Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation

  1. Margarida Dantas
  2. Andreia Oliveira
  3. Paulo Aguiar
  4. Helder Maiato
  5. Jorge G. Ferreira

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Abstract

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

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  1. Version published to 10.1083/jcb.202205051
  2. Review Commons

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    Our response to the reviewers comments as well as our revision plan has been included as a separate file in the submission.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Dantas and colleagues use mechanical confinement assays to demonstrate that both mitotic entry and the timing of prophase are sensitive to mechanical perturbations. They identify a novel mechanism that fine tunes the dynamics of cyclin B1 nuclear import during prophase whereby acto-myosin contractility leads to nuclear membrane unfolding, cPLA2 recruitment and cyclinB1 nuclear import. They show how mechanical confinement can accelerate this mechanism by independently inducing nuclear unfolding, and that this can go on to induce defects in mitotic spindle assembly and chromosome segregation.

    Major

    This work contains an impressive amount of data including some technically challenging experiments. The conclusions are convincing and for the most part well supported by experimental evidence (for exceptions see below). Appropriate controls are presented and statistical analysis is adequate. The methods are mostly described well but some important details are omitted (see below). The methods and figure legends would benefit from expansion, particularly in describing how the images presented relate to quantification in graphs. Although generally the manuscript is well written, there are parts when both the experimental logic and conclusions are hard to follow, particularly in the description of figures 1 and 5 (see below for details). With a large amount of data, including important experiments relegated to supplementary figures, this work would benefit from expansion into a longer article format to allow for more clarity. Particularly:

    • Figure 1A-C: here the authors show that non-adherent cells only enter mitosis when confined. There is some key information lacking here, including the experimental timeframe. How long were the cells plated on pll-peg before imaging and for how long were they imaged? In 1C, 80% of confined cells enter mitosis, which implies that cells were filmed for a relatively long time (given an average cell cycle length of 20-24 hours). Unless of course cells were previously synchronised in G2 but the authors do not state that this is the case. In the legend it states that images were acquired every 20s. Imaging cells for 20+ hours every 20s with multiple zs is likely to have a very deleterious effect on cells and to disrupt mitotic entry itself. The authors need to explicitly explain the experimental set-up used to generate the graphs in figure 1. In 1C, it would also be good to see the equivalent adherent control included in the graph (ie % cells that enter mitosis on fibronectin in the same timeframe). The authors use the data in 1A-C to claim that 'the G2-M transition requires contact with external stimuli'. However they haven't shown this, only that non adherent cells don't enter mitosis. To show that the G2/M transition is affected, they need to look at the cell cycle phase of cells on PLL-PEG and show that cells become arrested specifically in G2.
    • Figure 5: The explanation of the conclusions here was hard to follow. It's not immediately clear why a faster prophase would lead to chromosome attachment delays in metaphase or segregation errors in anaphase since these events occur only after NEP. I think the authors' hypothesis is that a faster prophase results in less time for centrosome separation and that this is responsible for later spindle defects but this is not very clearly stated. If this is the case, then one might expect cells in which centrosome separation is delayed to also be the cells with lagging chromosomes. Did the authors observe such a correlation? It's also not clear why the authors expected confinement to rescue the spindle defects imposed by STLC treatment (supp figure 5). An alternative hypothesis that the authors neglect to mention is that faster cyclinB1 entry into the nucleus could also induce defects through changes to nuclear events such as chromosome condensation? Did they also see any changes to the rate of chromosome condensation in the confined prophase? Either way, the authors should explain more clearly in the text what they think is happening here.

    Minor

    • No reference is cited for the endogenous tagged CyclinB1 RPE1 line nor are any details about its construction given. Has this cell line been previously published by the Pines lab? Are one or both alleles tagged? N or C terminus?
    • Figure 1C: presumably n in this case is number of experiments, not cells. How many cells were analysed in each case?
    • Figure 1H. Why do the graphs have different scales on the x axis? Where does 101+-12s for confined cyclin B translocation mentioned in the text come from? From the graph, it looks longer than this?
    • Figure 3 J, K. Confinement is able to rescue the effect of Y27 on cyclin B dynamics but not shROCK1. Why this difference? The authors should discuss this discrepancy in the text.

