Aurora B‐dependent polarization of the cortical actomyosin network during mitotic exit

Abstract The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA‐mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.

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We would also welcome the submission of cover suggestions, or motifs to be used by our Graphics Illustrator in designing a cover. During cytokinesis, cells assemble an equatorial contractile ring that pinches the cell into two daughters. The generally round shape of the metaphase cell is transformed into an elongated shape as the two ends of the cell expand and move away from each other. How this process is regulated is not well understood. The process of polar relaxation contributes to cytokinesis by "relaxing" the polar cortices, often measured by the removal of F-actin and anillin at the two poles. Polar relaxation has a microtubule dependent component and also microtubule independent mechanism which is thought to involve signals from chromosomes.
In this manuscript, the authors test the role of two regulators of cytokinesis, centralspindlin and aurB kinase in driving cell shape changes in anaphase. They confirm that centralspindlin depletion (siRacGAP) prevents cytokinesis but not the cell shape changes observed in anaphase and the reduction in F actin. Similarly, inhibition of the CPC component aurora B kinase also prevents cytokinesis and blocks polarization of cells undergoing monopolar cytokinesis. Double inhibition, however, has a more profound effect, and cells remain rounded. This is an interesting observation; however, a mechanistic basis for this is not provided. Comments: 1. The abundance of cortical Factin and the presence of actin filopodia, ruffles, blebs at the poles mean that it is not completely cleared in anaphase. A better description of how actin measurements were made -central slice from Z stack?-is needed. A line scan along the cortex (as done in fig 2) would show precisely where actin is cleared. Alternatively selecting a different probe (anillin? ERM?) for the bipolar cells is recommended. Measurement of aspect ratio for fig 1 would be good.
2. chromosomes have been shown to play a role in polar relaxation due to their proximilty to the cortex. The authors need to measure the distance of the chromosomes to the cortex under the various treatments; is the change in midzone microtubules following loss of CS or CPC changing chromosome position relative to cortex? What happens in cells that are doubly inhibited? Imaging the midzone microtubules would also be good (or referencing others that have done this).
3. Perhaps it is beyond the scope of the current work but using a probe that shows kinase activity would be a nice addition.
4. An aurA inhibitor is used on monopolar cytokinesis in suppl fig2 (8 cells) and the polarization is not blocked, as happens with ZM. additional controls would be nice to see, to be sure that ZM is acting as expected. Depletion of another CPC component would be another experimental approach to try.

the experiment in suppl Fig 4 should be expanded to
show what happens to aspect ratio and cortical protein levels. The authors might also perform these expts (siEct2, with and without ZM treatment) on the hela cells, as the RPE cells can divide without the Rho activation.

Minor
Introduction reference in 3rd paragraph is wrong.

