Actin contraction controls nuclear blebbing and rupture independent of actin confinement

  1. Mai Pho
  2. Yasmin Berrada
  3. Aachal Gunda
  4. Anya Lavallee
  5. Katherine Chiu
  6. Arimita Padam
  7. Marilena L. Currey
  8. Andrew D. Stephens

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Abstract

The nucleus is a mechanically stable compartment of the cell that contains the genome and performs many essential functions. Nuclear mechanical components chromatin and lamins maintain nuclear shape, compartmentalization, and function by resisting antagonistic actin contraction and confinement. Studies have yet to compare chromatin and lamins perturbations side-by-side as well as modulated actin contraction while holding confinement constant. To accomplish this, we used nuclear localization signal green fluorescent protein to measure nuclear shape and rupture in live cells with chromatin and lamin perturbations. We then modulated actin contraction while maintaining actin confinement measured by nuclear height. Wild type, chromatin decompaction, and lamin B1 null present bleb-based nuclear deformations and ruptures dependent on actin contraction and independent of actin confinement. Actin contraction inhibition by Y27632 decreased nuclear blebbing and ruptures while activation by CN03 increased rupture frequency. Lamin A/C null results in overall abnormal shape also reliant on actin contraction, but similar blebs and ruptures as wild type. Increased DNA damage is caused by nuclear blebbing or abnormal shape which can be relieved by inhibition of actin contraction which rescues nuclear shape and decreases DNA damage levels in all perturbations. Thus, actin contraction drives nuclear blebbing, bleb-based ruptures, and abnormal shape independent of changes in actin confinement.

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  1. Version published to 10.1091/mbc.e23-07-0292
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    Reply to the reviewers

    Reviewer 1

    In this manuscript Pho et al., characterize the effects of actin confinement versus acto-myosin contractility on nuclear deformation and nuclear envelope (NE) rupture/blebs. They use both genetic and pharmacological perturbations that impact nuclear shape and integrity such as chromatin decompaction and genetic deletion of nucleo-skeleton components (LaminA and LaminB). First, the authors show that modulation of acto-myosin contractility does not affect nuclear height (a proxy for the effects of actin confinement on the nucleus). This finding allowed them to study the effects of acto-myosin contractility on nuclear shape and integrity independently of actin-mediated nuclear confinement. Using fibroblasts expressing a NLS-GFP construct to assess nuclear shape and integrity, the authors show that increased acto-myosin contractility by treatment with Rho activator (CN03) leads to nuclear blebs and NE rupture events (curiously, they show that in LaminA-/- fibroblasts the NE rupture events were not bleb-based and were not affected by CN03). With these results, authors conclude that actin contraction is a major determinant of bleb-based NE rupture, independently of actin confinement. Next, since many studies have associated loss of nucleo-cytoplasmic compartmentalization with increased DNA damage, the authors addressed the occurrence of DNA damage foci in several contexts/perturbations. They find that increased NE rupture frequency by CN03 treatment does not lead to increased DNA damage. Based on the sole finding, authors then claim that DNA damage is mostly associated with abnormal shaped nuclei instead of ruptured nuclei. The data presented is overall convincing and well controlled but a more in-depth characterization of the DNA damage events in VPA-treated cells, LaminB-/- and LaminA-/- cells is needed to support the claim that DNA damage is not associated with NE events in their system. More details below.

    We thank the reviewer for careful consideration of our work. We are happy that the reviewer found, “__The data presented is overall convincing and well controlled…”. __While the author appears positive on the paper as a whole, what follows is the Reviewer’s largest concern with the paper.

    “ a more in-depth characterization of the DNA damage events…is needed to support the claim that DNA damage is not associated with NE events in their system.” We appreciate the Reviewer’s feedback and agree that this one statement was an overstatement. To address this concern,

    1. We removed this disagreeable/unsupported conclusion,

    2. We replaced it with more measured statement supported by the data (red text highlights changes to the original manuscript),

    3. because we have removed the unsupported conclusion, we prefer not to carry out the suggested experiments because they constitute a completely new study beyond the scope of this manuscript.

    Major points 1,2,3 all focus on the removed unsupported conclusion. They outline numerous new experiments which we feel constitute a new study and separate manuscript and thus beyond the scope of the current manuscript. Instead, we tone down or conclusions clearly labeled by red text.

