how polarity is initiated. This mechanical input during cytokinesis may serve as an organizing signal for centrosome positioning. This idea is supported by recent work showing that the actin cytoskeleton can influence centrosome positioning (Jimenez et al., 2021), suggesting that contractile actin structures formed during cytokinesis may contribute to spatial organization in a manner that cannot be replicated by simple aggregation.

In our experiments, we simply captured two cells that were in contact within Matrigel. We cannot say for sure that it captures all the interaction mechanisms of non-daughter cells, but it does provide a contrast to daughter cells produced by cytokinesis.

Reviewer #3 (Public review):

Here, Wang et al. aim to clarify the role of the centrosome and conserved polarity regulators in apical membrane formation during the polarization of MDCK cells cultured in 3D. Through well-presented and rigorous studies, the authors focused on the emergence of polarity as a single MDCK cell divided in 3D culture to form a two-cell cyst with a nascent lumen. Focusing on these very initial stages, rather than in later large cyst formation as in most studies, is a real strength of this study. The authors found that conserved polarity regulators Gp135/podocalyxin, Crb3, Cdc42, and the recycling endosome component Rab11a all localize to the centrosome before localizing to the apical membrane initiation site (AMIS) following cytokinesis. This protein relocalization was concomitant with a repositioning of centrosomes towards the AMIS. In contrast, Par3, aPKC, and the junctional components E-cadherin and ZO1 localize directly to the AMIS without first localizing to the centrosome. Based on the timing of the localization of these proteins, these observational studies suggested that Par3 is upstream of centrosome repositioning towards the AMIS and that the centrosome might be required for delivery of apical/luminal proteins to the AMIS.

To test this hypothesis, the authors generated numerous new cell lines and/or employed pharmacological inhibitors to determine the hierarchy of localization among these components. They found that removal of the centrosome via centrinone treatment severely delayed and weakened the delivery of Gp135 to the AMIS and single lumen formation, although normal lumenogenesis was apparently rescued with time. This effect was not due to the presence of CEP164, ODF2, CEP120, or Pericentrin. Par3 depletion perturbed the repositioning of the centrosome towards the AMIS and the relocalization of the Gp135 and Rab11 to the AMIS, causing these proteins to get stuck at the centrosome. Finally, the authors culture the MDCK cells in several ways (forced aggregation and ECM depleted) to try and further uncouple localization of the pertinent components, finding that Par3 can localize to the cell-cell interface in the absence of cell division. Par3 localized to the edge of the cell-cell contacts in the absence of ECM and this localization was not sufficient to orient the centrosomes to this site, indicating the importance of other factors in centrosome recruitment.

Together, these data suggest a model where Par3 positions the centrosome at the AMIS and is required for the efficient transfer of more downstream polarity determinants (Gp135 and Rab11) to the apical membrane from the centrosome. The authors present solid and compelling data and are well-positioned to directly test this model with their existing system and tools. In particular, one obvious mechanism here is that centrosome-based microtubules help to efficiently direct the transport of molecules required to reinforce polarity and/or promote lumenogenesis. This model is not really explored by the authors except by Pericentrin and subdistal appendage depletion and the authors do not test whether these perturbations affect centrosomal microtubules. Exploring the role of microtubules in this process could considerably add to the mechanisms presented here. In its current state, this paper is a careful observation of the events of MCDK polarization and will fill a knowledge gap in this field. However, the mechanism could be significantly bolstered with existing tools, thereby elevating our understanding of how polarity emerges in this system.

We agree that further exploration of microtubule dynamics could strengthen the mechanistic framework of our study. In our initial experiments, we disrupted centrosome function through genetic perturbations (e.g., knockout of PCNT, CEP120, CEP164, and ODF2). However, consistent with previous reports (Gavilan et al., 2018; Tateishi et al., 2013), we found that single-gene deletions did not completely eliminate centrosomal microtubules. Furthermore, imaging microtubule organization in 3D culture presents technical challenges. Due to the increased density of microtubules during cell rounding, we were unable to obtain clear microtubule filament structures—either using α-tubulin staining in fixed cells or SiR-tubulin labeling in live cells. Instead, the signal appeared diffusely distributed throughout the cytosol.

To overcome this, we employed a recently reported approach by co-expressing the centrosome-targeting carboxy-terminal domain (C-CTD) of CDK5RAP2 and the γtubulin-binding domain (gTBD) of NEDD1 to completely deplete γ-tubulin and abolish centrosomal microtubule nucleation (Vinopal et al., 2023). In our new data presented in the revised manuscript (Page 8, Paragraph 4; Figure 4—figure supplement 3), we found that cells lacking centrosomal microtubules were still able to polarize and position the centrioles apically. However, the efficiency of polarized transport of Gp135 vesicles to the apical membrane was reduced. These findings suggest that centrosomal microtubules are not essential for polarity establishment but may contribute to efficient apical transport.