Referee #3:
Ramkumar and colleagues present a concise and focussed manuscript reporting a role for Aurora B in clearance of DNA-proximal actin that is necessary for polar relaxation in dividing cells. The experiments are well performed, the data are clear and the interpretations made are sensible. The observations are novel, but mechanistically underdeveloped, which I think fit EMBO Reports' remit. As such, I am supportive of publication, with some minor changes as outlined below. I think most can be performed with textual changes: 1. The authors report in control cells: 'During this early anaphase, we observed a modest but reproducibly and statistically significant clearance of actin filaments from cell poles.', but I couldn't see the data or quantification that would permit this statement, nor is it clear to which panel the reader should look to find this data.
2. In the markup for Fig 1C, Aurora B is labelled as GFP, but I think it is mNG 3. In the RACGAP depleted cells (4D) mNG-AuroraB seems dimmer -is this consistent and are there any differences in Aurora B expression in the absence of centralspindlin components? Similarly, no change in RACGAP localisation is claimed in ZM-treated cells (4E), but there is much more RACGAP in the cytoplasm and the relative enrichment at spindle midzone is reduced. Is this not evidence of reciprocal communication between these components? 4. In figure 2B, the cortical polarisation and pERM-rich pseudofurrow is clear and correlates with the pseudofurrow forming on the DNA-distal pole of the cell. The failure of actin to clear from the DNAproximal side in ZM-treated cells is subtle, but clear. The red dashed line in 2F makes seeing the quantification hard -could it underlay the data, rather than overlaying it? 5. Figure 3 is clear and convincing and evidences proximal DNA contributing to actin clearance in an Aurora B dependent manner using RAP1 overexpression to flatten the cell. It is perhaps worth commenting that the sphere-of-influence of the DNA-dependent actin clearance appears smaller in these RAP1 overexpressing cells -it is only a small DNA-proximal actin patch that is cleared, rather than a pole, as per the spherical monopolar assays. Stats on 3E are missing.
6. Statistical analysis, and the letters 'ngressuion' are missing from Figure S4C 7. Call outs for references, particularly those involving citation of book chapters, are improperly formatted (e.g., Rappaport  Successful execution of cytokinesis, the last step of the cell division programme, requires the coordination of the tubulin and actin cytoskeleton with the membrane trafficking machinery, and many aspects of this are still incompletely understood. The mitotic kinase Aurora B has an important role in the regulation of the microtubule-based central spindle during anaphase and cytokinesis but also regulates other aspects of the process. Ramkumar and colleagues, here, specifically investigate the role of Aurora B in regulating actomyosin behaviour. They find that clearance of actomyosin from the poles during cytokinesis requires Aurora B activity, and that this an event that is independent of the presence of the central spindle. Furthermore, they show that the distance of the cortex to the chromatin seems to be relevant for actomyosin clearance suggesting an involvement of DNA derived signals. Lastly, the authors demonstrate that the combined inhibition of Aurora B activity and depletion of RacGAP1, important for central spindle formation, has the most drastic effect on cell shape changes during cytokinesis, in line with the idea that these two pathways act in parallel. The main drawback of this work is that the manuscript is completely descriptive with no insight into the molecular biology underpinning the reported observations. Even the text is very vague, at multiple points in the text the authors refer to "DNA-dependent cues" without ever specifying or testing what this could be. The same applies for the role of Aurora B; it remains completely unclear whether the observed reliance of cortical actomyosin clearance on Aurora B is actually a direct effect and what possible targets of Aurora B may mediate this effect. A further issue is that the manuscript is confusing to read because the authors start out with the reasonable hypothesis that Aurora B mediates cortical actomyosin clearance but then don't pursue this at a molecular level. Instead they move on to look at the effect of the distance of chromatin from the cortex, and then from there jump to the combined effect of Aurora B inhibition and RacGAP1 depletion. In the end, the reader is left with the feeling that none of these potentially important aspects have been satisfactorily addressed.
Minor points: Canman et al., Nature, 2003 should be cited as the first paper to use a monopolar spindle system to look at the localisation of the chromosomal passenger proteins in the absence of a central spindle.
As the authors point out themselves, the central spindle is critical for cytokinesis, yet is never 10th Feb 2021 Additional Referee Report visualised in the manuscript. For the correct interpretation of the events under investigation, it is important that there are no indirect effects on the microtubule cytoskeleton that may confound the analysis.

