Mitosis sets nuclear homeostasis of cancer cells under confinement

Reviewer #1 (Public Review):

Summary
In this work, Mouelhi et al investigated how the nucleus responds to long term confinement. They find that short-term confinement does not affect nuclear volume, whereas long-term confinement leads to a decrease in volume. The authors propose this decrease occurs after mitosis and relies on cPLA2 and myosin contractility.

Strengths

The ability to accurately control cell confinement allows authors to determine its effects on cellular function with high resolution. This provides a good addition to the existing collection of tools used for cellular micromanipulation. The results provided are relevant and timely and could help understand how cancer cells adapt to conditions of confinement.

Weaknesses

I have a few concerns which I believe should be addressed:

(1) It is unclear whether the authors took into consideration the contribution of nuclear blebs for nuclear volume measurements. This would be particularly relevant in situations of very strong confinement. Blebs were previously shown to affect volume (Mistriotis et al., JCB 2019). One could argue that the decreased nuclear volume was due to the increased blebbing observed in very strong confinements.

(2) From their experimental setup, it is unclear whether the reduced nuclear volume observed after confined cell division arises from a geometrical constraint or is due to an intrinsic nuclear feature. One could argue that cells exiting mitosis under confinement have clustered chromosomes and, therefore, will have decreased volume. This would imply that the nucleus is not "reset" but rather that a geometrical constraint is forcing nuclei to be smaller. One way to test this would be to follow individual cells under confinement, let them enter mitosis, and then release the confinement. If, under these conditions, the daughter nuclei are smaller, then it supports their model. If daughter nuclei recover to their initial value, then it´s simply due to a geometrical constraint that forces the clustering of chromosomes and the reassembly of the NE in a confined space.

(3) The authors claim that the nucleus adapts to confinement based on evidence that the nucleus no longer shrinks in the second division following the first division. I would argue no further decrease is possible because the DNA is already compacted in the smallest possible volume. If indeed nuclei are in a new homeostatic state as the authors claim, then one would expect nuclei to remain smaller even after confinement is removed. This analysis is missing.

(4) Also, if the authors want to claim that this is a mechanism used for cancer cells to adapt to confined situations as the title says, they need to show that normal, near-diploid cells do not behave in the same way. This analysis is missing.

(5) Authors state that "Loss of nuclear blebs is clearly linked to mitosis, suggesting that nuclear volume and nuclear envelope tension are tightly coupled, and supports the hypothesis that mitosis is a key regulator of nuclear envelope tension". I have a few issues with the way this sentence is written. Firstly, one could say that all nuclear structures (and not only blebs) are lost during mitosis because the nucleus disassembles. Hence, the new homeostatic state could be determined by envelope reassembly after mitosis and not mitosis itself. Secondly, I don´t understand why the loss of nuclear blebs suggests that volume and tension are tightly coupled. Thirdly, how can mitosis be a key regulator of nuclear envelope tension when the nucleus is disassembled during the process? These require clarification.

(6) The authors claim that, unlike previous studies (Lomakin et al), this work shows a "gradual nuclear adaptation". From their results, this is difficult to conclude simply because they do not analyse cPLA2 levels. This is solely based on indirect evidence obtained from cPLA2 inhibition. A gradual adaptation would mean that based on the level of confinement we would expect to have increasingly higher levels of cPLA2 (and therefore nuclear tension).

(7) The authors should refrain from saying that the mechanism behind DNA repair is coupled to the nuclear adaptation they show. There are several points regarding this statement. Firstly, increased DNA damage could be due to nuclear ruptures imposed by confinement at 2h. In fact, the authors show leakage of NLS from the nucleus after confinement (Figure S3A). Secondly, the decrease in DNA damage at 24h could be because these nuclei did not rupture. How can they ensure that cells with low DNA damage at 24h had increased DNA damage at 2h? Finally, one needs to confirm if the nuclei they are analysing at 24h did undergo a round of cell division previously. From the evidence provided, the authors cannot conclude that DNA damage regulation is occurring in confined cells. Moreover, cell cycle arrest is a known effect of DNA damage. Cells with high damage at 2h most likely are arrested or will present with increased mitotic errors (which the authors exclude from their analyses).

