A Novel Role for CSA in the Regulation of Nuclear Envelope Integrity: Uncovering a Non-Canonical Function

  1. Denny Yang
  2. Austin Lai
  3. Amelie Davies
  4. Anne FJ Janssen
  5. Delphine Larrieu

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Abstract

Cockayne syndrome (CS) is an autosomal recessive premature ageing condition mainly characterized by microcephaly, growth failure, and neurodegeneration. It is caused by mutations in ERCC6 or ERCC8 genes which encode for Cockayne Syndrome B (CSB) and Cockayne Syndrome A (CSA) proteins, respectively. CSA and CSB have well-characterised roles in transcription-coupled nucleotide excision repair (TC-NER), responsible for the removal of bulky DNA lesions, including those caused by UV irradiation. Here, we report that CSA knockout cells and CSA patient cells (CS-A) carrying a loss-of-function mutation in the ERCC8 gene exhibit defects in nuclear envelope (NE) integrity. NE dysfunction is a characteristic phenotype of cells from progeroid disorders caused by mutation in NE proteins, such as Hutchinson-Gilford Progeria Syndrome (HGPS). However, it has never been reported in Cockayne Syndrome. We observed that CS-A cells displayed reduced levels of LAP2-emerin-MAN1 (LEM)-domain 2 (LEMD2) at the NE resulting in decreased formation of LEMD2-lamin A/C complexes. In addition, loss of CSA function caused increased actin stress fibers that contributed to enhanced mechanical stress to the NE. Altogether, these led to NE blebbing and ruptures in interphase, causing activation of the innate/immune cGAS/STING signaling pathway. Disrupting the linker of the nucleoskeleton and cytoskeleton (LINC) complex that is responsible for anchoring the cytoskeleton to the NE, rescued the NE phenotypes and reduced the activation of cGAS/STING pathway. This work has revealed a previously uncharacterized role for CSA in regulating NE integrity and shed light on mechanisms that may further explain some of the clinical phenotypes observed in CS patients such as neuroinflammation. This is to our knowledge, the first study showing NE dysfunction in a progeroid syndrome caused by mutations in a DNA damage repair protein, reinforcing the connection between NE deregulation and ageing.

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    Reply to the reviewers

    Reviewer #1

    • Albeit the link between CSA and NE integrity the work is in my eyes too preliminary. Although the data presented are well done and carefully evaluated they mostly (except Fig 1A) rely on direct comparisons of one patient cell line (CS-A or CS-B) to the same cells expression the wildtype protein. It remains thus open whether the effects seen on LEM2 expression, LEM2-LaimA/C interaction, stress fibre formation, cGAS/STING signaling pathway activation in the CS-A cells are representative for a number of different CS patient derived cells. This is especially important given the small changes observed. Please note that there are alos clear differences between the CSA-wt and CSB-wt cells. Would the HPA CSA KO cells show in addition to NE irregularities (not even quantified) the same phenotypes and can they be reverted by re-expression of the wildtype CSA protein? We would like to thank the reviewer for this comment. Indeed we have previously observed nuclear circularity defects in CSA KO HAP1 cells but we haven’t investigated the other phenotypes in this cell line. One of the main reasons behind this is the technical difficulties associated with performing immunofluorescence with HAP1 cells who are very small and tend to grow in aggregates.

    Proposed experimental plan: To address this reviewer’s comment, we will attempt again to use HAP1 cells (WT and CSA KO) and look at nuclear circularity (with quantification), stress fiber formation and cGas foci. If we don’t succeed, we will use an alternative isogenic cell model consisting of fibroblasts in which we will knock out CSA using CRISPR/Cas9. We will then repeat the same experiments as proposed above for the HAP1 cells.

    • The link between CSA and SUN1 is not well worked out. What is the effect of SUN2 downregulation and that of nespirins? It remains unclear whether the observed effects are indeed LINC mediated. Proposed experimental plan: To address this point, we will downregulate SUN2 and nesprins using siRNAs in two different cell models (as described above) and assess nuclear shape as well as cGas/STING pathway activation.