    __

    __

    __ __

    __Major comments:____

    1-In fig. 4, increased NE rupture frequency by CN03 treatment did not lead to an increase in gH2AX foci, so authors claim that DNA damage is not associated with NE events. However, the increase observed in NE rupture frequency is rather very subtle (from 1.5 to 3 in WT cells; from 2.5 to 4 in VPA treated ells and from 2 to 2.5 in LaminB-/- cells) so I wonder if such a minor increase might be sufficient to cause an increase in DNA damage foci. Moreover, DNA damage events are repaired within minutes and the analysis throughout this manuscript is made on snapshots. Therefore it becomes difficult to draw any conclusion regarding the absence of a link between NE rupture and DNA damage. To assess DNA damage events in a quantitative manner and show that abnormal shape (as opposed to NE rupture) is the main driver of DNA damage, authors should perform live cell imaging of cells co-expressing a DNA damage marker and a NE rupture marker to track DNA damage events following NE rupture.__

    The reviewer raises concerns that we have too strongly stated a conclusion of nuclear rupture affects DNA damage less than shape. Overall, we understand this concern and have revised the text to remove this conclusion and in general tone down our conclusion from this data. We agree with the reviewers point that doubling the frequency either might not be sufficient to increase DNA damage OR a single rupture is sufficient to increase DNA damage. We now state: “ DNA damage measured by γH2AX foci did not significantly increase upon CN03 activation of actin contraction, suggesting that the slight yet significant increase in nuclear blebbing, rupture, and rupture frequency from CN03 did not have a significant impact on DNA damage (Figure 4, C and B).

    We prefer not to. The reviewer’s request for live cell nuclear rupture and DNA damage data is now beyond the scope of the paper because we have revised the manuscript by removing this conclusion. A full analysis of live cell DNA damage analysis in conjunction with nuclear rupture for bleb-based, non-bleb-based normal circularity, and non-bleb-based normal circularity across 4 conditions (WT, VPA, LMNB1-/-, and LMNA-/-) is a paper in and of itself.

    2-Fig. 4C: to show that frequency of NE rupture is less important than abnormal nuclear shape for DNA damage appearance, authors need to quantify gH2AX in normal x blebbed x abnormal nuclei in each one of the conditions (untreated, CN03 and Y27). But again, this analysis must be complemented by live cell imaging experiments to track DNA damage foci appearance (or not?) following NE rupture events.

    We prefer not to Similar to point (1) we have removed this disagreeable conclusion and revised the manuscript. Further extensive studies of DNA damage for nuclear rupture and shape, while interesting, are beyond the scope of the current manuscript.

    We now clearly state this in the Discussion section: “Our data in wild type, VPA, and LMNB1-/- cannot decouple the roles of nuclear shape and ruptures, which are intertwined, causing increased DNA damage (Figure 4). However, we provide novel data that actin contraction is necessary for the behaviors of nuclear blebbing, rupture, and increased DNA damage, independent of changes in actin confinement.

    __ 3- Since VPA treatment increases both the percentage of bleb-based NE rupture and NE rupture frequency in LaminA-/- cells, authors should show that gH2AX foci in this sample remain unaltered to further support their claim that frequency of NE rupture is less important than abnormal nuclear shape.__

    We prefer not to Similar to points (1) and (2) we have revised the manuscript to remove this disagreeable conclusion. Instead, Figure 6 is focused on the interesting phenomena where LMINA-/- nuclei display abnormal nuclear shape and majority non-bleb-based nuclear ruptures. To determine if LMNA-/- nuclei have the capacity to increase nuclear blebbing and bleb-based ruptures, we treated with VPA, which causes both. LMNA-/- with VPA shows that these calls lacking both lamin A and C can form blebs and increase in bleb-based ruptures.

    __ 4-Does methylstat treatment rescue LaminB-/- phenotypes (blebs, ruptures, ...)?__

    We do not see how this relates to a specific change to the manuscript but instead is a direct question of interest.

    The reviewer would like us to clarify how our past work rescued Lamin B1 null (LMNB1-/-) phenotype which we cited in the original manuscript as a supporting point (top of page 16).

    “This new data agrees with our previous data showing that increased heterochromatin levels via histone demethylase inhibition by methylstat treatment (Stephens et al., 2018) and mechanotransduction (Stephens et al., 2019a) rescues nuclear shape in LMNB1-/- nuclei.”