Referee #1:
During cytokinesis, cells assemble an equatorial contractile ring that pinches the cell into two daughters. The generally round shape of the metaphase cell is transformed into an elongated shape as the two ends of the cell expand and move away from each other. How this process is regulated is not well understood. The process of polar relaxation contributes to cytokinesis by "relaxing" the polar cortices, often measured by the removal of F-actin and anillin at the two poles. Polar relaxation has a microtubule dependent component and also microtubule independent mechanism which is thought to involve signals from chromosomes.
In this manuscript, the authors test the role of two regulators of cytokinesis, centralspindlin and aurB kinase in driving cell shape changes in anaphase. They confirm that centralspindlin depletion (siRacGAP) prevents cytokinesis but not the cell shape changes observed in anaphase and the reduction in F actin. Similarly, inhibition of the CPC component aurora B kinase also prevents cytokinesis and blocks polarization of cells undergoing monopolar cytokinesis. Double inhibition, however, has a more profound effect, and cells remain rounded. This is a good summary of our main findings. This is an interesting observation; however, a mechanistic basis for this is not provided.
In the revised manuscript we have strengthened our analysis. While the precise mechanistic details by which Aurora B acts remain to be resolved, we think our paper contributes to our understanding of this previously unappreciated role for Aurora B. Comments: 1. The abundance of cortical Factin and the presence of actin filopodia, ruffles, blebs at the poles mean that it is not completely cleared in anaphase. A better description of how actin measurements were made -central slice from Z stack?-is needed. A line scan along the cortex (as done in fig 2) would show precisely where actin is cleared.
We thank the reviewer for their suggestion. As mentioned by the reviewer-we have used a projection of 2-z slices of the central region of the cell for our measurements and have amended our description to include this. We have also included line scans as suggested by the reviewer to better illustrate the loss of actin from polar regions in Figure 1 and Figure 5.
Alternatively selecting a different probe (anillin? ERM?) for the bipolar cells is recommended.
Use of an alternative probe is challenging as we would have to generate stable cell lines with live reporters to show relative clearance from metaphase to anaphase. The -Aspect ratio for siRacgap is included in Fig 4E where we have compared cell shape changes in all treatment conditions. 2. chromosomes have been shown to play a role in polar relaxation due to their proximilty to the cortex. The authors need to measure the distance of the chromosomes to the cortex under the various treatments; is the change in midzone microtubules following loss of CS or CPC changing chromosome position relative to cortex?
We thank reviewer for this suggestion and have now included this quantification of chromatin position with different treatment conditions ( Figure 5F-G). Proximity of chromatin to cortical actomyosin network remains unaffected upon loss of CS but is affected upon inhibition of CPC. This effect is enhanced in the double treatment.
What happens in cells that are doubly inhibited? Imaging the midzone microtubules would also be good (or referencing others that have done this).
We thank the reviewer for this suggestion and have included a panel showing midzone microtubules and midzone PLK1 accumulation with different treatments (Figure 4C,  EV4A) 3. Perhaps it is beyond the scope of the current work but using a probe that shows kinase activity would be a nice addition.
We thank the reviewer for their suggestion. We tried to use membrane targeted Aurora B-FRET sensor to assess the activity of the kinase. In order to visualize region specific Aurora B activity, the sensor needs to be targeted to-centromere, microtubule, membrane, etc. as the cytoplasmic FRET sensor is unable to recapitulate the tagged sensor activity. Owing to the dynamic re-localization of Aurora B, we found the results not very useful for this study.
4. An aurA inhibitor is used on monopolar cytokinesis in suppl fig2 (8 cells) and the polarization is not blocked, as happens with ZM. additional controls would be nice to see, to be sure that ZM is acting as expected. Depletion of another CPC component would be another experimental approach to try.
We thank the reviewer for this suggestion. We have included knockdown of INCENP, a core member of the CPC complex and performed our monopolar exit analysis. Similar to MKLP2 knockdown, we find that INCENP knockdown also leads to a loss in polarization of pERM during monopolar exit. Fig 4 should be expanded to show what happens to aspect ratio and cortical protein levels. The authors might also perform these expts (siEct2, with and without ZM treatment) on the hela cells, as the RPE cells can divide without the Rho activation.

the experiment in suppl
We thank the reviewer for their suggestions. 1. We have now expanded the figure to include the cell shape quantifications for RPE1 cells under different treatment ( Figure S5). RPE1 cells are migratory cells and therefore, unlike Hela cells, the lamellipodia and other actin dependent projections make is harder to definitively quantify cortical protein levels during anaphase. However, we think cell shape changes, quantified as aspect ratio is a good indicator of cell polarization.
2. We have also included data on Hela cells treated with siEct2 and siEct2+ZM ( Figure  S4). Similar to RPE1 cells, double treatment of cells with siEct2 and ZM completely abrogates any shape changes.

Minor
Introduction reference in 3rd paragraph is wrong.
We thank the reviewer and have amended the reference.

Supp 3 add control for top panel of A
We have corrected the figure to reflect this.