Reviewer #2 (Public Review):

Summary:

Extensive previous research has shown that cell confinement, e.g., vertical compression of cells to a height smaller than the height of the unconfined cells, results in the unfolding of nuclear membrane invaginations, calcium and membrane tension mediated recruitment of cPLA2 to the nuclear membrane (which triggers increased cortical myosin accumulation and activity, among other effects), nuclear blebbing, and DNA damage. However, the long-term effects of confinement, and how cells adapt to such confined conditions, have remained largely unexplored.

In this work, the authors use custom-built cell confinement devices that enable precise control of confinement for prolonged periods of time (up to several days), along with live cell and fixed cell imaging to compare short-term (2 hours) and long-term (24+ hours) effects of confinement on nuclear structure. The authors report that while vertical confinement results in a short-term increase in nuclear cross-sectional area, associated with an increase in nuclear surface area due to unfolding of nuclear envelope invaginations while maintaining nuclear volume, long-term confinement results in a decrease in nuclear volume, reduced cross-sectional area, and re-appearance of nuclear envelope invaginations. Using time-lapse imaging, the authors demonstrate that these effects are associated with a reduction in nuclear volume upon completion of the first mitosis under confinement. Pharmacological inhibition experiments indicate a requirement of cPLA2, calcium signaling, and actomyosin contractility in this process. Although it is not surprising that nuclear blebs disappear following mitosis, as the nuclear envelope breaks down at the onset of mitosis and subsequently reforms as the chromatin decondenses, the observed change in nuclear volume upon prolonged confinement is intriguing. Notably, the nuclear adaptation following prolonged confinement was also associated with a reduction in DNA damage when comparing cells at 2h and 24h of confinements, measured by the presence of gamma-H2AX foci in the nucleus. By fitting their experimental data of nuclear surface area measurements, the authors arrive at the conclusion that cells have an intrinsic nuclear envelope tension set-point and that completing mitosis enables cells to reset nuclear envelope tension to this set-point.

Strengths:

The use of an agarose confinement system with precise control over vertical confinement enables the authors to apply long-term confinement without depriving cells of nutrients while performing live cell imaging or immunofluorescence analysis following fixation. The live cell imaging is a powerful tool to assess the effect of confinement not only on nuclear morphology, but also on cell cycle progression (using the FUCCI fluorescent reporter) and to compare nuclear volume between mother and daughter cells. The data presented by the authors to demonstrate changes in nuclear volume and surface area are convincing and supported by several independent measurements. The model comparing total and apparent nuclear surface area nicely complements the experimental measurements and helps to make the point that cells have a nuclear envelope tension set-point, even though the authors were unable to directly measure nuclear envelope tension. The inhibitor experiments targeting cPLA2 (using AACOCF3), intracellular calcium (using BAPTA-Amand 2APB), and myosin contractility (using blebbistatin) identify key players in the underlying cellular mechanism.

Weaknesses:

Although the findings by the authors will be of interest to a broad community, several weaknesses limit the mechanistic insights gained from this study. One major limitation is that all experiments are performed in a single cell line, H-29 human colorectal cancer cells, which has an unusual nuclear envelope composition as it has no lamin B2, low lamin B1 levels, and contains a p53 mutation. Because lamins B1 and B2 play important functions in protecting the nuclear envelope from blebs and confinement-induced rupture, and p53 is crucial in the cellular DNA damage response, it remains unclear whether other cell lines exhibit similar adaptation behavior.

Furthermore, although the time-lapse experiments suggest that reduction in nuclear volume occurs primarily during mitosis, the authors do not address whether prolonged confinement, even in the absence of apoptosis, could also result in cells adjusting their nuclear volume, or alternatively normalizing nuclear envelope tension by recruiting additional membrane from the endoplasmic reticulum, which is continuous with the nuclear membranes.

Additionally, the molecular mechanisms underlying the observed loss in nuclear volume and the regulation of this process remain to be identified. The pharmacological studies implicate cPLA2, intracellular calcium, and actomyosin contractility in this process, but do not include validation to confirm the efficiency of the drug treatment or to rule out off-target effects. Regarding the proposed role of cPLA2, previous studies have shown that cPLA2 recruitment to the nuclear membrane, which is essential to mediate its nuclear mechanotransduction function, requires both an increase in nuclear membrane tension and intracellular calcium. However, the current study does not include any data showing the recruitment of cPLA2 to the nuclear membrane upon confinement, or the disappearance of nuclear membrane-associated cPLA2 during prolonged confinement, leaving unclear the precise function and dynamics of cPLA2 in the process.