    Minor comments:

    Fig 1B: Why is HA-Tagged CSA not shown on the CSA western? This would be helpful to compare to the endogenous levels at least in CSB cells. A western showing an housekeeping marker would allow better comparison. Judging from the proteins markers HA-tagged CSA seems much larger as endogenous CSA (first versus second row). Again, less cropped western blots would help.*

    We are sorry for the confusion and we have realised that the molecular weights on the western blots were incorrectly labeled on this figure. This will be modified, and the full, uncropped WB will be provided as a supplementary file.

    Fig 3A: Is CSA FLAG or HA-tagged? Or both? If both are expressed the question raises of why the CSA-LEM 2 interaction is only seen in an overexpression situation.

    CSA is HA tagged. To address this reviewer’s comment we will try performing immunoprecipitation on endogenous proteins.

    Fig 5: Inconsistency between figure and figure legend: 20 vs 25 nM Jasplakinolide. I assume Latrunculin A should read Cytochalasin D?

    Thank you for pointing this out, and yes this is indeed a mistake on our labeling. We will rectify this on the figure legend.

    Fig5B: Not clear why in "CSA-wT cells" Cytochalasin D and Jasplakinolide have the same effect on nuclear envelope shape yet only Jasplakinolide increases the number of blebs.

    Cyt D inhibits actin polymerization while jasplakinolide increases polymerization. Likely actin polymerization increases blebs through extra force being put on the nucleus through actin cables/ actin based motility. Both drugs decrease nuclear roundness as they disrupt the normal actin network leading to worsening of nuclear shape through different mechanisms.

    Page 10: Method for IF: 4% (v/v) paraformaldehyde and 2% /v/v) should likely read (w/v).* Page 19: replace "withl" by "with".*

    We will rectify both these points.

    Reviewer #2

    The paper is well-written and for the most part, the data support the conclusions of the authors. Some minor caveats could be addressed to improve the quality of the manuscript.

    We would like to thank the reviewer for their positive feedback on our manuscript.

    • The phenotype of decreased LEMD2 incorporation into the NE in CS-A cells is minor. Only ~20% and thus, it is not clear whether this is causal of any of the NE abnormalities.* It should be better explained how these data add to the story.*

    To address this point, we will overexpress LEMD2 in CS-A cells and assess whether the NE phenotype can be significantly rescued. This will add value to this part of story.

    • Inducing actin polymerization and depolarization impact nuclear morphological abnormalities and nuclear blebbing. Do these treatments impact nuclear fragility and cGAS accumulation at NE break sites?* This is a good point indeed. To address this question, we will include cGas foci staining and quantification upon treatment with these chemicals.

    • Depletion of SUN1 in CS-A cells increased nuclear circularity, decreased blebbing, and phosphorylation of TBK1. The impact of SUN1 depletion in cGAS foci formation at NE break sites and phosphorylation of STING is not shown. Such experiments will provide stronger evidence that CS-A activates the cGAS-STING pathway in a SUN1 (mechanical stress)-dependent manner.

    We will address this question by analysing cGas foci and cGas-STING pathway activation upon SUN1 depletion by siRNA.

    Reviewer #3

    The data are generally clear, well performed and well interpreted with some exceptions:*

    1. I appreciate the use of isogenic cell lines (a big plus when dealing with patient-derived cell lines). However, these lines were established 30 years ago and the reported phenotypes might be due to genetic drifts. To exclude this, I suggest to complement the HAP-1 ERCC8 KO cell line with exogenously expressed CSA and assess if this rescues the phenotypes reported. Validation of the KO in these lines, either by western blotting or sequencing is needed.*

    This point has also been raised by the first reviewer, and will be addressed as described above (and pasted below):

    We would like to thank the reviewer for this comment. Indeed we have previously observed nuclear circularity defects in CSA KO HAP1 cells but we haven’t investigated the other phenotypes in this cell line. One of the main reasons behind this is the technical difficulties associated with performing immunofluorescence with HAP1 cells who are very small and tend to grow in aggregates.