Reference

Betleja, E., Nanjundappa, R., Cheng, T., & Mahjoub, M. R. (2018). A novel Cep120-dependent mechanism inhibits centriole maturation in quiescent cells. Elife, 7. doi:10.7554/eLife.35439

Gavilan, M. P., Gandolfo, P., Balestra, F. R., Arias, F., Bornens, M., & Rios, R. M. (2018). The dual role of the centrosome in organizing the microtubule network in interphase. EMBO Rep, 19(11). doi:10.15252/embr.201845942

Jimenez, A. J., Schaeffer, A., De Pascalis, C., Letort, G., Vianay, B., Bornens, M., . . . Thery, M. (2021). Acto-myosin network geometry defines centrosome position. Curr Biol, 31(6), 1206-1220 e1205. doi:10.1016/j.cub.2021.01.002

Martin, M., Veloso, A., Wu, J., Katrukha, E. A., & Akhmanova, A. (2018). Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules. Elife, 7. doi:10.7554/eLife.33864

Meitinger, F., Anzola, J. V., Kaulich, M., Richardson, A., Stender, J. D., Benner, C., . . . Oegema, K. (2016). 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. J Cell Biol, 214(2), 155-166. doi:10.1083/jcb.201604081

Schmoranzer, J., Fawcett, J. P., Segura, M., Tan, S., Vallee, R. B., Pawson, T., & Gundersen, G. G. (2009). Par3 and dynein associate to regulate local microtubule dynamics and centrosome orientation during migration. Curr Biol, 19(13), 1065-1074. doi:10.1016/j.cub.2009.05.065

Tateishi, K., Yamazaki, Y., Nishida, T., Watanabe, S., Kunimoto, K., Ishikawa, H., & Tsukita, S. (2013). Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains. J Cell Biol, 203(3), 417-425. doi:10.1083/jcb.201303071

Vasquez-Limeta, A., & Loncarek, J. (2021). Human centrosome organization and function in interphase and mitosis. Semin Cell Dev Biol, 117, 30-41. doi:10.1016/j.semcdb.2021.03.020

Vinopal, S., Dupraz, S., Alfadil, E., Pietralla, T., Bendre, S., Stiess, M., . . . Bradke, F. (2023). Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain. Neuron, 111(8), 1241-1263 e1216. doi:10.1016/j.neuron.2023.01.020.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Figures:

(1) Figure 3 B+C - Although in comparison to Figure 2 it appears the p53 mutation does not affect θN-C, or Lo-c. the figure would benefit from direct comparison to control cells.

We appreciate your suggestion to improve the clarity of the figure. In response, we have revised Figure 3B+C to include control cell data, allowing for clearer side-by-side comparisons in the updated figures.

(2) Figure 3D - Clarify if both were normalized to time point 0:00 of the p53 KO. The image used appears that Gp135 intensity increases substantially between 0:00 and 0:15 in the figure, but the graph suggests that the intensity is the same if not slightly lower.

Figure 3D – The data were normalized to the respective 0:00 time point for each condition. Because the intensity profile was measured along a line connecting the two nuclei, Gp135 signal could only be detected if it appeared along this line. However, the images shown are maximum-intensity projections, meaning that Gp135 signals from peripheral regions are projected onto the center of the image. This may create the appearance of increased intensity at certain time points (e.g., Figure 3A, p53-KO + CN, 0:00–0:15).

(3) Figure 4A: The diagram does not accurately represent the effect of the mutations, for example, PCNT mutation likely doesn't completely disrupt PCM (given gamma-tubulin is still visible in the staining), but instead results in its disorganization, Cep164 also wouldn't be expected to completely ablate distal appendages.

Thank you for your comment. We have modified the figure in the revised manuscript (Figure 4A) to more clearly depict the defective DAs.

(4) Figure 4 + Supplements: A more in-depth characterization of the mutations would help address the previous comment and strengthen the manuscript. Especially as these components have previously been implicated in centrosome transport.

Thank you for your valuable suggestion. As noted in previous studies, CEP164 is essential for distal appendage function and basal body docking, with its loss resulting in blocked ciliogenesis (Tanos et al., 2013); CEP120 is required for centriole elongation and distal appendage formation, and its loss also results in blocked ciliogenesis (Comartin et al., 2013; Lin et al., 2013; Tsai, Hsu, Liu, Chang, & Tang, 2019); ODF2 functions upstream in the formation of subdistal appendages, and its loss eliminates these structures and impairs microtubule anchoring (Tateishi et al., 2013); and PCNT functions as a PCM scaffold, necessary for the recruitment of PCM components and for microtubule nucleation at the centrosome (Fong, Choi, Rattner, & Qi, 2008; Zimmerman, Sillibourne, Rosa, & Doxsey, 2004).