Lastly, it remains unclear (1) whether the reduction in nuclear volume is caused by a reduction in nuclear water content, by chromatin compaction, e.g. associated with an increase in heterochromatin, or through other mechanisms, (2) whether the change in nuclear volume is reversible, and if so, how quickly, and (3) what functional consequences the substantial reduction in nuclear volume has on nuclear function, as one would expect that this reduction would be associated with a substantial increase in nuclear crowding, affecting numerous nuclear processes.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors discover that nuclear volume decreases after mitotic exit following cell confinement in a manner that scales with the extent of confinement. This adaptation appears to protect the cells from adverse outcomes of critical confinement such as nuclear blebs and DNA damage. The evidence to support these claims is strong.

The authors also provide a model in which argue that what they call the "apparent nuclear surface area" is modulated by confinement through a mechanism regulated by cPLA2 and myosin II activities. Here there are weaknesses in that the manuscript relies on a single approach, measurements are indirect, and alternative models are not explored. Similarly, additional considerations need to be addressed so that the reader can interpret the data presented - for example whether cell volume is also changing coincident with nuclear volume changes, and whether other aspects of cell physiology such as cytokinesis are altered.

Considerations that could support the manuscript further:

One essential consideration that goes unaddressed is whether the nuclear volume alone is changing under compression (resulting in a higher nuclear to cytoplasmic ratio) or if the cell volume is changing and the nuclear volume is following suit (no change in the N:C ratio). Depending on which of these is the case, the overall model would likely shift. In particular, interpreting the effect of disrupting myosin II activity given its different distribution at the cortex in response to the higher confinement would be influenced by which of these conditions are at play.

A key approach used and interpreted by the investigators is an assessment of the folding of the "inner lamin envelope", which they derive from an image analysis routine of lamin staining that they developed and argue reflects "nuclear envelope tension". I am not convinced of the robustness of this approach or what it mechanistically reveals. It may or may not reflect the contour of the inner nuclear membrane, which (perhaps) is the most relevant to the authors' interpretation of nuclear envelope tension. Given the major contribution of this data to the model, which is based on the "unfolding" of the nuclear envelope, an orthogonal approach (e.g. electron microscopy - which one needs to truly address the high-frequency undulations of the nuclear envelope) is needed to support the larger conclusions.

The authors argue that nuclear tension is lost after mitosis in the confined devices because nuclear volume has decreased. While a smaller nuclear volume might indeed translate to less compressive force from the device on the nucleus, one would imagine that the chromosomes still have to be accommodated and that confining them in a smaller volume could increase the tension. Although arguable, the potential alternative possibilities suggest that actual measurements of nuclear envelope tension are needed to robustly test the model. The authors cite the observation that blebs are less prevalent after mitosis as additional support for this model, but this is expected as nuclear envelope breakdown and reformation will "reset" the nuclear contour while the appearance of blebs at mitotic entry is essential a "memory" of all blebs and ruptures over the entire preceding cell cycle.

Representative images for the pharmacological perturbations other than blebbistatin are notably absent - only the analyzed data are presented in the manuscript or the supplemental material. How these perturbations (e.g. to cPLA2) also affect the cortex is important to interpret the data given the point raised above. Orthogonal approaches would also strengthen the conclusions (for example, the statement that "nuclear adaptation observed during mitosis requires nuclear tension sensing through cPLA2" requires more evidence to be convincing - it is not sufficiently supported by the data presented). Even if this is the case, the authors acknowledge that cPLA2 is likely not the answer to the adaption observed under the lower degrees of confinement. Thus, the mechanisms underlying the adaptive changes to nuclear volume remain enigmatic.

One more consideration that seems to go without comment is that the cells under confinement do not appear to successfully complete cytokinesis (Fig. 5b). At a minimum this seems like a major perturbation to cell physiology and needs to be more fully discussed by the authors as playing a role in the observed changes in nuclear volume.