Referee #3:
Ramkumar and colleagues present a concise and focussed manuscript reporting a role for Aurora B in clearance of DNA-proximal actin that is necessary for polar relaxation in dividing cells. The experiments are well performed, the data are clear and the interpretations made are sensible. The observations are novel, but mechanistically underdeveloped, which I think fit EMBO Reports' remit. As such, I am supportive of publication, with some minor changes as outlined below. I think most can be performed with textual changes: We thank the reviewer for their assessment.
1. The authors report in control cells: 'During this early anaphase, we observed a modest but reproducibly and statistically significant clearance of actin filaments from cell poles.', but I couldn't see the data or quantification that would permit this statement, nor is it clear to which panel the reader should look to find this data.
We thank the reviewer for their suggestion and have amended the text to reflect this. We have also included statistical analysis to show that there is significant reduction of actin levels in both siControl and siRacGAP treated cells. (Figure1) We amended the text -"During this early anaphase, we observed a modest but significant clearance of actin filaments from cell poles ( Figure 1A-E)

as seen by actin intensity levels clustering below 1 (normalized to metaphase levels)."
We have also added line scan plots to better illustrate the loss of actin filaments from the poles.
2. In the markup for Fig 1C, Aurora B is labelled as GFP, but I think it is mNG We have amended the figure. 3. In the RACGAP depleted cells (4D) mNG-AuroraB seems dimmer -is this consistent and are there any differences in Aurora B expression in the absence of centralspindlin components?
We thank the reviewer for their suggestion. We quantified the levels of Aurora B upon centralspindlin depletion and found a moderate decrease in its accumulation at the midzone. We have included a figure panel to reflect this quantification ( Figure S1D). However, we do not see a correlation between the level of Aurora B accumulation at the spindle midzone and the ability of cells to perform cytokinesis. Irrespective of the levels of Aurora B at the midzone, the cells fail to form a furrow.
Similarly, no change in RACGAP localisation is claimed in ZM-treated cells (4E), but there is much more RACGAP in the cytoplasm and the relative enrichment at spindle midzone is reduced. Is this not evidence of reciprocal communication between these components?
We thank the reviewer for their careful observation. We have amended the text to reflect this. figure 2B, the cortical polarisation and pERM-rich pseudofurrow is clear and correlates with the pseudofurrow forming on the DNA-distal pole of the cell. The failure of actin to clear from the DNA-proximal side in ZM-treated cells is subtle, but clear. The red dashed line in 2F makes seeing the quantification hard -could it underlay the data, rather than overlaying it?

In
We thank the reviewer for this suggestion and have moved the dashed line as an underlay. Figure 3 is clear and convincing and evidences proximal DNA contributing to actin clearance in an Aurora B dependent manner using RAP1 overexpression to flatten the cell. It is perhaps worth commenting that the sphere-of-influence of the DNA-dependent actin clearance appears smaller in these RAP1 overexpressing cells -it is only a small DNA-proximal actin patch that is cleared, rather than a pole, as per the spherical monopolar assays. Stats on 3E are missing.

5.
We thank the reviewer for this suggestion. The reviewer is correct in pointing out that the effect of DNA on the relaxation is localized to the region directly in close proximity to it. It perhaps works in a similar manner in the polar regions. We currently do not have the ability to splay out the polar region to visualize this local effect and therefore in the tangential view, visualize it as a gradient.
We had included statistical analysis -Chi-square test to compare distribution of cells in Figure 3E and Figure EV3B, both of which show significant difference.
6. Statistical analysis, and the letters 'ngressuion' are missing from Figure S4C We have amended the figure and also included statistical analysis for Figure EV4C, currently Figure EV5G. Chi-square test shows significant difference. We have amended the text accordingly.