    Significance

    This work identifies a novel mechanical mechanism that regulates the timing of cyclin B1 nuclear import in early mitosis. The role of nuclear unfolding in controlling cyclinB1 import is particularly interesting. How important this new mechanism will be in controlling the duration of prophase or mitotic fidelity in a 'normal' mitosis within a tissue is not yet clear. However, it raises many intriguing questions about how cells' mechanical environment could impact mitotic entry, which could be relevant to disease situations where mechanics is altered such as fibrosis or cancer. The work is likely to be of interest to a wide range of cell and molecular biologists including those interested in cell cycle, mitosis, mechano-biology and nuclear biology.

    I am a cell biologist working on mitosis and the cell cycle.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Dantas and colleagues report that confinement is sufficient to restore G2/M transition in cells than can't adhere to their matrix. Exploring further the mechanisms involved, they show that confinement (dynamic cell compression) stimulates nuclear import of cyclin B1 and nuclear envelope permeability using cells in 2D culture. The authors observed that actomyosin contractility increases NE tension in cells preparing for prophase, leading to an increase in nuclear translocation of cyclin B1. However, a few inconsistencies between the data and the conclusion make the current report too preliminary for publication. It may require significant additional work to consolidate the authors' model.

    • The specific contribution of Nuclear Envelope tension. The authors conclude that confinement acts through increasing NE tension, although confinement may affect cytoplasmic signaling, which could contribute to G2/M transition. The authors should test whether compressing the nucleus versus compressing the cytoplasm have distinct effects on cyclin B1 nuclear translocation and G2/M, as it has been done by others when addressing nuclear mechanosensitive mechanisms (Elosegui-Artola et al. or Lomakin et al.). To consolidate their model, the authors should also test whether decreasing NE tension (independently of actomyosin tension) has opposite effect on G2/M (for example using LBR overexpression). Increase in nuclear membrane tension has been shown to trigger cPLA2 recruitment to the NE (Enyeidi et al, 2013; Lomakin et al. 2020), although the authors show here that confinement does not induce cPLA2 recruitment (but still increases NE tension figure 4G) in the absence of Rock activity or when the LINC complex is disrupted. This is surprising considering that confinement should increase NE tension independently of actomyosin contractility and should increase cPLA2 recruitment at the NE, unless in this case cPLA2 recruitment is not mediated by an increase in NE tension.
    • NPC transport versus NE permeability. The authors suggest that confinement increases cyclin B1 transport via NPC-mediated transport and rule out that confinement may affect NE permeability based on the absence of NE rupture using the INM marker lap2. However, the sample size for this observation is missing and NE permeability could be altered even in the absence of major INM rupture observed by confocal. The authors should use a reporter of nuclear permeability (fluorescent cytoplasmic marker or nuclear marker as previously used by Denais et al or, 2016 or Raab et al., 2016) to make sure that NE permeability is not affected by confinement. In addition, NPC function should be tested in parallel with other fluorescent reporter (such as NLS-GFP constructs) to test whether global NPC-mediated transport is changed during prophase (with or without confinement).
    • Effect of confinement on cyclin B transport (NEP) in adherent cells. In figure 1D, we can see that confinement enhances cyclin B1 nuclear translocation in cells adhering on fibronectin. Although it is unclear whether confinement has a significant effect in other figures, for example in figure 2F: DMSO is not significantly different from confiner+CDKi (same thing in 3i and 3j with Rock inhibitor and Kash construct). In these figures the untreated+confiner (or control in 3j) is missing, and the absence of difference between treated+confiner and control is puzzling. Either there is no difference between confiner and CDKi+confiner and it means there is no difference between control and confiner (surprising considering figure 1D); or there is a difference between CDKi+confiner and confiner, indicating that CDK inhibition affects confinement-induced cyclin B import. Both possibilities suggest that the authors should significantly revisit their model. In any case, all control (untreated, treated +/- confiner should be in all figures to avoid any misunderstanding).
    • Consequences of cPLA2 recruitment at the NE. The authors state that "Active cPLA2 then stimulates actomyosin contractility creating a positive feedback loop" But the NE is already unfolded and distance between NPR is increased before cPLA2 recruitment. Does PLA2 inhibition affect nuclear irregularity (or distance between NPC)? Or does cPLA2 impact cyclin B1 transport via a distinct mechanism? Did the author analyze CDK1 phosphorylation in presence of PLA2 inhibitor?
    • Robustness of the main observation. On page 4, the authors report that cells enter mitosis after 140 sec (+/- 80 sec) of confinement, although in the example showed in figure 1b, the cell enters at least 420 sec min after confinement, as we can see that the cell is already confined -420 sec (compressed shape) and NEP occurs at 0. Did the author showed a cell that was not included in their statistics? This would be very surprising considering the very low sample size used for this experiment (n=6 and 10). In addition, many observations have been made on small sample size (n=6 for figure 1) or/and not from independent experiments. The authors should increase their sample size and compare results from independent experiments to consolidate their model.
    • 2h shows nuclear signal (cyclin in grayscale), while 2e does not, why?
    • starting point to quantify cyclin entry is the lowest intensity, which may depend on many factors (and could be affected by experimental design). It would be necessary to have synchronized cells to homogenize the starting point of these experiments.
    • DN-KASH have been transiently transfected for single cell experiments, how does the authors unsure that cell observed are transfected? Does it have a fluorescent tag, if so which one?
    • "requires contact with external stimuli" or "that mechanical confinement is sufficient to overcome the lack of external stimuli." (page 4): external stimuli is vague here and it could be better to replace it with a more specific description