    In our previous publication we showed that methylstat, which increases heterochromatin levels, can suppress nuclear blebbing in LMNB1-/- (Stephens 2018 MBoC). In another manuscript we increased heterochromatin levels via a mechantransduction pathway. This resulted in decreased nuclear blebbing, ruptures, and DNA damage (Stephens 2019 MBoC). However, this manuscript goes on to show that loss of facultative heterochromatin alone via GSK126 can recapitulate the LMNB1-/- which shows loss of facultative heterochromatin (Figure 5).

    __ 5-Auhors mention throughout the manuscript the "actin confinement" component (actin cables localized at the top of the nucleus-not convincingly shown in fig. 2C). Some studies have reported the occurrence of perinuclear actin caps that surround the entire nucleus (DOI: 10.1038/s41467-018-04404-4; DOI: 10.1038/srep40953; DOI: 10.1038/ncb3387). Can the authors investigate the existence of such perinuclear actin ring on the LaminB-/-, LaminA-/- and VPA-treated cells? Could this ring affect NE rupture and shape? Also, if these perinuclear actin rings can be observed, what would they look like in CN03- and Y27-treated cells?__

    The reviewer is requesting that we address possible changes to actin perinuclear cap and surrounding structure. We plan to address this concern by closer analysis of our current data and gather more data if needed.

    __ Minor comments:__

    We will address all the minor comments during revision.__

    1-Please mention/discuss CytoD treatment in main text (Fig 2C).__

    We plan to add text to address this concern.

    __ 2-Can the authors comment on why CN03 treatment on VPA cells does not cause changes in blebs or NE rupture (fig. 3A, B)?__

    We plan to comment on this point.

    __ 3-I'd move fig. 2B to supplement.__

    We prefer to keep this material in the main manuscript Figures as it supports a major support of the title and major conlcusion.

    __ 4-In my opinion schematics on figs. 1A, 2A, 3E are confusing and do not add anything to the manuscript.__

    We prefer to keep this material in the manuscript.

    __ 5-There is something missing on the following sentence, please revise it: "We hypothesized that LMNA-/- nuclei do not show bleb-based behaviors because this perturbation cannot, due to reported disrupted nuclear-actin connections (Broers et al., 2004; Vahabikashi et al., 2022)."__

    We have revised this sentence to read: “We hypothesized that LMNA-/- nuclei do not show bleb-based behaviors because of the reported disruption of nuclear-actin connections (Broers et al., 2004; Vahabikashi et al., 2022).”

    __ 6-Can the authors explain in the Discussion why there is a decrease in gH2AX foci in VPA-treated cells and LaminA-/- cells upon CN03 treatment?__

    We do not have an explanation at this time.

    Significance

    Nuclear deformation is a common event observed in homeostasis and disease and both extra-cellular physical cues and different cellular components play critical roles in nuclear morphology and integrity. It is well known that the actin cytoskeleton exerts a wide range of forces on the nucleus and causes nuclear deformation (via LINC complex) as cells migrate, grow or spread within complex microenvironments. The contribution of mutations of nucleo-skeleton components to nuclear abnormalities and rupture are well described. Additionally, more recently, the contribution of the actin cytoskeleton to nuclear integrity and morphology has also been characterized. However, the role played by actin contraction on nuclear shape and integrity, independently of actin confinement (exerted by the actin cables localized at the top of the nucleus), remain elusive. In this manuscript Pho et al., address this question using cells expressing NLS-GFP to detect nuclear rupture events and potential nuclear deformations. There is no real conceptual advance in this study but there is a novel finding as it shows that acto-myosin contraction affects nuclear integrity and morphology independently of dorsal actin cables (actin confinement). Moreover, the experiments were performed on flat 2D surfaces, a distant scenario from the 3D in vivo landscapes. As a classic cell biology study this manuscript has the potential to be of interest to basic researchers in the field of cell migration, crosstalk of nucleo-skeleton/cytoskeleton and nuclear mechano-sensing.