Given that the phenotypes of these mutants have been well characterized in the literature. Here, we further focus on their roles in centrosome migration and polarized vesicle trafficking within the specific context of our study.

(5) Figure 4: It would be interesting to measure the Gp135 intensity at the centrosomes, given that the model proposes it is trafficked from the centrosomes to the AMIS.

Thank you for your suggestion. We have included measurements of Gp135 intensity at the centrosomes during the Pre-Abs stage in the revised figure (Figure 4I). Our data show no significant differences in Gp135 intensity between wild-type (WT) and CEP164-, ODF2-, or CEP120-knockout (KO) cell lines. However, a slight decrease in Gp135 intensity was observed in PCNT-KO cells.

(6) Figure 6F shows that in suspension culture polarity is reversed, however, in Figure 6G gp135 still localizes to the cytokinetic furrow prior to polarity reversal. Given this paper demonstrates Par-3 is upstream of centrosome positioning, it would be important to have temporal data of how Par-3 localizes prior to the ring observed in 6F.

Thank you for your comment. We have included a temporal analysis of Par3 localization using fixed-cell staining in the revised figure (Figure 6—figure supplement 1D). This analysis shows that Par3 also localizes to the cytokinesis site during the Pre-Abs stage, prior to ring formation observed during the Post-CK stage (Figure 6F). Interestingly, during the Pre-Abs stage, the centrosomes also migrate toward the center of the cell doublets in suspension culture, and Gp135 surrounding the centrosomes is also recruited to a region near the center (Figure 6—figure supplement 1E). These data suggest that Par3 also is initially recruited to the cytokinesis site before polarity reversal, potentially promoting centrosome migration. The main difference from Matrigel culture is the peripheral localization of Par3 and Gp135 in suspension, which is likely due to the lack of external ECM signaling.

Results:

(1) Page 7 Paragraph 1 - consistently use AMIS (Apical membrane initiation site) rather than "the apical site".

Thank you for your helpful comment. We have revised the manuscript (Page 7, Paragraph 1) and will now use "AMIS" (Apical Membrane Initiation Site) instead of "the apical site" throughout the text.

(2) Page 7 Paragraph 4 - A single sentence explaining why the p53 background had to be used for the Cep120 deletion would be beneficial. Did the cell line have a reduced centrosome number? Does this effect apical membrane initiation similar to centrinone?

We have revised the text (Page 7, Paragraph 4) to clarify that we were unable to generate a CEP120 KO line in p53-WT cells for unknown reasons. CEP120-KO cells have a normal number of centrosome, but their centrioles are shorter. Because this KO line still contains centrioles, the effect is different from centrinone treatment, which results in a complete loss of centrioles.

(3) Page 10 paragraph 4 - This paragraph is confusing to read. I understand that in the cysts and epithelial sheet the cytokinetic furrow is apical, therefore a movement towards the AMIS could be due to its coincidence with the furrow. However, the phrasing "....we found that centrosomes move towards the apical membrane initiation site direction before bridge abscission. Taken together these findings indicate the position is strongly associated with the site of cytokinesis but not with the apical membrane" is confusing to the reader.

We have revised the manuscript (Page 11, paragraph 4) to change the AMIS as the center of the cell doublet. During de novo epithelial polarization, the apical membrane has not yet formed at the Pre-Abs stage. However, at the Pre-Abs stage, the centrosome has already migrated toward the site of cytokinesis, suggesting that centrosome positioning is correlated with the site of cell division. A similar phenomenon occurs in fully polarized epithelial cysts and sheets, where the centrosomes also migrate before bridge abscission. Thus, we propose that the position of the centrosome is closely associated with the site of cytokinesis and is independent of apical membrane formation.

Discussion

(1) Page 11, Paragraph 2 - citations needed when discussing previous studies.

Thank you for your suggestion. We have included the necessary references to the discussion of the previous studies in the revised manuscript (Page 12, Paragraph 2).

(2) Page 12, Paragraph 2 - This section of the discussion would be strengthened by discussing the role of the actomyosin network in defining centrosome position (Jimenez et al., 2021). It seems plausible that the differences observed in the different conditions could be due to altered actomyosin architecture. Especially where the cells haven't undergone cytokinesis.