    Significance

    While the physiological relevance of these findings remain to be determined, the authors report an interesting observation that could have a significant impact in the field. The authors do not comment the potential overlap of their findings with other reports involving the LINC complex (Booth et al., ELife) or CDK-mediated actin remodeling (Ramanathan et al., NCB 2015) during prophase.

  6. Version published to 10.1101/2021.09.23.461473v2 on bioRxiv
  7. ASAPbio crowd review

    This review reflects comments and contributions by Madison Bolger-Munro, Rachel Lau, Arthur Molines, Sónia Gomes Pereira, Mafalda Pimentel, Mugdha Sathe, and Wasim Sayyad

    In this paper the authors test how mitotic entry is controlled by mechanical confinement or mechanical stimulation. Using a set of quantitative fluorescence experiments, they conclude that confinement triggers the dissolution of the nuclear envelope and eventually allows the cyclin B1 to translocate into the nucleus, thus triggering the G2/M transition. Further, using a set of small molecule inhibitors, the authors demonstrate that this mechanochemical pathway involves the LINC complex, cPLA2 and actomyosin contractility, while microtubule-related signalling is dispensable. The study is well-controlled and convincing.

    Regarding the terminology, in the text the terms 'confinement', 'mechanical stimulation', and 'mechanical force' are used interchangeably, but these are not always necessarily equal to each other. For instance, the majority of the experiments are performed using long-term confinement of cells in PDMS chambers which flatten the nucleus. Does the mechanical stimulation on the nucleus persist through this long-term confinement, instead of being gradually dissipated? In other words, is it possible to separate the effect of confinement from the effect of mechanical stimulation? Figure 5 partly addresses this issue while testing for mitotic defects after 4 minute-long confinement. If the import of cyclin B1 into the nucleus could be tested after a short confinement like this, it would strengthen the case for mechanical stimulation triggering the mitotic entry as opposed to long-term confinement itself.

    ‘This signal relies on nuclear unfolding during the G2-M transition, which activates the stretch-sensitive cPLA2 on the nuclear envelope. This activation upregulates actomyosin contractility, determining the spatiotemporal translocation of cyclin B1 in the nucleus.’ - There is a question as to whether the causality between these events has been shown. The study reports interesting discoveries: artificial confinement induces a faster rate of nuclear cyclinB1 import, dependent on importin, but independent of KASH, actomyosin, cPLA2 and Ca2+. Because cPLA2 is recruited to the "unfolding" NE in prophase and in confined cells, and because inhibition of KASH, actomyosin, and cPLA2 decreases the rate of cyclin B1 import in the absence of confinement, the paper extrapolates that this pathway is generally used as mechanosensing pathway to determine mitotic timing. If cPLA2 was indeed transducing nuclear deformability into lipid second messengers, the inhibition of cPLA2 should probably not be rescued by mechanical confinement. Moreover, AAOCF3 treatment should significantly impact cyclin B1 nuclear import, which does not seem to be the case (only relative fluorescence values are shown, but NEP does not seem to be affected).

    In the Abstract or early in the main text when the concept and players are introduced it would be nice to say what organism or kind of cells are referred to as these proteins are conserved across eukaryotes.

    ‘its mainly cytoplasmic localization’ - could be written as "is mainly cytoplasmic" or is "mainly localized in the cytoplasm".

    ‘RPE-1 cells expressing H2B-GFP/tubulin-RFP’ - The experiments demonstrate how the mechanical signal may regulate cyclin B1 translocation to the nucleus via cPLA2 and actomyosin contractility. Would this mechanism be universal across human cell lines or is it just specific to RPE1? Could this be discussed as future work.