    We appreciate that Reviewer states

    • “role played by actin contraction on nuclear shape and integrity, independently of actin confinement (exerted by the actin cables localized at the top of the nucleus), remain elusive.”
    • “In this manuscript Pho et al., address this question…” and “[our manuscript] is a novel finding as it shows that acto-myosin contraction affects nuclear integrity and morphology independently of dorsal actin cables (actin confinement).” Thus our manuscript provides a novel finding of interest to the cell biology community

    While individually, chromatin decompaction via VPA, lamin B1 knockout (LMNB1-/-), and lamin A/C knockout (LMNA-/-) have been well-studied, there is no other publication that directly compares them and furthermore decouples the role of actin contraction from confinement.

    __ __

    Reviewer 2

    The authentication of cell lines was not clear. The origin of the cell lines used was not clear. Were they immortalized? Did they examine primary MEFs? The authors did not seem to be aware of convincing data showing that Lamin B1 increases nuclear dispensability, whereas lamin B1 deficiency has the opposite effect.

    Cell lines have been previously published multiple times, but originated from Shimi et al. 2011. We have revised the text to clarify that these are immortalized MEFs used in many previous studies cited in the main manuscript and the materials and methods, and not primary MEFs.

    “ MEFs were immortalized with SV40 large T antigen by retroviral transduction of the gene encoding the SV40 large T antigen as previously described (Shimi et al., 2011, 2015).”

    We also included citations to (Vahabikashi et al., PNAS 2022) which has recently compared many lamin knockouts and knockdowns.

    The reviewer makes an unclear statement about lamin B1 levels and “dispensability” but provides no citations. As cited in the original manuscript lamin B1 null nuclei are one of the most studied models of nuclear blebbing and rupture (Vagas et al., Nucleus 2012; Hatch et al., JCB 2016; Young et al., MBoC 2020). __

    The paper reads like a rough draft. Nomenclature inappropriate.__

    The nomenclature used in this manuscript follows previous publications in the field including for chromatin notation (VPA, Stephens et al., 2017 and all previous manuscripts using this drug), lamin KOs (LMNB1-/- and LMNA-/-, Shimi et al. 2011.), and actin contraction and confinement are field appropriate.

    We will revise the text to clarify nomenclature.

    Significance

    The impact of actin on blebs and nuclear shape is well established. The implications of these findings for distinct roles of the nuclear lamin proteins were not clear. The impact of the interventions on nuclear stiffness was not measured.

    We agree with the reviewer that the impact of actin on nuclear shape is well established. However, the differential roles of actin contraction vs. confinement are not clear, as we state in the intro of the original manuscript. We believe our paper shows for the first time the separate role of actin contraction from actin confinement, where actin contraction is modulated, and we find that actin confinement measured by nuclear height remains the same. Reviewer #1 agrees that our data separating the roles of actin contraction and confinement is a novel finding (See Reviewer #1 Significance above).

    The reviewer states that the distinct roles of lamins are not clear. We use different lamin knockout mutants as phenotypes of nuclear blebbing (LMNB1-/-, along side VPA) and abnormal nuclear shape measured as decreased nuclear circularity with no change in nuclear blebbing (LMNA-/-). These two different phenotypes of nuclear blebbing and abnormal shape also coincide with bleb-based nuclear ruptures and non-bleb-based nuclear ruptures, respectively (Figure 1). We do not examine the full role of lamins in this manuscript, as it is well beyond the scope of this work.

    As cited in the original manuscript, interventions on chromatin (VPA) and lamins (LMNB1-/- and LMNA-/-) have been previously published and thus were not the focus. Nuclear stiffness measurement of perturbation of chromatin compaction via VPA has been published numerous times by our lab (Stephens et al., 2017,2018, 2019 MBoC; Berg et al., 2022 biorxiv) and others (Hobson et al., 2020 MBoC, Shimanoto et al., 2017). Nuclear stiffness in LMNB1-/- and LMNA-/- has been published in (Vahabikashi et al., PNAS 2022). We also have previously shown that depolymerization of actin does not impact nuclear stiffness (Stephens et al., 2017 MBoC), which would suggest that changes in actin contraction would not influence nuclear stiffness. This is supported by the fact that changes in actin contraction to not alter actin confinement pushing down on the nucleus resisting it (force balance) in all conditions except VPA Y27632 (Figure 2).