    Proposed experimental plan: To address this reviewer’s comment, we will attempt again to use HAP1 cells (WT and CSA KO) and look at nuclear circularity (with quantification), stress fiber formation and cGas foci. If we don’t succeed, we will use an alternative isogenic cell model consisting of fibroblasts in which we will knock out CSA using CRISPR/Cas9. We will then repeat the same experiments as proposed above for the HAP1 cells.

    2) Related to the complementation of patient cell lines, the exogenous HA-CSA is not recognised by the anti-CSA in the CSA-null patient cell lines (Fig 1B, second blot). Shouldn't you be able to see this exogenous protein? HA-GFP-CSB in the complemented CSB-null patient cell line runs at the same weight as endogenous CSB (Fig 1B, fourth blot). This is also unexpected. I think you need better characterisation of your cell lines and need to demonstrate the level of exogenous transgenes that have been used to complement the cells and that they localise appropriately, presumably to the nucleus. You should also make sure to cite the paper where they were isolated and describe that they were immortalised (Troelstra et al., 1992) and the paper in which transgenes were stably overexpressed (Qiang et al., 2021).*

    As mentioned above, we have realised that we made some mistakes with the labeling of the molecular weight on this western blot. This will be corrected and the full uncropped western blot will be provided as a supplementary figure.

    We will also cite the suggested papers accordingly.

    3) Immunolocalisation of INM proteins is notoriously tricky and the permeabilisation steps include only 0.2% Tx100, which can be insufficient to permeabilise the INM. I appreciate the Emerin and Lamin immunostaining seems to have worked, but in many cases successful immunostaining can be antibody-specific. Can you try harsher permeabilisation to expose LEM2 epitopes? I'm somewhat uncomfortable with the suggestion that there is a cytosolic (ER?) pool of endogenous LEM2 as this runs counter to the literature and feel that your antibody or fixation conditions are illuminating a non-specific protein. The WB in Fig 2E shows that there is virtually no LEM2 in the "soluble" fraction. I would be more cautious on this cytoplasmic/nuclear pool interpretation. Biochemical nuclear and cytoplasmic fractionation would help clarify the signal in a NE vs a non-NE pool.*

    As suggested by the reviewer, we will try harsher permeabilisation conditions to test the LEMD2 antibody. As we suggest in the manuscript however, we think that the “cytoplasmic” LEMD2 pool we observed by IF in the absence of pre-extraction is indeed unspecific. This is why we have performed the rest of the experiments with a pre-extraction step, that we have shown to give a specific LEMD2 signal that disappear upon depleting LEMD2 by siRNA.

    4) Page 15: "Using a Proximity Ligation Assay (PLA), we showed a significant reduction in the number of PLA foci in CS-A cells compared to the WT(HA-CSA) cells, reflecting a reduced number of LEMD2-lamin A/C complexes (Figure 2G, 2H). This data suggests defects in the incorporation of LEMD2 into the NE and lamin protein complexes in CS-A cells". If you have less LEM2 in the NE, it is quite expected that you will have less "LEM2-laminA/C" complexes. To me the logic doesn't hold and this data does not suggest that there is an underlying defect in LEM2-lamin interaction. To ascertain whether there is such a defect one could perform an IP against LEM2 and quantify laminA/C, normalizing by the amount of LEM2 in the input.

    We feel we may not have been clear in how we interpreted this data. What we mean is that in each individual cell, the number of Lamin-LEMD2 complexes is decreased, probably indeed due to the fact that there is less LEMD2 altogether within the nucleus in the absence of CSA. We will clarify this in the text.

    5) "We overexpressed LEMD2-GFP and Flag-CSA constructs, followed by GFP pulldown in WT(HA-CSA) cells". Since the co-IP data are obtained in overexpression conditions (of both HA-CSA and Flag-CSA?), the authors should validate the interaction between LEM2 and CSA using an orthogonal approach. Perhaps anti-HA capture of the WT(HA-CSA) cells would allow you to immunoblot for endogenous LEM2?*

    To address this point, we will try to immunoprecipitate HA-CSA and look at endogenous LEMD2.