    ‘Under these conditions, cells failed to enter mitosis’ - This aspect could be documented more as it is the baseline for the rest of the experiments. It is unclear from the result or the methods how long the cells were in the PLL-g-PEG before they were imaged. Could it be that they take longer to enter mitosis after the transfer? Fig 1G suggests that cyclin B1 accumulates in the nucleus in CTRL cells at a similar rate to the confined cells. The confinement ‘boosts’ the accumulation at the beginning of the experiment (in the first minute or so) but after that rates look similar. Is there a possibility that CTRL cells eventually divide but after a longer time? If that is the case, then it challenges the statement ‘cells failed to enter mitosis’ and the idea that adhesion is necessary. Would it be possible to perform some cell counting experiments over multiple days to show that cell concentration does not change?

    ‘acute mechanical stimulation’ - ‘mechanical stimulation’ is conceptually different from ‘confinement’. Can more details be provided here to clarify? From the figure it seems that the "suction cup" method was used to squeeze the cells. What was the control parameter in the experiments? Cell height? Applied pressure?

    'mechanical confinement is sufficient to overcome the lack of external stimuli’ - Here ‘external stimuli’ can be replaced by ‘the adhesion’ as mechanical confinement is also an external stimulus.

    ‘after stimulation’ - What happens if the stimulation is transient? Let's say squeeze for 5 minutes then release? Is this enough to induce mitosis?

    ‘normal and confined conditions (fig. 1d,e)’ - Would it be possible to clarify when dynamic or static confinement set ups are used? Based on the ‘confinement’ label on top of some time lapse panels, it seems like the dynamic set up is used most often. Please report the pillar height for the dynamic confinement set up. There is little difference in nucleus shape and size when confinement is applied vs before (or vs control cells in most cases), except for Fig1a-b and supp Fig1a. Is it certain that the confiner without vacuum is not exerting any effect on cells already? An ideal control should be in the confinement device without vacuum, but it seems cells in 2D FBN dishes are used. Can this comparison and set up be discussed?

    ‘We defined time zero as the lowest fluorescence intensity levels of nuclear cyclin B1 and quantified its increase as cyclin B1 translocated to the nucleus, up until NEP’ - This statement seems to refer to the graphs in Fig1g but in Fig1d and e, Time 0 is at NEP. It might be useful to align the time indicated on the images in d and e with the time used for quantification so that it is easier to connect the images to the graphs. Also, the experimental timings could be clarified: If fluorescence was quantified until NEP, it is NEP that sets the 300ms timing (instead of 0ms being set by ‘lowest fluorescence intensity levels of nuclear cyclin B1’). These data imply that control cells had a lower fold increase (slower) in terms of nuclear cyclinB1 in the 300ms preceding NEP. How different are the absolute levels of nuclear cyclinB1 in CT vs confined before NEP?

    ‘This was not due to a rupture of the nucleus, as we could not detect any obvious tears or gaps in the NE when RPE-1 cells expressing Lap2β-mRFP were mechanically stimulated’ - It is difficult to tell for sure that the nucleus does not rupture from the 2D image in Supplementary Fig 1a. Perhaps it may be possible to try expressing NLS-GFP to show there is no leakage of the GFP signal into the cytoplasm during confinement.

    (Supplementary Fig. 1a) - Were these cells also in late G2? How long does it take to unfold after confinement? How do the timings relate with nuclear cyclinB1 increase? In the 300ms quantifications, the increase seems to happen >200ms before NEP. How long is that after the confinement?

    ‘and resulted in an increase in the distance between neighbouring NPCs (Supplementary Fig. 1b, c; ***p<0,001), when compared to unconfined cells’ - Did the nuclear volume change? If so, it would also change the concentration of everything in the nucleus and may affect the signaling pathway?

    Figure 1

    • Figure 1a - it would help to include in Fig1a a schematic of the confinement chamber and the following treatment, to make the concept easier to grasp for non-specialists.
    • Figure 1b - the middle panel seems to have a misplaced scale bar in yz.
    • It is confusing to see the H2B-GFP shown in magenta and tubulin-mRFP in green. Please clearly mention in the legends, and also in the legend of subsequent figures.
    • Figure 1h ‘RPE-1 cells expressing cyclinB1-Venus/tubulin-mRFP dividing on a FBN coated substrate, treated with DMSO (top panel; n=15), a CDK1 inhibitor (middle panel, CDK1i; RO-3306; n=23) or with CDK1i and confinement (bottom panel; n=17). Time is in min:sec and time zero corresponds to NEP. Images were acquired with a 20 sec interval. Scale bar corresponds to 10μm’- Figure 1h demonstrates that confinement reversed the effects of CDK1 inhibition as cyclin B1 translocated to the nucleus. It may be nice to allow for visual comparison if the timepoints for CDKi (middle panel) and CDKi and confinement (bottom panel) were the same. Similarly, it may be nice to have the same timepoints for Supplementary Figure 2 and other figures after time zero (e.g Figure 4) for comparison.
    • Figure 1k - This Y axis starts on 0.5. Since intensities are normalized to time 0ms, graph visualization and comparison could be strengthened if all Y axis started closer to 1.
    • How CDK1 is blocked by inhibitor RO3306 needs a brief explanation as given for Importazole treatment.