Author Response

Reviewer #1 (Public Review):

(1) It is unclear whether the authors took into consideration the contribution of nuclear blebs for nuclear volume measurements. This would be particularly relevant in situations of very strong confinement. Blebs were previously shown to affect volume (Mistriotis et al., JCB 2019). One could argue that the decreased nuclear volume was due to the increased blebbing observed in very strong confinements.

As stated in the main text: “[Nuclear Blebs] had a limited contribution to the increase in nuclearprojected area, as the increase remained significantly different even if protrusions were dismissed to compute the projected area (Fig S3C)”. In addition, a decrease in the nuclear volume was also observed for slight and intermediate confinement (height = 7 and 9 µm), while in these two conditions, no blebs are observed.

(2) From their experimental setup, it is unclear whether the reduced nuclear volume observed after confined cell division arises from a geometrical constraint or is due to an intrinsic nuclear feature. One could argue that cells exiting mitosis under confinement have clustered chromosomes and, therefore, will have decreased volume. This would imply that the nucleus is not "reset" but rather that a geometrical constraint is forcing nuclei to be smaller. One way to test this would be to follow individual cells under confinement, let them enter mitosis, and then release the confinement. If, under these conditions, the daughter nuclei are smaller, then it supports their model. If daughter nuclei recover to their initial value, then it´s simply due to a geometrical constraint that forces the clustering of chromosomes and the reassembly of the NE in a confined space.

We agree with the reviewer. As stated in the discussion, “For now, the mechanisms involved remain elusive”, and “Our results call for an in-depth analysis of the molecular pathways at play”. The experiments suggested by the reviewer are definitely important experiments that we plan to carry out. Indeed, it is important to know if cells that were ‘born’ under confinement will retain smaller nuclei in the next generation if confinement is released, or whether the next generation will recover their initial larger nuclei.

(3) The authors claim that the nucleus adapts to confinement based on evidence that the nucleus no longer shrinks in the second division following the first division. I would argue no further decrease is possible because the DNA is already compacted in the smallest possible volume. If indeed nuclei are in a new homeostatic state as the authors claim, then one would expect nuclei to remain smaller even after confinement is removed. This analysis is missing.

As mentioned above, we agree that “deconfinement experiments” are indeed important. Nevertheless, we respectfully want to point out that the DNA is not compacted to its maximum level during confinement.

First, we observed that the nuclei of the second generation of cells born in confinement no longer shrink for all investigated confinement conditions, including for slight confinement (height of 9 µm, corresponding to an initial nuclear deformation of 41%), where DNA is less confined compared to the very strong confinement condition (height of 3 µm, corresponding to an initial nuclear deformation of 70%).

Second, the total uncompressible volumetric fraction of a cell is smaller than 30% (Roffay et al. PMID: 34785592, Cell Biology by the Numbers ISBN: 9780815345374) this allows a nucleus to be compressed to over 70% of its size, as we observed in the extreme scenario.

(4) Also, if the authors want to claim that this is a mechanism used for cancer cells to adapt to confined situations as the title says, they need to show that normal, near-diploid cells do not behave in the same way. This analysis is missing.

We agree with the reviewer. For the revised version, we have planned to analyze cell response to confinement using the RPE-1 cell line, as a model of a diploid and untransformed cell line. This will be important experiments to know if the nuclear mechanism identified in the HT-29 cell line is also at stake for normal cells.

(5) Authors state that "Loss of nuclear blebs is clearly linked to mitosis, suggesting that nuclear volume and nuclear envelope tension are tightly coupled, and supports the hypothesis that mitosis is a key regulator of nuclear envelope tension". I have a few issues with the way this sentence is written. Firstly, one could say that all nuclear structures (and not only blebs) are lost during mitosis because the nucleus disassembles. Hence, the new homeostatic state could be determined by envelope reassembly after mitosis and not mitosis itself. Thirdly, how can mitosis be a key regulator of nuclear envelope tension when the nucleus is disassembled during the process? These require clarification.