Reviewer 2
Successful execution of cytokinesis, the last step of the cell division programme, requires the coordination of the tubulin and actin cytoskeleton with the membrane trafficking machinery, and many aspects of this are still incompletely understood. The mitotic kinase Aurora B has an important role in the regulation of the microtubule-based central spindle during anaphase and cytokinesis but also regulates other aspects of the process. Ramkumar and colleagues, here, specifically investigate the role of Aurora B in regulating actomyosin behaviour. They find that clearance of actomyosin from the poles during cytokinesis requires Aurora B activity, and that this an event that is independent of the presence of the central spindle. Furthermore, they show that the distance of the cortex to the chromatin seems to be relevant for actomyosin clearance suggesting an involvement of DNA derived signals. Lastly, the authors demonstrate that the combined inhibition of Aurora B activity and depletion of RacGAP1, important for central spindle formation, has the most drastic effect on cell shape changes during cytokinesis, in line with the idea that these two pathways act in parallel. This is a good summary of our work.
The main drawback of this work is that the manuscript is completely descriptive with no insight into the molecular biology underpinning the reported observations. Even the text is very vague, at multiple points in the text the authors refer to "DNA-dependent cues" without ever specifying or testing what this could be. The same applies for the role of Aurora B; it remains completely unclear whether the observed reliance of cortical actomyosin clearance on Aurora B is actually a direct effect and what possible targets of Aurora B may mediate this effect.
We respectfully disagree that this is "completely descriptive". We describe a causal mechanistic role for Aurora B in polar relaxation. We would note that there are almost no systems in which we know the complete molecular and biophysical processes that underpin division.
A further issue is that the manuscript is confusing to read because the authors start out with the reasonable hypothesis that Aurora B mediates cortical actomyosin clearance but then don't pursue this at a molecular level. Instead they move on to look at the effect of the distance of chromatin from the cortex, and then from there jump to the combined effect of Aurora B inhibition and RacGAP1 depletion. In the end, the reader is left with the feeling that none of these potentially important aspects have been satisfactorily addressed.
Our work addresses an important cell biological problem -anaphase polarization. Successful completion of cytokinesis requires both the formation of a contractile actomyosin ring and the softening of the polar region. Using a range of perturbationscell shape, monopolar spindles, small molecular inhibition and RNAi, we identify a novel role for Aurora B, in regulating polar relaxation. This function of Aurora B is independent of its role in regulating centralspindlin. Therefore, Aurora B emerges as a central player in cytokinesis by coordinating the changes in actomyosin at the equatorial and polar regions to ensure a successful cytokinesis. While much more work needs to be done to identify the precise molecular details by which Aurora B executes this functionour work helps open up a new avenue of research that we believe will help to stimulate research into this understudied aspect of cytokinesis.
Minor points: Canman et al., Nature, 2003 should be cited as the first paper to use a monopolar spindle system to look at the localisation of the chromosomal passenger proteins in the absence of a central spindle.
We have amended the text accordingly and referenced the paper.
As the authors point out themselves, the central spindle is critical for cytokinesis, yet is never visualised in the manuscript. For the correct interpretation of the events under investigation, it is important that there are no indirect effects on the microtubule cytoskeleton that may confound the analysis.
We thank the reviewer for this suggestion and have now included two figure panels showing immunostaining of microtubules ( Figure 4C) and spindle midzone component PLK1 ( Figure EV4A). They both show that midzone is formed normally in early anaphase upon RacGAP knockdown, but fails to be stabilized soon after. In contrast, ZM treatment leads to a disperse midzone which persists through mitotic exit. In the double treatment, there is no recognizable spindle midzone. We demonstrate that cells are able to clear actin from their poles under various treatments where the midzone is altered --upon RacGAP knockdown-where the midzone is unstable, -in monopolar cytokinesis, where there is no over-lapping microtubules or spindle midzone structure.
This suggests that the ability of cells to relax their poles must be independent of the function of central spindle during cytokinesis.