    ‘Interestingly, confinement was sufficient to overcome the inhibition of CDK1 and force translocation of cyclin B1 to the nucleus (Fig. 1h, j; p<0.001). However, these cells failed to enter mitosis, as nuclear envelope permeabilization (NEP) was blocked by CDK1 inhibition31,32.This observation further strengthens the idea that mechanical stimulation per se does not affect the barrier function of the nucleus’ - If we understood correctly, under confinement cyclin B1 translocates into the nucleus independently of CDK1 activity. However, without CDK1 activity NEP doesn't take place and cells don't undergo mitosis. So, although there might not be a general effect on nucleus permeabilization, there is clearly an effect on the entry of certain proteins (like cyclin B1). How is CDK1 getting translocated in the presence of the inhibitor + confinement? Especially so when the NEP is blocked by the inhibitor. Does this mean that CDK1 translocation to the nucleus doesn't depend on its activation? Could these aspects be discussed further, or perhaps a scheme added to the figure, to guide the readers through the mechanism of CKD1-Cyclin B1-NEP-mitosis, and how this is changed by CDK1 inhibition and confinement.

    Supplementary Figure 2 - Panel b lacks a scale-bar. Additionally, tubulin is not as visible in panel b as in a, for comparison purposes it could be a good idea to apply the same threshold to both images.

    ‘​​Similar delays in cyclin B1 translocation’ - The control experiment (30 min DMSO) is not shown in the graphs. Nevertheless, it is understandable that the treatments slightly decrease the rate of cyclinB1 import compared to the controls in other panels. It may be useful to calculate the rate over time, for comparison across experiments.

    Fig. 2d; supplementary Fig 3a.p<0.01 - The p-values seem to compare with and without confinement, not with and without drug. They are mentioned again below (‘Importantly, mechanical stimulation not only reverted … but also accelerated both cyclin B1 accumulation and NEP in cells where actomyosin contractility was inhibited Fig. 2d-h; **p<0.01; ***p<0.001’).

    Supplementary Figure 3 - Panel d lacks a scale-bar. In some of the conditions it is a bit hard to check exactly when CyclinB1 is considered to have been translocated to the nucleus. Would it be possible to add a small panel on the figures, as in Figure 1?

    Figure 2

    • Figure 2b ‘in control non-confined cells expressing the DN-KASH mutant (black) and confined cells expressing the DN-KASH mutant (green)’ - It is not clear that the colors from the legend and graph correspond, can this be clarified?
    • Figure 2h ‘Time lag between cyclin B1 and tubulin nuclear translocation in DMSO-treated, non-confined cells (black), cells with different treatments (green), and confined cells in the same conditions (magenta).’ - It may be clearer to say confined cells with different treatments? Please also clarify what comparisons the statistical analyses correspond to. It is assumed that this is the non-confined vs. confined cells in each treatment, but it could be added to the figure legend.

    ‘and an actomyosin-dependent translocation of cyclin B1 into the nucleus’ - While in normal conditions there's a delay in cyclin B1 translocation to the nucleus upon actomyosin perturbation/inhibition, under confinement this translocation appears to go faster when actomyosin is perturbed/inhibited. So, it is expected that the confinement can somehow overcome the need for the actomyosin contractility and lead to independent translocation. In the absence of actomyosin, cyclinB1 import rate is lower, but it is not clear if there is a connection to mechanical cues for the translocation of cyclinB1. Can this fragment be clarified?

    Figure 3 - What is C-Pla2 tagged to?

    ‘​​Indeed, inhibition of cPLA2 activity with AAOCF3 led to a significant decrease in cyclin B1 nucleoplasmic shuttling (Fig. 3f, h, i; p<0.01’ - This p value is for AAOCF3 with and without confinement, not for AAOCF3 vs DMSO.