    6) Related to the CSA-LEM2 binding in the above experiment, the procedure involves combining a native detergent-extracted cytoplasmic pool with a denatured (RIPA-extracted) nuclear pool for performing the GFP-trap. From which pool was the tagged CSA bound to LEM2 in?*

    We are sorry about the confusion. We didn’t try to run the IP from the different pools but instead from the combined pools, to ensure we were looking in the whole cell extract. We would expect however that the interaction occurs in the nuclear pool as both CSA and LEMD2 are nuclear proteins.

    7) "The absence of CSA in CS-A patient cells does not affect the mobility of LEMD2 at the NE but instead decreases its interaction with A-type lamins". To me the fact that loss of CSA decreases LEM2-lamin interaction is not well supported (see point 3).

    See our response to point 4

    8) "Here, we showed by immunoprecipitation that LEMD2 also interacts with CSA. This suggests that the recruitment and stabilization of LEMD2 to the NE is mediated by an interaction with CSA, although the mechanism remains unclear". I think this is an overstatement: there are no data suggesting that CSA recruits or stabilises LEM2 at the NE.

    *We will tone down this statement in the text

    9) As the authors suggest in the discussion, it would be worth checking whether LEM2 overexpression is able to rescue some of the NE defects reported, strengthening the hypothesis that LEM2 levels are at least in part responsible for the phenotypes reported.

    To address this point, we will perform LEMD2 overexpression in the CSA cells, and analyse the nuclear envelope defects and ruptures (shape and cGas foci quantification)

    10) To me it is not clear how the reported phenotypes are interrelated. The first part of the manuscript shows that CSA interacts with LEM2, and that loss-of-function CSA impacts on LEM2 levels and LEM2-lamin interaction, suggesting a direct role for CSA at the nuclear envelope. The second part of the manuscript shows that cells with defective CSA have more actin stress fibres and releasing the cytoskeleton-nuclear tethering is able per se to rescue the nuclear membrane and cGAS phenotypes. How do the authors reconciliate these two parts? Is CSA directly involved in both inner nuclear membrane homeostasis and actin cytoskeleton modulation or is this latter role upstream and the NE defects a mere consequence of increased cytoskeleton rigidity?

    At this point indeed we cannot draw definitive conclusions as to whether the two described phenotypes are inter-related. However, by addressing the other points raised by the reviewers, we hope this will help clarifying the mechanism.

    11) It is not clear how or why actin stress fibres are elevated in the CS-A cells. Can the authors provide any insight based on their RNAseq analysis? Demonstrating a link to ROCK, LIMK or Rho signalling would be interesting and verifying ppMLC2 levels would help explain why contractility is enhanced. Additionally, is the increase in contractility dependent upon any of the genes identified as up- or downregulated in RNAseq? Presently, the manuscript is missing a link between its two halves.*

    We would like to reiterate that the RNASeq analysis we performed was done on previously published data from another group (as described in the text). To address the point raised by the reviewer, we will look more specifically into our analysis to look at ROCK, LIMK or Rho signalling to see if any of these pathways appear to be modulated by the absence of CSA.

    12) Related to point 1, the RNAseq comparison was performed on patient cells lacking CS-A and patient cells lacking CS-A and later over-expressing HA-CSA, and this comparison is used extensively for phenotype description in the manuscript. In isn't clear to me that this is the most insightful comparison to make; the rescue by overexpression is not as elegant as CRISPR reversion and the ko fibroblasts have presumably been surviving well in culture without CS-A before this protein was overexpressed. Can you validate the differential expression of any identified proteins in the acute HAP1 ko? Can you validate any of the differentially expressed proteins in comparison to normal fibroblasts (e.g., 13O6, as per Qiang et al., 2021)?

    As we will validate our experiments in an additional cell model (as described above), we will also indeed validate the level of expression of cytoskeletal proteins upon CSA KO/rescue.