We agree with the reviewer that the formulation used required clarification that will be made in the revised version: for now, we only have evidence that nuclear volume regulation is at stake at mitosis. The most probable hypothesis is that confinement perturbed NE reassembly after mitosis, and that this perturbed reassembly leads to a change in nuclear volume. Complementary experiments are needed to test such a hypothesis, using cell lines stably expressing LAP2/LAP2b-GFP for instance. It is however delicate experiments that will require a dedicated study on its own.

Secondly, I don´t understand why the loss of nuclear blebs suggests that volume and tension are tightly coupled.

Nuclear Blebs appear once nuclei have reached a critical NE tension (Srivastava, et al PMID: 33662810). The fact that cells “born” under confinement have no nuclear blebs means that their nuclei are no longer under tension. This is a direct consequence of the decrease in nuclear volume, implying a coupling between volume and tension.

(6) The authors claim that, unlike previous studies (Lomakin et al), this work shows a "gradual nuclear adaptation". From their results, this is difficult to conclude simply because they do not analyse cPLA2 levels. This is solely based on indirect evidence obtained from cPLA2 inhibition. A gradual adaptation would mean that based on the level of confinement we would expect to have increasingly higher levels of cPLA2 (and therefore nuclear tension).

We thank the reviewer for his/her comment. Indeed, we have no direct evidence of gradual cPLA2 recruitment in our study, as we did not analyze cPLA2 levels.

However, of note, in our study, nuclear volume and tension adaptation occur in the entire range of confinement height (from 3 to 9 µm), with a decrease in nuclear volume inversely correlated with the imposed initial nuclear deformation (fig S2C). On the contrary, in Lomakin et al., for HeLa cells, a threshold of 5 µm confinement is needed to trigger a cell motility response mediated by cPLA2. Such a difference suggests that other parameters are used as a confinement readout by cells during the reassembly of the NE after mitosis.

(7) The authors should refrain from saying that the mechanism behind DNA repair is coupled to the nuclear adaptation they show. There are several points regarding this statement. Firstly, increased DNA damage could be due to nuclear ruptures imposed by confinement at 2h. In fact, the authors show leakage of NLS from the nucleus after confinement (Figure S3A). Secondly, the decrease in DNA damage at 24h could be because these nuclei did not rupture. How can they ensure that cells with low DNA damage at 24h had increased DNA damage at 2h? Finally, one needs to confirm if the nuclei they are analysing at 24h did undergo a round of cell division previously. From the evidence provided, the authors cannot conclude that DNA damage regulation is occurring in confined cells. Moreover, cell cycle arrest is a known effect of DNA damage. Cells with high damage at 2h most likely are arrested or will present with increased mitotic errors (which the authors exclude from their analyses).

We need to clarify our analysis workflow: it was only in live experiments that we excluded cells with abnormal cell division, as cell division was visible in the timelapse. For immuno-staining analysis on fixed samples, all non-apoptotic cells were taken into account in the analysis. The decrease in DNA damage observed at 24h thus applies to all cells under confinement. There is a clear difference between 2h and 24h in the 2AX immunostaining (that is used as a proxy for DNA damage): whereas at 2h almost all cells have several foci (10-15 foci per cells on average fig. 3H), the number of foci in the entire cell population decreases to 1-2 foci per cell at 24h. The population at 24h mainly includes cells that have undergone a round of cell division, with >80 % of normal cells, as quantified in Fig. 3 E. In the revised version, we will include as a supplementary figure, a quantification of the percentage of cells having more than 5 foci at 2h and 24h, as well as large field of views for -2AX immunostaining to illustrate the distribution.

Reviewer #2 (Public Review)

One major limitation is that all experiments are performed in a single cell line, HT-29 human colorectal cancer cells, which has an unusual nuclear envelope composition as it has no lamin B2, low lamin B1 levels, and contains a p53 mutation. Because lamins B1 and B2 play important functions in protecting the nuclear envelope from blebs and confinement-induced rupture, and p53 is crucial in the cellular DNA damage response, it remains unclear whether other cell lines exhibit similar adaptation behavior.

We agree that including other cell lines would help generalize our findings. It would be interesting in the future to analyze if a similar regulation exists for other cell types. In particular, as stated in the discussion, it would be very interesting to investigate whether this nuclear adaptation is universal, or if it is a consequence of a dysregulation in a specific cancer pathway. Our current manuscript is relevant as it uncovers the existence of this highly interesting phenomenon.