10th Jun 2021 1st Revision -Editorial Decision
Dear Dr. Ramkumar, Thank you for submitting your revised manuscript. It has now been seen by two of the original referees.
As you can see, the referee finds that the study is significantly improved during revision and recommends publication. However, I need you to address the editorial points below before I can accept the manuscript.
• Please address the remaining minor concerns of referee #1. • As per our format requirements, manuscripts containing 5 main figures are classified as 'reports', however, your manuscript format is 'article'. Therefore, please convert your manuscript format into a 'report' and thus combine the results and the discussion sections. Please see https://www.embopress.org/page/journal/14693178/authorguide#researcharticleguide • Please provide 3-5 keywords for your study. These will be visible in the html version of the paper and on PubMed and will help increase the discoverability of your work. • As per our guidelines, please add a 'Data Availability Section', where you state that no data were deposited in a public database. • Please rename the 'Author statement' section as 'Conflict of Interest'. • Please rename the 'Methods' section as 'Materials and Methods' • Please reorder the manuscript sections in the order shown here: https://www.embopress.org/page/journal/14693178/authorguide#textformat • Please include the funding information also in the manuscript text -i.e., in the Acknowledgements section.
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Thank you again for giving us to consider your manuscript for EMBO Reports, I look forward to your minor revision. The authors provide data consistent with a role for aurora B activity in cell polarization and cortical actin clearance during the events of late anaphase. They do not cite Mangal JCB 2018 who showed that clearance of anillin from the polar cortex requires Aurora A activation by TPXl1 in C. elegans. The current Ms shows that a chemical inhibitor of Aurora A has no effect on cortical polarization. The authors should cite Mangal et al and add a statement of the reasons that might give rise to the different results.
On page 5 the authors mention that depletion of MKLP2 prevented pERM cortical polarization "suggesting a role for Aurora B localization and activity". MKLP2 should alter the localization of the CPC to the midzone, but not AurB activity, so one might expect that loss of MKLP2 would not show the same effect as ZM. Please clarify.
In Figure 5, quantification in C. please show the quantification for the siR and DMSO on the graph.

My concerns have been addressed -all looks fine to me
We thank the reviewer for their comments and have revised the manuscript to include the suggestions.
In the Figures the authors show the actin (or ERM) fluorescence intensity around the cell cortex by graphing distance around the cortex vs the relative intensity (see for example figure 2). In other cases (i.e., Figure 5) they show a single trace of normalized intensity vs normalized distance. The first method does a much better job of showing the trend around the cell, as compared with the single lines (see fig 5E). Can the authors please use the average of several cells instead of the single trace?
We thank the reviewer for their suggestion. We have replaced graphs for single cells with graphs showing the trend for average population of cells under different treatments ( Figure  5E).
The authors provide data consistent with a role for aurora B activity in cell polarization and cortical actin clearance during the events of late anaphase. They do not cite Mangal JCB 2018 who showed that clearance of anillin from the polar cortex requires Aurora A activation by TPXl1 in C. elegans. The current Ms shows that a chemical inhibitor of Aurora A has no effect on cortical polarization. The authors should cite Mangal et al and add a statement of the reasons that might give rise to the different results.
We thank the reviewer for their suggestion and have included this in our discussion.
"Although we did not observe a defect in DNA-dependent clearance upon Aurora A inhibition in human cells ( Figure EV2A On page 5 the authors mention that depletion of MKLP2 prevented pERM cortical polarization "suggesting a role for Aurora B localization and activity". MKLP2 should alter the localization of the CPC to the midzone, but not AurB activity, so one might expect that loss of MKLP2 would not show the same effect as ZM. Please clarify. Our data with MKLP2 silencing suggests that Aurora B activity needs to be spatially regulated to induce polar relaxation. Therefor we hypothesize that the switch in Aurora B localization from centromeres to midzone at early anaphase controls the spatial region of its activity which in turn regulates polar relaxation. We have amended the text to reflect this. "Interestingly, cortical polarization was affected in a similar way by depletion of MKLP2 ( Figure  EV2C-D), a kinesin required for Aurora B re-localization during anaphase (Serena et al, 2015). This suggests a role for spatial control of Aurora B activity in polarizing the cortex at mitotic exit." 17th Jul 2021 2nd Authors' Response to Reviewers In Figure 5, quantification in C. please show the quantification for the siR and DMSO on the graph.
We have amended Figure 5C to include the above conditions. At the end of this email I include important information about how to proceed. Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.
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