We appreciate the suggestion of a role for the actomyosin network in determining centrosome positioning. Recent studies have indeed highlighted the role of the actomyosin network in regulating centrosome centering and off-centering (Jimenez et al., 2021). During the pre-abscission stage of cell division, the actomyosin network undergoes significant dynamic changes, with the contractile ring forming at the center and actin levels decreasing at the cell periphery. In contrast, under aggregated cell conditions—meaning cells that have not undergone division—the actomyosin network does not exhibit such dynamic changes. The loss of actomyosin remodeling may therefore influence whether the centrosome moves. Thus, alterations in actomyosin architecture may contribute to the differences observed under various conditions, particularly when cells have not yet completed cytokinesis. We have revised Paragraph 2 on Page 13 to briefly mention the referenced study and to propose that the actomyosin network may influence centrosome positioning, contributing to our observed results. This addition strengthens the discussion and clarifies our findings.

(3) Page 12 paragraph 3 - Given that centrosome translocation during cytokinesis in MDCK cells (this study) appears to be similar to that observed in HeLa cells and the zebrafish Kupffers vesicle (Krishnan et al., 2022) it would be interesting to discuss why Rab11a and PCNT may not be essential to centrosome positioning in MDCK cells.

Thank you for your insightful comment. We agree that it is interesting that centrosome translocation during cytokinesis in MDCK cells (as observed in our study) is similar to that observed in HeLa cells and zebrafish Kupffer's vesicle (Krishnan et al., 2022). However, there are notable differences between these systems that may help explain why Rab11a and PCNT are not essential for centrosome positioning in MDCK cells.

Our study used 3D culture of MDCK cells, while the reference study examined adherent culture of HeLa cells. In the adherent culture, cells attached to the culture surface form large actin stress fibers on their basal side, which weakens the actin networks in the apical and intercellular regions. In contrast, the 3D culture system used in our study better preserves cell polarity and the integrity of the actin network, which might contribute to centrosome positioning independent of Rab11a and PCNT. Differences in culture conditions and actin network architecture may explain why Rab11a and PCNT are not required for centrosome positioning in MDCK cells.

Furthermore, the referenced study focused on Rab11a and PCNT in zebrafish embryos at 3.3–5 hours post-fertilization (hpf), a time point before the formation of the Kupffer’s vesicle. At this stage, the cells they examined may not yet have become epithelial cells, which may also influence the requirement of Rab11a and PCNT for centrosome positioning. We hypothesize that during the pre-abscission stage, centrosome migration toward the cytokinetic bridge occurs primarily in epithelial cells, and that the polarity and centrosome positioning mechanisms in these cells may differ from those in other cell types, such as zebrafish embryos.

Furthermore, data from Krishnan et al. (2022) suggest that cytokinesis failure in pcnt+/- heterozygous embryos and Rab11a functional-blocked embryos may be due to the presence of supernumerary centrosomes. Consistent with this, our data show that blocking cytokinesis inhibits centrosome movement in MDCK cells. However, in our MDCK cell lines with PCNT or Rab11a knockdown, we did not observe significant cytokinesis failure, and centrosome migration proceeded normally.

Reviewer #2 (Recommendations for the authors):

Suggestions for experiments:

(1) A description of the organization of microtubules in the absence of centriole, or in the absence of ECM would be interesting to understand how polarity markers end up where you observed them. This easy experiment may significantly improve our understanding of this system.

Previous studies have shown that in the absence of centrioles, microtubule organization undergoes significant changes. Specifically, the number of non-centrosomal microtubules increases, and these microtubules are not radially arranged, leading to the absence of focused microtubule organizing centers in centriolar-deficient cells (Martin, Veloso, Wu, Katrukha, & Akhmanova, 2018). This disorganized microtubule network reduces the efficiency of vesicle transport during de novo epithelial polarization at the mitotic preabscission stage.

In contrast, the organization of microtubules under ECM-free conditions remains less well characterized. Here, we show that while the ECM plays a critical role in establishing the direction of epithelial polarity, it does not influence the positioning of the centrosome, the microtubule-organizing center (MTOC).

(2) Would it be possible to knock down ODF2 and pericentrin to completely disconnect the centrosome from microtubules?