Investigating if other cell types have the same capacity to adapt would provide insights into the molecular mechanisms involved. In the revised version, we specifically plan to analyze nuclear response under prolonged confinement in 2 types of cells :(1) normal cells with near diploid characteristics (RPE-1 cell line, as a model of a diploid and untransformed cell line); (2) other colorectal cancer cell lines presenting higher levels of lamin B2 and B1, and no P53 mutation (HCT-116).

Furthermore, although the time-lapse experiments suggest that reduction in nuclear volume occurs primarily during mitosis, the authors do not address whether prolonged confinement, even in the absence of apoptosis, could also result in cells adjusting their nuclear volume, or alternatively normalizing nuclear envelope tension by recruiting additional membrane from the endoplasmic reticulum, which is continuous with the nuclear membranes.

Even if we cannot completely ruin the hypothesis raised by the reviewer, we respectfully want to stress that if additional membrane from the endoplasmic reticulum were recruited, we should observe an increase in nuclear volume at S/G2, which is the case only for the strongest imposed confinment (h=3 µm, corresponding to an initial nuclear deformation of 70 % Figure S2E). It should be however very interesting in the future to directly assess nuclear envelope tension and to follow with high resolution live experiments the eventual recruitment of additional membrane.

Regarding the proposed role of cPLA2, previous studies have shown that cPLA2 recruitment to the nuclear membrane, which is essential to mediate its nuclear mechanotransduction function, requires both an increase in nuclear membrane tension and intracellular calcium. However, the current study does not include any data showing the recruitment of cPLA2 to the nuclear membrane upon confinement, or the disappearance of nuclear membrane-associated cPLA2 during prolonged confinement, leaving unclear the precise function and dynamics of cPLA2 in the process.

We agree with the reviewer that it would be very informative to analyze the recruitment of cPLA2 in live experiments. We plan to do this in future experiments using cPLA2 immunostaining at different time points or the cPLA2-mKate construct. This will be the subject of a dedicated study, together with possible changes in nuclear pores size and organization, as well as nuclear tension analysis. For this article, we plan to add the analysis of the effect of cPLA2 inhibition in live experiments.

Lastly, it remains unclear (1) whether the reduction in nuclear volume is caused by a reduction in nuclear water content, by chromatin compaction, e.g. associated with an increase in heterochromatin, or through other mechanisms, (2) whether the change in nuclear volume is reversible, and if so, how quickly,

We thank the reviewer for his/her comment. This point was also mentioned by Reviewer #1. It is important to know if cells that were ‘born’ under confinement will retain smaller nuclei in the next generation if confinement is released, or whether the next generation will recover their initial larger nuclei. We plan to perform such “deconfinement” experiments and add the results in the revised version. In addition, we also plan to investigate in more detail the DNA compaction state during confinement.

and (3) what functional consequences the substantial reduction in nuclear volume has on nuclear function, as one would expect that this reduction would be associated with a substantial increase in nuclear crowding, affecting numerous nuclear processes.

We agree with the reviewer that such a reduction in nuclear volume would most probably affect numerous nuclear processes that would be highly interesting to decipher in the future. Especially, as pointed out in the discussion, “the regulation of nuclear size identified in this study could have important consequences on resistance to classical chemotherapeutic treatments that target proliferation”. This question merits an entire study and is outside the scope of our current manuscript.

Reviewer #3 (Public Review)

(1) One essential consideration that goes unaddressed is whether the nuclear volume alone is changing under compression (resulting in a higher nuclear to cytoplasmic ratio) or if the cell volume is changing and the nuclear volume is following suit (no change in the N:C ratio). Depending on which of these is the case, the overall model would likely shift. In particular, interpreting the effect of disrupting myosin II activity given its different distribution at the cortex in response to the higher confinement would be influenced by which of these conditions are at play.

We agree with the reviewer. As stated in the discussion, “the nuclear to cytoplasmic volume ratio, which is constant within a given population, is most likely to be impacted by confinement and changes in nuclear envelope tension (24, 45, 46), and might be at play in the regulation we describe herein”.