    Minor comments

    • Page 14: "To characterize the NE phenotypes further, we obtained CS patient-derived cell lines carrying loss-of-function mutations in CSA (CS-A cells) or CSB (CS-B cells), and their respective isogenic control cell lines (WT(HACSA) and WT(HA-GFP-CSB))." What type of loss-of-function? Is the mutant protein still produced? In Fig 6A there seem to be a band in the CS-A blot (second lane), but in Fig 1B, there isn't. I think this is important to know to interpret the phenotype related to LEM2 interaction.*

    We can clarify that in the text. Indeed, the loss of function mutation leads to the absence of CSA protein.

    - Figure 1B is poorly annotated. What do - and + stand for? In general, I find a bit confusing how the WB are presented throughout the manuscript, specifically how the antibodies are reported (e.g., HA-CSA instead of HA). Please mark up all western blots with antisera used. Please make sure all expected bands are within the crops - e.g., Fig 3B, the anti-LEM2 blot should be expanded vertically to show the LEM2-GFP relative to endogenous LEM2.

    We will correct these on the figures

    *- From the methods, it appears that you obtained a *Please provide clarity on which construct was used in which figure, and verify that an N-terminally tagged LEM2 still localises to the NE.

    We actually cloned LEMD2 into an empty pEGFP vector but still maintained LEM2-GFP. We will remove the C1plasmid from the methods to avoid confusion as we removed the MCS and GFP and just used the blank vector and inserted lem2-gfp as we obtained it.

    - Fig 1I: there is some text on top of the upper panels (DAPI, cGAS, Merge).

    • "Through gene ontology analysis, we found that genes involved in endoplasmic reticulum (ER) stress were differentially expressed (Figure 4B)". I don't think that the way data are shown in Fig 4B is effective. Since GO has been performed, I would replace the table with a GO enrichment analysis graph. Ensure to report all the data in a supplementary .xls so that others can see and reuse it. Is there a mandated repository that accepts RNAseq data?*

    The RNAseq experiment and data was performed by another group and reported in a previous study, as referenced in the main text of the manuscript (Epanchintsev A, Costanzo F, Rauschendorf MA, Caputo M, Ye T, Donnio LM, et al. Cockayne’s Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation. Mol Cell. 2017 Dec;68(6):1054-1066.e6.). Here, we only re-analysed their data using STRING pathway analysis, as detailed in the Material and Methods. However, as suggested by the reviewer, we will replace the table by a GO enrichment graph.

    - The volcano plot looks weird with many values at the maximum log10 (P-value) - is the data processed appropriately?

    As mentioned above, the RNA Seq analysis was performed and published in a different study. We think this is because the Y axis shows adjusted P values.

    - Figure 5B: the legend says "Latrunculin A". Please correct.

    We will correct this

    - For a Wellcome funded researcher, I'm surprised that the mandated OA statement and RRS is absent from the acknowledgements.

    We will of course comply with the open access policy of the Wellcome Trust. However, and based on the WT requirements detailed on their website, we believe the acknowledgement section complies with the funder’s policy: “All research publications must acknowledge Wellcome's support and list the grant reference number which funded the research reported.”

    Maybe mention the changes in nuclear shape is not a causative of nuclear blebbing. But maybe not say that they are completely mutually exclusive phenotype to each other.

    suggestion

    Maybe say that we will overexpress LEMD2 in CS-A cells and show that the NE phenotype can be significantly rescued. This will add value to this part of story. I remember when I did the FRAP experiment, CS-A cells with expression of LEMD2-GFP (that doesn’t form aggregates) looks better in term of shape.

    I think Anne, please check the plasmid map? According to the lab inventory (Plasmids Anne), it is LEMD2-GFP. So probably GFP is at C-terminus.

    I think there was a part in discussion was LEMD2-GFP was mistakenly written as GFP-LEMD… But I am sure I used LEMD2-GFP throughout the work

    We cloned it into an empty pEGFP vector but still maintained LEM2-GFP. Maybe remove the C1 in the methods to avoid confusion as we removed the MCS and GFP and just used the blank vector and inserted lem2-gfp as we obtained it.

    Same construct was used for GFP pulldown and for FRAP. And we can see in FRAp that they localise to the NE. SO it should localise to the NE. Maybe mention that we will do a LEMD2-GFP over expression experiment in CS-A cells and show that they do localise to the NE.