    ‘This likely occurs due to confinement-induced unfolding of the NE (Supplementary Fig. 1), that is sufficient to bypass the pharmacological inhibition of contractility and still induce an increase in NPC distance’ - The data suggest that mechanically induced cyclin B1 import is dependent of importin and independent of actomyosin, cyclin B1 activation, Nesprin, Calcium and cPLA2. How could NE unfolding and NPC distance explain the increased rate of cyclin B1 import? Perhaps reference 23 should be cited here, instead of above regarding the cPLA2 hypothesis, which the authors proved to be uncoupled.

    ‘To confirm this hypothesis, we seeded cells in a soft hydrogel (5kPa) or in a rigid glass, inducing low or high nuclear tension, respectively’ - Is this known from the literature (if so could a reference be provided) or was it established in this study?

    ‘indicating that increased tension facilitates mitotic entry’ - The study used glass vs hydrogel, instead of hydrogels with different stiffness. This should be taken into consideration when stating claims that the difference here is due to the change in NE tension.

    Figure 4 - Please add a scale-bar to the lower panel.

    ‘Fig. 5e; 24±7 min for controls vs. 36±20 min’ - should this be 5f? These values don't seem to match the graph in panel F.

    ‘promote cyclin B1 nuclear translocation by permeabilizing the NE with laser microsurgery, therefore anticipating mitotic entry’ -Does this relate to prophase cells? In this experiment, rupturing the nucleus gives the same result as the mechanical perturbations. Additional evidence may be valuable to show that the confinement/mechanical perturbations do not cause nuclear rupture.

    Supplementary Figure 5 - The lower panels are missing scale-bars

    Figure 5

    • 'Centrosomes angle at the time of NEP' - angles between centrosomes and long nuclear axis at the moment of NEP.
    • ‘​​laser surgery was performed on the cytoplasm’ - For clarity, the top panel could say "cytoplasm surgery" and the bottom panel "NE surgery".
    • ‘mechanical pathway based on nuclear tension’- Showing that nuclear tension changes would strengthen the claims. Could this be done with a FRET sensor to show that nuclear tension occurs when cyclin B1 translocation occurs? It is important to show that all the mechanical perturbations tried directly change nuclear tension.
    • It is not clear that the experiments performed directly test the role of the NE tension as most perturbations used are pleiotropic in nature and NE tension is not measured directly (admittedly very hard to do). Is there a way to alter NE tension more directly? Maybe a lamina mutant, or a mutant with excess NE, or a transient hypotonic shock?

    ‘cyclin B1 translocation’ - How could cyclin B1/CDK activity be altered by mechanical stimulations (other than with the inhibitor)? Would it be possible to show that cyclin B1 is activated across the different mechanical stimulations with a western blot? It would be nice to address if the mechanical perturbations activate cyclinB1/CDK1 to induce its translocation into the nucleus or if the mechanical perturbations cause the translocation of not active cyclinB1/CDK1. Some more discussion on this idea would be useful.

    ‘In agreement, we were able to rescue cyclin B1 shuttling to the nucleus by mechanical stimulation, even in the absence of cPLA2 activity or actomyosin contractility’ -According to refs 24 and 28, mechanical constriction is upstream of unfolding, NPC spacing, and cPLA2 increase, which will later result in cortical (not perinuclear) actomyosin activation. The authors show that cyclin B1 is translocated under confinement independently of every condition tested, except for importin. Can the order in which the described events take place, and their interdependence, be clarified?

    ‘Establishing mechanical forces as a determinant for cyclin B1 nuclear translocation and mitotic entry raises the interesting possibility that the nucleus might act as a sensor for external forces’ - Could the level of conservation of the proteins involved across eukaryotes and the generality of this mechanism be discussed? Could such a mechanism work in organisms with a cell-wall like plants or yeast or organisms that have no lamina?

    Methods

    • Recommend using concentrations (M, v/v, g/v) instead of dilutions and volumes; can the cell cycle stage of cells imaged, height of pillars, type of confinement and controls be reported?
    • ‘RPE-1 parental and RPE-1 H2B-GFP and tubulin-mRFP were already available in our lab’ - Were these previously published? If so, please include the citation. If not, could you report where these were obtained or how they were generated.
    • Cell confinement setup - a scheme of these manipulations would be very useful for non-experts to understand the experimental set-up and design.
    • ‘correlate the angle between the centrosomes and the nucleus, at the moment of NEP.’ - the nucleus and long nuclear axis.
  8. Version published to 10.1101/2021.09.23.461473v1 on bioRxiv