ODF2 is the base of subdistal appendages. When ODF2 is knocked out, it affects the recruitment of all downstream proteins to the subdistal appendages (Mazo, Soplop, Wang, Uryu, & Tsou, 2016). One study has shown that ODF2 knockout cells almost completely lost subdistal appendage structures and significantly reduced the microtubule asters surrounding the centrioles (Tateishi et al., 2013). However, although pericentrin (PCNT) is the main scaffold of the pericentriolar matrix (PCM) of centrosomes, the microtubule organization ability of centrosomes can be compensated by AKAP450, a paralog of PCNT, after PCNT knockout. A previous study has even shown that in cells with a double knockout of PCNT and AKAP450, γ-tubulin can still be recruited to the centrosomes, and centrosomes can still nucleate microtubules (Gavilan et al., 2018). This suggests that there are other proteins or pathways that promote microtubule nucleation on centrosomes. We are unsure whether the triple knockout of ODF2, PCNT, and AKAP450 can completely disconnect the centrosome from microtubules. However, a recent study reported a simpler approach involving the expression of dominant-negative fragments of the γ-tubulinbinding protein NEDD1 and the activator CDK5RAP2 at the centrosome (Vinopal et al., 2023). In our revised manuscript (Page 8, Paragraph 4; Figure 4—figure supplement 3), we applied this strategy, which resulted in the depletion of nearly all γ-tubulin from the centrosome. This indicates a strong suppression of centrosomal microtubule nucleation and an effective disconnection of the centrosome from the microtubule network.

(3) The study does not distinguish the role of cytokinesis from the role of tight junctions, which form only after cytokinesis and not simply by bringing cells into contact. Would it be feasible and interesting to study the polarization after cytokinesis in cells that could not form tight junctions (due to the absence of Ecad or ZO1 for example)?

Studying cell polarization after cytokinesis in cells unable to form tight junctions is a promising area of research.

Recent studies have shown that mouse embryonic stem cells (mESCs) cultured in Matrigel can form ZO-1-labelled tight junctions at the midpoint of cell–cell contact even in the absence of cell division. However, in the absence of E-cadherin, ZO-1 localization is significantly impaired. Interestingly, despite the loss of E-cadherin, the Golgi apparatus and centrosomes remain oriented toward the cell–cell interface (Liang, Weberling, Hii, Zernicka-Goetz, & Buckley, 2022). These findings suggest that cell polarity can be maintained independently of tight junction formation, highlighting the potential value of studying cell polarization that lack tight junctions.

Furthermore, while studies have explored the effects of knocking down tight junction components such as JAM-A and Cingulin on lumen formation in MDCK 3D cultures (Mangan et al., 2016; Tuncay et al., 2015), the role of ZO-1 in this context remains underexplored. Cingulin knockdown has been shown to disrupt endosome targeting and the formation of the AMIS, while both JAM-A and Cingulin knockdown result in actin accumulation at multiple points, leading to the formation of multi-lumen structures rather than a reversal of polarity. However, previous research has not specifically investigated centrosome positioning in JAM-A and Cingulin knockdown cells, an area that could provide valuable insights into how polarity is maintained in the absence of tight junctions.

Writing details:

(1) The migration of the centrosome in the absence of appendages or PCM is proposed to be ensured by compensatory mechanisms ensuring the robustness of microtubule anchoring to the centrosome. It could also be envisaged that the centrosome motion does not require this anchoring and that other yet unknown moving mechanisms, based on an actin network for example, might exist.

Thank you for your valuable comments. We agree that there may indeed be some unexpected mechanisms that allow centrosomes to move independently of microtubule anchoring to the centrosome, such as mechanisms based on actin filaments or noncentrosomal microtubules; these mechanisms are worth further investigation.

In response to your suggestion, in the Paragraph 5 of the discussion section, we further clarified that while a microtubule anchoring mechanism might be one explanation, other mechanisms could also influence centrosome movement in the absence of appendages or PCM. Additionally, we revised the Paragraph 4 regarding the possibility of actin network-driven centrosome movement and emphasized the importance of future research for a deeper understanding of these processes.

(2) The actual conclusion of the study of Martin et al (eLife 2018) is not simply that centrosome is not involved in cell polarization but that it hinders cell polarization!

Thank you for your valuable feedback. We agree with the findings of Martin et al. (eLife 2018) that centrosome is not irrelevant to cell polarity, but rather they inhibit cell polarization. Therefore, we have revised the manuscript (Page 2, Paragraph 2) to more accurately reflect this viewpoint.

(3) This study recalls some conclusions of the study by Burute et al (Dev Cell 2017), in particular the role of Par3 in driving centrosome toward the intercellular junction of daughter cells after cytokinesis. It would be welcome to comment on the results of this study in light of their work.

Thank you for your valuable feedback. The study by Burute et al. (Dev Cell, 2017) showed that in micropattern-cultures of MCF10A cells, the cells exhibit polarity and localize their centrosomes towards the intercellular junction, while downregulation of Par3 gene expression disrupts this centrosome positioning. This result is similar to our findings in 3D cultured MDCK cells and consistent with previous studies in C. elegans intestinal cells and migrating NIH 3T3 cells (Feldman & Priess, 2012; Schmoranzer et al., 2009), indicating that Par3 indeed influences centrosome positioning in different cellular systems. However, Par3 does not directly localize to the centrosome; rather, it localizes to the cell cortex or cell-cell junctions. Therefore, Par3 likely regulates centrosome positioning through other intermediary molecules or mechanisms, but the specific mechanism remains unclear and requires further investigation.