    I don’t remember fully if Denny did this and what came out. I thought he did and ER stress and cytoskeleton regulation came out as enriched terms?

    Denny will have to check this but I think this is because the Y axis shows adjusted P values?? I have the same in my data and Jack told me this is an artefact of the analysis if you adjust for multiple comparisons and is something more often seen in mass spec data

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    In this manuscript, Yang and colleagues report that the absence of CSA (a protein with a well-characterised role in DNA damage repair and whose mutations cause a premature ageing syndrome) leads to defects in nuclear envelope (NE) integrity. Using a Cockayne syndrome patient-derived cell line they show that CSA deficiency correlates with the presence of an aberrant nuclear morphology, nuclear blebbing and increased cGAS-signalling. Moreover, they suggest that this CSA defective cell line presents decreased LEM2 at the NE and a consequent reduction in LEM2-laminA/C interaction. They were able to observe interaction betweenCSA and LEM2, andsuggested that CSA might interfere with LEM2 functionality at the nuclear envelope. Starting from the analysis of an already published RNAseq dataset, the authors go on investigating the cytoskeleton status in the CSA mutant cell line and identify an increase in actin, which they suggest leads to an increase in actin stress fibres. By promoting general actin depolymerisation and by disanchoring the cytoskeleton from the nuclear envelope, Yang et al are able to rescue the dysfunctional phenotypes reported above, suggesting that increased mechanical forces transmitted from the actin cytoskeleton to the nuclear envelope might cause defects in nuclear envelope integrity. Finally, the authors report that alleviating the nuclear envelope defects in CSA mutant cells does not decrease their sensitivity to a UV mimetic, hinting that CSA role in maintaining nuclear integrity is independent from its DNA damage repair function.

    Major comments

    The key conclusions of the manuscript are convincing and supported by the data. However, there are some conceptual concerns that limit my enthusiasm.

    Firstly, that the actin cytoskeleton can transmit forces to the NE via the LINC complex is established. That these forces that lead to NE deformation, and in some cases, rupture, is somewhat unsurprising. The interesting observation in this manuscript is that CSA mutant patient cells exhibit more stress fibres. This leads to more LINC-dependent transmission of forces to the NE, with consequential effects on NE morphology and rupture. However, the mechanism by which actomyosin contractility is elevated in CSA-mutant patient cells is unexplored.

    Secondly, it is unclear whether CSA controls the quality, or simply the number, of LEM2/Lamin interactions. As such, a mechanistic link between elevated stress fibres and CSA-dependent NE/lamina interactions is not provided and the paper sits as two presently unconnected observations.

    The data are generally clear, well performed and well interpreted with some exceptions:

    1. I appreciate the use of isogenic cell lines (a big plus when dealing with patient-derived cell lines). However, these lines were established 30 years ago and the reported phenotypes might be due to genetic drifts. To exclude this, I suggest to complement the HAP-1 ERCC8 KO cell line with exogenously expressed CSA and assess if this rescues the phenotypes reported. Validation of the KO in these lines, either by western blotting or sequencing is needed.
    2. Related to the complementation of patient cell lines, the exogenous HA-CSA is not recognised by the anti-CSA in the CSA-null patient cell lines (Fig 1B, second blot). Shouldn't you be able to see this exogenous protein? HA-GFP-CSB in the complemented CSB-null patient cell line runs at the same weight as endogenous CSB (Fig 1B, fourth blot). This is also unexpected. I think you need better characterisation of your cell lines and need to demonstrate the level of exogenous transgenes that have been used to complement the cells and that they localise appropriately, presumably to the nucleus. You should also make sure to cite the paper where they were isolated and describe that they were immortalised (Troelstra et al., 1992) and the paper in which transgenes were stably overexpressed (Qiang et al., 2021).
    3. Immunolocalisation of INM proteins is notoriously tricky and the permeabilisation steps include only 0.2% Tx100, which can be insufficient to permeabilise the INM. I appreciate the Emerin and Lamin immunostaining seems to have worked, but in many cases successful immunostaining can be antibody-specific. Can you try harsher permeabilisation to expose LEM2 epitopes? I'm somewhat uncomfortable with the suggestion that there is a cytosolic (ER?) pool of endogenous LEM2 as this runs counter to the literature and feel that your antibody or fixation conditions are illuminating a non-specific protein. The WB in Fig 2E shows that there is virtually no LEM2 in the "soluble" fraction. I would be more cautious on this cytoplasmic/nuclear pool interpretation. Biochemical nuclear and cytoplasmic fractionation would help clarify the signal in a NE vs a non-NE pool.
    4. Pag 15: "Using a Proximity Ligation Assay (PLA), we showed a significant reduction in the number of PLA foci in CS-A cells compared to the WT(HA-CSA) cells, reflecting a reduced number of LEMD2-lamin A/C complexes (Figure 2G, 2H). This data suggests defects in the incorporation of LEMD2 into the NE and lamin protein complexes in CS-A cells". If you have less LEM2 in the NE, it is quite expected that you will have less "LEM2-laminA/C" complexes. To me the logic doesn't hold and this data does not suggest that there is an underlying defect in LEM2-lamin interaction. To ascertain whether there is such a defect one could perform an IP against LEM2 and quantify laminA/C, normalizing by the amount of LEM2 in the input.
    5. "We overexpressed LEMD2-GFP and Flag-CSA constructs, followed by GFP pulldown in WT(HA-CSA) cells". Since the co-IP data are obtained in overexpression conditions (of both HA-CSA and Flag-CSA?), the authors should validate the interaction between LEM2 and CSA using an orthogonal approach. Perhaps anti-HA capture of the WT(HA-CSA) cells would allow you to immunoblot for endogenous LEM2?
    6. Related to the CSA-LEM2 binding in the above experiment, the procedure involves combining a native detergent-extracted cytoplasmic pool with a denatured (RIPA-extracted) nuclear pool for performing the GFP-trap. From which pool was the tagged CSA bound to LEM2 in?
    7. "The absence of CSA in CS-A patient cells does not affect the mobility of LEMD2 at the NE but instead decreases its interaction with A-type lamins". To me the fact that loss of CSA decreases LEM2-lamin interaction is not well supported (see point 3).
    8. "Here, we showed by immunoprecipitation that LEMD2 also interacts with CSA. This suggests that the recruitment and stabilization of LEMD2 to the NE is mediated by an interaction with CSA, although the mechanism remains unclear". I think this is an overstatement: there are no data suggesting that CSA recruits or stabilises LEM2 at the NE.
    9. As the authors suggest in the discussion, it would be worth checking whether LEM2 overexpression is able to rescue some of the NE defects reported, strengthening the hypothesis that LEM2 levels are at least in part responsible for the phenotypes reported.
    10. To me it is not clear how the reported phenotypes are interrelated. The first part of the manuscript shows that CSA interacts with LEM2, and that loss-of-function CSA impacts on LEM2 levels and LEM2-lamin interaction, suggesting a direct role for CSA at the nuclear envelope. The second part of the manuscript shows that cells with defective CSA have more actin stress fibres and releasing the cytoskeleton-nuclear tethering is able per se to rescue the nuclear membrane and cGAS phenotypes. How do the authors reconciliate these two parts? Is CSA directly involved in both inner nuclear membrane homeostasis and actin cytoskeleton modulation or is this latter role upstream and the NE defects a mere consequence of increased cytoskeleton rigidity?
    11. It is not clear how or why actin stress fibres are elevated in the CS-A cells. Can the authors provide any insight based on their RNAseq analysis? Demonstrating a link to ROCK, LIMK or Rho signalling would be interesting and verifying ppMLC2 levels would help explain why contractility is enhanced. Additionally, is the increase in contractility dependent upon any of the genes identified as up- or downregulated in RNAseq? Presently, the manuscript is missing a link between its two halves.
    12. Related to point 1, the RNAseq comparison was performed on patient cells lacking CS-A and patient cells lacking CS-A and later over-expressing HA-CSA, and this comparison is used extensively for phenotype description in the manuscript. In isn't clear to me that this is the most insightful comparison to make; the rescue by overexpression is not as elegant as CRISPR reversion and the ko fibroblasts have presumably been surviving well in culture without CS-A before this protein was overexpressed. Can you validate the differential expression of any identified proteins in the acute HAP1 ko? Can you validate any of the differentially expressed proteins in comparison to normal fibroblasts (e.g., 13O6, as per Qiang et al., 2021)?