As mentioned in the results section, “the distance between the cell membrane and the nuclear envelope was significantly reduced with confinement (Fig. 1D, Fig. S1B) and accompanied by the relocalization of the contractility machinery (Phosphorylated Myosin Light Chain (p-MLC) staining) from above the nucleus to the side, indicating a cortex rearrangement (Fig. S1C)”. For the revised version, we plan to investigate if such relocalization is accompanied by a change in the nuclear to cytoplasmic ratio using the p-MLC and nuclei immunostaining performed at 2h and 24h under the entire range of confinement investigated.

(2) -A key approach used and interpreted by the investigators is an assessment of the folding of the "inner lamin envelope", which they derive from an image analysis routine of lamin staining that they developed and argue reflects "nuclear envelope tension". I am not convinced of the robustness of this approach or what it mechanistically reveals. It may or may not reflect the contour of the inner nuclear membrane, which (perhaps) is the most relevant to the authors' interpretation of nuclear envelope tension. Given the major contribution of this data to the model, which is based on the "unfolding" of the nuclear envelope, an orthogonal approach (e.g. electron microscopy - which one needs to truly address the high-frequency undulations of the nuclear envelope) is needed to support the larger conclusions.

We agree with the reviewer that the precise measurement of NE surface area is challenging because of the NE folds, and that our approach is provides semi-quantitative information. Higher-resolution approaches would be necessary to investigate that point in more details, using 3D super-resolution. However, we want to point out that even with our limited resolution, the differences observed in lamin A/C staining are striking (Fig. 3A): while lamin folds are completely absent at 2h under strong confinement, inner lamin folds are massively observed at 24h, showing a pattern very similar to the control condition. In the revised version, we will add more representative images to strengthen that our analysis is representative of our observations.

(3) The authors argue that nuclear tension is lost after mitosis in the confined devices because nuclear volume has decreased. While a smaller nuclear volume might indeed translate to less compressive force from the device on the nucleus, one would imagine that the chromosomes still have to be accommodated and that confining them in a smaller volume could increase the tension. Although arguable, the potential alternative possibilities suggest that actual measurements of nuclear envelope tension are needed to robustly test the model. The authors cite the observation that blebs are less prevalent after mitosis as additional support for this model, but this is expected as nuclear envelope breakdown and reformation will "reset" the nuclear contour while the appearance of blebs at mitotic entry is essential a "memory" of all blebs and ruptures over the entire preceding cell cycle.

We agree with the reviewer that assessing the nuclear envelope tension would enable a better description of the underlying process. It will be the subject of a dedicated study, together with possible changes in nuclear pore size and organization, as well as the analysis of cPLA2 recruitment.

The proposed model in the current study is for the moment simply a geometrical model. Given the simplicity of the model, the fit with our experimental points is striking.

(4) Representative images for the pharmacological perturbations other than blebbistatin are notably absent - only the analyzed data are presented in the manuscript or the supplemental material. How these perturbations (e.g. to cPLA2) also affect the cortex is important to interpret the data given the point raised above. Orthogonal approaches would also strengthen the conclusions (for example, the statement that "nuclear adaptation observed during mitosis requires nuclear tension sensing through cPLA2" requires more evidence to be convincing - it is not sufficiently supported by the data presented). Even if this is the case, the authors acknowledge that cPLA2 is likely not the answer to the adaption observed under the lower degrees of confinement. Thus, the mechanisms underlying the adaptive changes to nuclear volume remain enigmatic.

We thank the reviewer for this insightful comment, and we plan to add representative images for the pharmacological perturbation in the revised version of the manuscript.

(5) One more consideration that seems to go without comment is that the cells under confinement do not appear to successfully complete cytokinesis (Fig. 5b). At a minimum this seems like a major perturbation to cell physiology and needs to be more fully discussed by the authors as playing a role in the observed changes in nuclear volume.

We agree that in the image chosen for Fig. 5b, cytokinesis does not seem to be complete. This is not representative of the entire cell population as 80% of the cell population showed a normal phenotype under very strong confinement with no drug (Fig. 5C and 3E, as well as fig S3D for a representative large field of view). Live experiments using the FUCCI cell lines also show that cells are capable of making several complete divisions under confinement (Fig. 2). Complementary experiments under pharmacological treatments and confinement are planned to extend our analysis of such processes.