(4) Could the term apico-basal be used in the absence of a basement membrane to form a basal pole?

We understand that using the term "apico-basal" in the absence of a basement membrane might raise some questions. Traditionally, the apico-basal axis refers to the polarity of epithelial cells, where the apical surface faces the lumen or external environment, and the basal surface is oriented toward the basement membrane. However, in the absence of a basement membrane, such as in certain in vitro systems or under specific experimental conditions, polarity along a similar axis can still be observed. In such cases, the term "apico-basal" can still be used to describe the polarity between the apical domain and the region where it contacts the substrate or adjacent cells.

(5) The absence of centrosome movement to the intercellular bridge in spread cells in culture is not so surprising considering the work of Lafaurie-Janvore et al (Science 2018) about the role of cell spreading in the regulation of bridge tension and abscission delay.

Thank you for your valuable comment. Indeed, previous studies have shown that in some cell types, the centrosome does move toward the intercellular bridge in spread cells (Krishnan et al., 2022; Piel, Nordberg, Euteneuer, & Bornens, 2001), but other studies have suggested that this movement may not be significant and it may not occur in universally observed across all cell types (Jonsdottir et al., 2010). In our study, we aim to demonstrate that this phenomenon is more pronounced in 3D culture systems compared to 2D spread cell culture systems. Previous studies and our work have observed that centrosome migration occurs during the pre-abscission stage, but whether this migration is directly related to cytokinetic bridge tension or the time of abscission remains an open question. Further research is needed to explore the potential relationship between centrosome positioning, cytokintic bridge tension, and the timing of abscission.

(6) GP135 (podocalyxin) has been proposed to have anti-adhesive/lubricant properties (hence its pro-invasive effect). Could it be possible that once localized at the cell surface it is systematically moved away from regions that are anchored to either the ECM or adjacent cells? So its localization away from the centrosome in an ECM-free experiment would not be a consequence of defective targeting but relocalization after reaching the plasma membrane?

Thank you for your valuable comment. We agree that GP135 may indeed move directly across the cell surface, away from the region where it interacts with the ECM or adjacent cells. This re-localization could be due to its anti-adhesive or lubricating properties, which may facilitate its displacement from these adhesive sites. To validate this, it is necessary to employ higher-resolution real-time imaging system to observe the dynamic behavior of GP135 on the cell surface.

However, this does not contradict our main conclusion. Under suspension culture conditions without ECM, the centrosome positioning in cell doublets is indeed decoupled from apical membrane orientation. This suggests that the localization of the centrosome and the apical membrane is regulated by different mechanisms. Specifically, the GP135 protein tends to accumulate away from areas of contact with the ECM or adjacent cells, possibly through movement within the cell membrane or by recycling endosome transport. In contrast, centrosome positioning is closely related to the cytokinesis site. Our study clearly elucidates the differences between these two polarity properties.

Reviewer #3 (Recommendations for the authors):

Major:

(1) To me, a clear implication of these studies is that Gp135, Rab11, etc. are delivered to the AMIS on centrosomal microtubules. The authors do not explore this model except to say that depletion of SD appendage or pericentrin has no effect on the protein relocalization to the AMIS. However, the authors do not observe microtubule association with the centrosome in these KO conditions. This analysis is imperative to interpret existing results since these are new KO conditions in this cell/culture system and parallel pathways (e.g. CDK5RAP2) are known to contribute to microtubule association with the centrosome. An ability to comment on the mechanism by which the centrosome contributes to the efficiency of polarization would greatly enhance the paper.

Microtubule requirement could also be tested in numerous additional ways requiring varying degrees of new experiments:

(a) faster live cell imaging at abscission to see if the deposition of those components appears to traffic on MTs;

(b) live cell imaging with microtubules (e.g. SPY-tubulin) and/or EB1 to determine the origin and polarity of microtubules at the pertinent stages;

For (a) and (b), because the cells were cultured in Matrigel, they tended to be round up, with a dense internal structure that made observation difficult. In contrast, under adherent culture conditions, the cells were flattened with a more dispersed internal structures, making them easier to observe. We had previously used SPY-tubulin to label microtubules for live cell imaging; however, due to the dense microtubule structure in 3D culture, the image contrast was reduced, and we could not clearly observe the microtubule network within the cells.

(c) acute nocodazole treatment at abscission to determine the effect on protein localization.