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    Referee #4

    Evidence, reproducibility and clarity

    This article examines the cellular processes that predispose cells to nuclear blebbing and DNA damage in response to lamin and chromatin perturbations. The authors show key differences in these two types of perturbation and demonstrate a role for actin contractility. The experiments are well controlled and the data analysis generally rigorous. However, prior to acceptance, a number of issues must be fixed to improve the manuscript. I do not know the field sufficiently well to judge the novelty of the data.

    Major issues:

    • page 7, bottom: The authors state that measuring nuclear height gives an indication of confinement and force balance. But, if the nuclear mechanical properties have changed, then the nuclear height could change without any change in contractility. So, the authors would need to also verify that the level of contractility hasn't changed and that the mechanical properties haven't changed to really confirm that the cell height is a good measure of confinement. The level of contractility can be assessed by staining for pMLC. The nuclear mechanical properties may have been measured by others.
    • In general, are the changes in contractility resulting from drug treatments sufficiently large to deform the nucleus? Can the authors show a time course of nuclear height in response to a treatment for WT for example? This would allow to link contractility to nuclear height.
    • Page 9: The authors do not find any change in nuclear shape. Can they measure shape pre/post treatment on the same cells? It could be that the effect is lost in variability unless you do paired measurements?
    • Page 11: the authors find nuclear ruptures unchanged in LMA -/- even when there is no contractility. They then state: "We hypothesized that LMNA-/- nuclei do not show bleb-based behaviors because this perturbation cannot, due to reported disrupted nuclear-actin connections". I do not understand this sentence.
    • To characterise actin contractility better, it would be good to present images of the actin cables in each condition and pre/post treatments. This would allow to visually assess whether the morphology of the F-actin cytoskeleton has changed. This is one of the main topics of the study and as such it should be examined.
    • On all bar charts, the authors should indicate: the number of independent experiments, the number of cells examined.
    • I find the diagrams on Fig 1A, 2A etc do not help to illustrate what the authors think is happening. Can they redraw them in a more informative way?
    • The abstract, introduction, and discussion are overly long and lack focus. These should be rewritten succinctly.

    Minor issues:

    • page 4: inhibitors of Rho-kinase will also modulate actin polymerisation indirectly through the action on Lim-kinase and cofilin.
    • page 5, second paragraph: the authors should state that they are measuring the frequency of ruptures. At first, I thought this might be a mechanical strain.
    • Page 7: In general, it may be useful to discuss the temporal evolution of the c/n and the circularity side by side. The change in circularity over time could be an indicator of mechanical strain, while the c/n would report on any transient loss of integrity of the nuclear membrane.
    • Fig 1B: it would be nice to present the time course of the c/n as well.
    • Fig S1: it might be interesting to characterise the dynamics/amplitude of the c/n for the different conditions. There doesn't appear to be any difference between the nuclear blebbing rupture and the non blebbing rupture. This suggests that the two phenomena (nuclear blebbing and nuclear rupture) are independent: i.e. rupture is not causally linked to blebbing.

    Significance

    This article examines the cellular processes that predispose cells to nuclear blebbing and DNA damage in response to lamin and chromatin perturbations. The authors show key differences in these two types of perturbation and demonstrate a role for actin contractility. The experiments are well controlled and the data analysis generally rigorous. However, prior to acceptance, a number of issues must be fixed to improve the manuscript. I do not know the field sufficiently well to judge the novelty of the data.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: In this manuscript, the authors set out to compare the contributions of chromatin and both A- and B-type lamins to the maintenance of nuclear shape and NE integrity, while also observing how actin contraction or confinement individually contribute to changes in nuclear shape and integrity. To do this, they used mouse embryonic fibroblasts (MEFs) expressing an NLS-GFP as a readout of nuclear shape and rupture, while perturbing chromatin compaction (using the HDAC inhibitor VPA) or using MEF lines devoid of A-type (LMNA -/-) or B-type (LMNB1 -/-) lamins, while simultaneously decreasing or increasing actin-mediating tensile forces but maintaining actin compressive forces by observing nuclear height. They found that increased actin contraction causes a higher instance of bleb-based nuclear shape changes and ruptures in WT and LMB1-/- cells, as well as cells with chromatin decompaction, but actin contraction did not impact the nuclear morphology or prevalence of bleb-based ruptures with a loss of LMNA. The authors also show that loss of LmnB1 causes chromatin decompaction phenotype, and loss of Lamin A/C creates and increase in bleb-based nuclear ruptures, but only under conditions of chromatin decompaction with VPA.

    Major critiques:

    1. Fig 2B- The authors use γMLC2 as a readout for actin contractility when CN03 and Y27632 is applied. While this method has been used previously as a readout for actin contractility, it would be more convincing if the authors included at least another technique to verify and increase or decrease in actin contractility in some of their conditions (e.g. traction force microscopy).
    2. Fig 2C- the authors suggest that the shape changes and blebbing are not due to actin confinement on the nucleus, because nuclear height does not change. This is an large assumption made from only a small amount of data, considering that there could be increased confinement on the nucleus during actin contractility, yet it is too small to be measured by the side imaging technique described in the paper. The manuscript would benefit greatly from more experiments that could show that the blebs are being made even when there is no confinement.

    Minor critiques:

    1. Fig 2B- the statistical significance asterisks above the MLC2 relative fluorescence graph do not seem to be aligned appropriately with the bars, and it is difficult to know which asterisk belongs to which bar. Same is true for the nuclear height graph. The "vs. WT" box above the nuclear height graph is also confusing in that it is hard to see which statistical

    Significance

    The manuscript creates value in the field, in that the major findings of the relationship between lamins, chromatin, and actin and their respective impacts on nuclear shape and rupture are in agreement with findings from previous studies and therefore bolsters the current model on NE mechanics. The novelty in the paper comes from the reported finding that actin contraction and not confinement is what controls nuclear blebbing and ruptures, although this evidence seems to be limited to a single method (nuclear height measurements) presented in Fig 2 and Fig S3A. In contrast to the author's suggestions, it is possible that during actin contractility, actin cables running over the nucleus could be confining the nucleus to height changes that are too subtle to be measured using a confocal microscope, and there are forces acting on to top of the nucleus that could contribute to the blebbing phenotype observed. In my opinion, the conclusions drawn by the authors in this matter rely too few evidence.

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    Referee #2

    Evidence, reproducibility and clarity

    The authentication of cell lines was not clear. The origin of the cell lines used was not clear. Were they immortalized? Did they examine primary MEFs? The authors did not seem to be aware of convincing data showing that Lamin B1 increases nuclear dispensability, whereas lamin B1 deficiency has the opposite effect.

    The paper reads like a rough draft. Nomenclature inappropriate.

    Significance

    The impact of actin on blebs and nuclear shape is well established. The implications of these findings for distinct roles of the nuclear lamin proteins were not clear. The impact of the interventions on nuclear stiffness was not measured.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript Pho et al., characterize the effects of actin confinement versus acto-myosin contractility on nuclear deformation and nuclear envelope (NE) rupture/blebs. They use both genetic and pharmacological perturbations that impact nuclear shape and integrity such as chromatin decompaction and genetic deletion of nucleo-skeleton components (LaminA and LaminB). First, the authors show that modulation of acto-myosin contractility does not affect nuclear height (a proxy for the effects of actin confinement on the nucleus). This finding allowed them to study the effects of acto-myosin contractility on nuclear shape and integrity independently of actin-mediated nuclear confinement. Using fibroblasts expressing a NLS-GFP construct to assess nuclear shape and integrity, the authors show that increased acto-myosin contractility by treatment with Rho activator (CN03) leads to nuclear blebs and NE rupture events (curiously, they show that in LaminA-/- fibroblasts the NE rupture events were not bleb-based and were not affected by CN03). With these results, authors conclude that actin contraction is a major determinant of bleb-based NE rupture, independently of actin confinement. Next, since many studies have associated loss of nucleo-cytoplasmic compartmentalization with increased DNA damage, the authors addressed the occurrence of DNA damage foci in several contexts/perturbations. They find that increased NE rupture frequency by CN03 treatment does not lead to increased DNA damage. Based on the sole finding, authors then claim that DNA damage is mostly associated with abnormal shaped nuclei instead of ruptured nuclei. The data presented is overall convincing and well controlled but a more in-depth characterization of the DNA damage events in VPA-treated cells, LaminB-/- and LaminA-/- cells is needed to support the claim that DNA damage is not associated with NE events in their system. More details below.