Regarding the method of using nocodazole to study microtubule requirements at the abscission stage, we believe that nocodazole treatment may lead to cytokinesis failure. Cell division failure results in the formation of binucleated cells, which are unable to establish cell polarity. Furthermore, nocodazole treatment cannot distinguish between centrosomal and non-centrosomal microtubules, making it unsuitable for studying the specific role of centrosomal microtubules in this process.

In our new data (Figure 4-figure supplementary 3) presented in the revised manuscript, we employed a recently reported method by co-expressing of the centrosome-targeting carboxy-terminal domain (C-CTD) of CDK5RAP2 and the γ-tubulin-binding domain (gTBD) of NEDD1 to completely deplete γ-tubulin and abolish centrosomal microtubule nucleation (Vinopal et al., 2023). We found that cells lacking centrosomal microtubules were still able to polarize and position the centrioles apically. However, the efficiency of polarized transport of Gp135 vesicles to the apical membrane was reduced. These findings suggest that centrosomal microtubules are not essential for polarity establishment but may contribute to facilitate efficient apical transport.

(2) Similar to the expanded analysis of the role of microtubules in this system, it would be excellent if the author could expand on the role of Par3 and the centrosome, although this reviewer recognizes that the authors have already done substantial work. For example, what are the consequences of Gp135 and/or Rab11 getting stuck at the centrosome? Do the authors have any later images to determine when and if these components ever leave the centrosome? Existing literature focuses on the more downstream consequence of Par3 removal on single-lumen formation.

Similarly, could the authors expand on the description of polarity disruption following centrinone treatment? It is clear that Gp135 recruitment is disrupted, but how and when do things get fixed and what else is disrupted at the very earliest stages of AMIS formation? The authors have an excellent opportunity to really expand on what is known about the requirements for these conserved components.

Regarding the use of centrinone in treatment, we speculate that Gp135 can still accumulate at the AMIS over time, although the efficiency of its recruitment may be reduced.

Furthermore, under similar conditions, other apical membrane components (such as the Crumbs3 protein) may exhibit similar characteristics to Gp135 protein.

(3) Perhaps satisfying both of the above asks, could the authors do a faster time-lapse at the relevant time points, i.e. as proteins are being recruited to the AMIS (time points between 1Aiv and v)? This type of imaging again might help shed light on the mechanism.

We believe the above questions are very important and may require further experimental verification in the future.

Minor:

(1) What is the green patch of Gp135 in Figure 2A that does not colocalize with the centrosome? Is this another source of Gp135 that is being delivered to the AMIS? This type of patch is also visible in Figure 3A 15 and 30-minute panels.

During mitosis, membrane-composed organelles such as the Golgi apparatus are typically dispersed throughout the cytoplasm. However, during the pre-abscission stage, these organelles begin to reassemble and cluster around the centrosome. Furthermore, they also accumulate in the region between the nucleus and the cytokinetic bridge, corresponding to the “patch” mentioned in Figure 2A.

Live cell imaging results showed that this Gp135 patch initially appears in a region not associated with the centrosome. Subsequently, they were either directly transported to the AMIS or fused with the centrosome-associated Gp135 and transported together. Notably, this patch was only observed when Gp135 was overexpressed in cells. No such distinct protein patches were observed when staining endogenous Gp135 protein (Figure 1A), suggesting that overexpression of Gp135 protein may lead to a localized increase in its concentration in that region.

(2) I am confused by the "polarity index" quantification as this appears to just be a nucleus centrosome distance measurement and wouldn't, for example, distinguish if the centrosomes separated from the nucleus but were on the basal side of the cell.

The position of the centrosome within the cell (i.e., its distance from the nucleus) can indeed serve as an indicator of cell polarity (Burute et al., 2017). We acknowledge that this quantitative method does not directly capture the specific direction in which the centrosome deviates from the cell center. To address this limitation, we have incorporated information about the angle between the nucleus and the centrosome, which allows for a more accurate description of changes in cell polarity (Rodriguez-Fraticelli, Auzan, Alonso, Bornens, & Martin-Belmonte, 2012).

(3) How is GP135 "at AMIS" measured? Is an arbitrary line drawn? This is important later when comparing to centrinone treatment in Figure 3D where the quantification does not seem to accurately capture the enrichment of Gp135 that is seen in the images.

To measure the expression level of Gp135 in the "AMIS" region of the cell, we first connected the centers of the two cell nuclei in three-dimensional space to form a straight line. Then, we used the Gp135 expression intensity at the midpoint of this line as the representative value for the AMIS region. This method is based on the assumption that the AMIS region is most likely located between the centers of the two cell nuclei. Therefore, this quantitative method provides a standardized assessment tool for comparing Gp135 expression levels under different conditions.