    Major comments:

    1. In fig. 4, increased NE rupture frequency by CN03 treatment did not lead to an increase in gH2AX foci, so authors claim that DNA damage is not associated with NE events. However, the increase observed in NE rupture frequency is rather very subtle (from 1.5 to 3 in WT cells; from 2.5 to 4 in VPA treated ells and from 2 to 2.5 in LaminB-/- cells) so I wonder if such a minor increase might be sufficient to cause an increase in DNA damage foci. Moreover, DNA damage events are repaired within minutes and the analysis throughout this manuscript is made on snapshots. Therefore it becomes difficult to draw any conclusion regarding the absence of a link between NE rupture and DNA damage. To assess DNA damage events in a quantitative manner and show that abnormal shape (as opposed to NE rupture) is the main driver of DNA damage, authors should perform live cell imaging of cells co-expressing a DNA damage marker and a NE rupture marker to track DNA damage events following NE rupture.
    2. Fig. 4C: to show that frequency of NE rupture is less important than abnormal nuclear shape for DNA damage appearance, authors need to quantify gH2AX in normal x blebbed x abnormal nuclei in each one of the conditions (untreated, CN03 and Y27). But again, this analysis must be complemented by live cell imaging experiments to track DNA damage foci appearance (or not?) following NE rupture events.
    3. Since VPA treatment increases both the percentage of bleb-based NE rupture and NE rupture frequency in LaminA-/- cells, authors should show that gH2AX foci in this sample remain unaltered to further support their claim that frequency of NE rupture is less important than abnormal nuclear shape.
    4. Does methylstat treatment rescue LaminB-/- phenotypes (blebs, ruptures, ...)?
    5. Auhors mention throughout the manuscript the "actin confinement" component (actin cables localized at the top of the nucleus-not convincingly shown in fig. 2C). Some studies have reported the occurrence of perinuclear actin caps that surround the entire nucleus (DOI: 10.1038/s41467-018-04404-4; DOI: 10.1038/srep40953; DOI: 10.1038/ncb3387). Can the authors investigate the existence of such perinuclear actin ring on the LaminB-/-, LaminA-/- and VPA-treated cells? Could this ring affect NE rupture and shape? Also, if these perinuclear actin rings can be observed, what would they look like in CN03- and Y27-treated cells?

    Minor comments:

    1. Please mention/discuss CytoD treatment in main text (Fig 2C).
    2. Can the authors comment on why CN03 treatment on VPA cells does not cause changes in blebs or NE rupture (fig. 3A, B)?
    3. I'd move fig. 2B to supplement.
    4. In my opinion schematics on figs. 1A, 2A, 3E are confusing and do not add anything to the manuscript.
    5. There is something missing on the following sentence, please revise it: "We hypothesized that LMNA-/- nuclei do not show bleb-based behaviors because this perturbation cannot, due to reported disrupted nuclear-actin connections (Broers et al., 2004; Vahabikashi et al., 2022)."
    6. Can the authors explain in the Discussion why there is a decrease in gH2AX foci in VPA-treated cells and LaminA-/- cells upon CN03 treatment?

    Significance

    Nuclear deformation is a common event observed in homeostasis and disease and both extra-cellular physical cues and different cellular components play critical roles in nuclear morphology and integrity. It is well known that the actin cytoskeleton exerts a wide range of forces on the nucleus and causes nuclear deformation (via LINC complex) as cells migrate, grow or spread within complex microenvironments. The contribution of mutations of nucleo-skeleton components to nuclear abnormalities and rupture are well described. Additionally, more recently, the contribution of the actin cytoskeleton to nuclear integrity and morphology has also been characterized. However, the role played by actin contraction on nuclear shape and integrity, independently of actin confinement (exerted by the actin cables localized at the top of the nucleus), remain elusive. In this manuscript Pho et al., address this question using cells expressing NLS-GFP to detect nuclear rupture events and potential nuclear deformations. There is no real conceptual advance in this study but there is a novel finding as it shows that acto-myosin contraction affects nuclear integrity and morphology independently of dorsal actin cables (actin confinement). Moreover, the experiments were performed on flat 2D surfaces, a distant scenario from the 3D in vivo landscapes. As a classic cell biology study this manuscript has the potential to be of interest to basic researchers in the field of cell migration, crosstalk of nucleo-skeleton/cytoskeleton and nuclear mechano-sensing.

  7. Version published to 10.1101/2022.12.01.518663v1 on bioRxiv