(4) The authors reference cell height (p.7) but no data for this measurement are shown

Thank you for the comment. Although we did not perform quantitative measurements, the differences in cell height are clearly visible in Figure 3E (p53-KO + CN), which visually illustrates this phenomenon.

(5) Can the authors comment on the seeming reduction of Par3 in p53 KO cells?

We did not observe a reduction of Par3 in p53-KO cells in our experiments.

(6) Can the authors make sense of the E-cad localization: Figure 5, Supplement 2.

Our study revealed that E-cadherin begins to accumulate at the cell-cell contact sites during the pre-abscission stage. Its appearance is similar to that of ZO-1, which also appears near the cell division site during this phase. Therefore, the behavior of E-cadherin contrasts sharply with that of Gp135, further highlighting the unique trafficking mechanisms of apical membrane proteins during this process.

(7) I find the results in Figure 6G puzzling. Why is ECM signaling required for Gp135 recruitment to the centrosome. Could the authors discuss what this means?

We appreciate the reviewer’s valuable comments and thank you for the opportunity to clarify this point. The data in Figure 6G do not indicate that ECM signaling is required for the recruitment of Gp135 to the centrosome. Rather, our findings suggest that even in the absence of ECM, the centrosomes can migrate to a polarized position similar to that in Matrigel culture. This suggests that centrosome migration and the orientation of the nucleus–centrosome axis may be independent of ECM signaling and are primarily driven by cytokinesis alone.

Regarding the localization of Gp135, previous studies have shown that ECM signaling through integrin promotes endocytosis, which is crucial for the internalization of Gp135 from the cell membrane and its subsequent transport to the AMIS (Buckley & St Johnston, 2022). Our study found that, prior to its accumulation at the AMIS, Gp135 transiently localizes around the centrosome. In the absence of ECM, due to reduced endocytosis, Gp135 primarily remains on the cell membrane and does not undergo intracellular trafficking.

(8) The authors end the Discussion stating that these studies may have implication for in vivo settings, yet do not discuss the striking similarities to the C. elegans and Drosophila intestine or the findings from any other more observational studies of tubular epithelial systems in vivo (e.g. mouse kidney polarization, zebrafish neuroepithelium, etc.). These models should be discussed.

Thank you for your valuable comment. Indeed, all types of epithelial tissues or tubular epithelial systems in vivo share some common features during cell division, which have been well-documented across various species.

These features include: during interphase, the centrosome is located at the apical surface of the cells; after the cell enters mitosis, the centrosome moves to the lateral side of the cell to regulate spindle orientation; and during cytokinesis, the cleavage furrow ingresses asymmetrically from the basal to the apical side, with the cytokinetic bridge positioned at the apical surface. Our study using MDCK 3D culture and transwell culture systems successfully mimicked these key features, demonstrating that these in vitro models are of significant value for studying cell polarization dynamics.

Based on our observations, we speculate that the centrosome may return to the apical surface after anaphase, just before bridge abscission. This is consistent with our findings from studies using MDCK 3D cultures and transwell systems, which showed that the centrosome relocates prior to the final stages of cytokinesis.

Additionally, we propose that de novo polarization of the kidney tubule in vivo may not solely depend on the aggregation and mesenchymal-epithelial transition (MET) of the metanephric mesenchyme. It may also be related to the cell division process, which triggers centrosome migration and polarized vesicle trafficking. These processes likely contribute to enhancing cell polarization, as we observed in our in vitro models.

We hope this will further clarity the potential implications of our findings for in vivo model studies, as well as and their broader impact on the field of tubular epithelial cell polarization research.

(9) There are several grammatical issues/typos throughout the paper. A careful readthrough is required. For example:

this sentence makes no sense "that the centrosome acts as a hub of apical recycling endosomes and centrosome migration during cytokinetic pre-abscission before apical membrane components are targeted to the AMIS"

We carefully reviewed the paper and made necessary revisions to address the issues raised. In particular, we revised certain sentences to improve clarity and readability (Page 5, Paragraph 3).

(10) P.8: have been previously reported [to be] involved in MDCK...

We appreciate the reviewer's valuable suggestions. We have revised the sentence accordingly (Page 9, Paragraph 2).

(11) This sentence seems misplaced: "Cultured conditions influence cellular polarization preferences."

The sentence itself is fine, but to improve the coherence and clarity of the paragraph, we adjusted the paragraph structure and added some transitional phrases (Page 13, Paragraph 1).

(12) "Play a downstream role in Par3 recruitment" doesn't make sense, this should just be downstream of Par3 recruitment.

Thank you for your suggestion. We have revised the wording accordingly, changing it to "downstream of Par3 recruitment" (Page 10, Paragraph 2).

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