    Minor comments

    • Pag 14: "To characterize the NE phenotypes further, we obtained CS patient-derived cell lines carrying loss-of-function mutations in CSA (CS-A cells) or CSB (CS-B cells), and their respective isogenic control cell lines (WT(HACSA) and WT(HA-GFP-CSB))." What type of loss-of-function? Is the mutant protein still produced? In Fig 6A there seem to be a band in the CS-A blot (second lane), but in Fig 1B, there isn't. I think this is important to know to interpret the phenotype related to LEM2 interaction.
    • Figure 1B is poorly annotated. What do - and + stand for? In general, I find a bit confusing how the WB are presented throughout the manuscript, specifically how the antibodies are reported (e.g., HA-CSA instead of HA). Please mark up all western blots with antisera used. Please make sure all expected bands are within the crops - e.g., Fig 3B, the anti-LEM2 blot should be expanded vertically to show the LEM2-GFP relative to endogenous LEM2.
    • From the methods, it appears that you obtained a LEM2-GFP, and cloned it into an expression vector (pEGFPC1) to make GFP-LEM2. Please provide clarity on which construct was used in which figure, and verify that an N-terminally tagged LEM2 still localises to the NE.
    • Fig 1I: there is some text on top of the upper panels (DAPI, cGAS, Merge).
    • "Through gene ontology analysis, we found that genes involved in endoplasmic reticulum (ER) stress were differentially expressed (Figure 4B)". I don't think that the way data are shown in Fig 4B is effective. Since GO has been performed, I would replace the table with a GO enrichment analysis graph. Ensure to report all the data in a supplementary .xls so that others can see and reuse it. Is there a mandated repository that accepts RNAseq data?
    • The volcano plot looks weird with many values at the maximum log10 (P-value) - is the data processed appropriately?
    • Figure 5B: the legend says "Latrunculin A". Please correct.
    • For a Wellcome funded researcher, I'm surprised that the mandated OA statement and RRS is absent from the acknowledgements.

    Referees cross-commenting

    I think the other reviews are fair and accurate

    Significance

    This work provides interesting insights on a possible new moonlighting role of the CSA protein. This could enhance the pathophysiological comprehension of some clinical manifestations in CS patients. The nuclear envelope defects are well described and convincing; however, there is no clear understanding of how (or even if) the reported cytoskeleton and nuclear envelope defects depend on CSA. Better characterising mechanistic roles of CSA in both nuclear envelope integrity and in stress fibre formation will boost the overall impact of the manuscript. At this stage, the manuscript could be of interest for a specialised audience (premature ageing syndromes) but with more mechanistical dissection it could become of broader interest (basic research, nuclear architecture/integrity readership).

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Yang et al. shows that ERCC8 (Cockayne syndrome A protein) mutations/loss of function causes nuclear envelope (NE) abnormalities and fragility, in addition to the well described deficiencies in transcription-coupled nucleotide excision repair (TC-NER). Cells from CS-A patients show decreased LEMD2-lamin A/C complexes at the NE and increased actin stress fibers that cause mechanical stress in the NE, in addition to more blebbing and ruptures of NE. This is turn causes activation of the innate/immune cGAS/STING pathway. Importantly, disrupting the LINC complex rescued NE problems and activation of the cGAS/STING pathway. This effect of ERCC8 dysfunction on NE integrity and activation of the cGAS/STING pathway may be behind patients' phenotypes of neuroinflammation, but not UV sensitivity.

    The paper is well-written and for the most part, the data support the conclusions of the authors. Some minor caveats could be addressed to improve the quality of the manuscript.

    • The phenotype of decreased LEMD2 incorporation into the NE in CS-A cells is minor. Only ~20% and thus, it is not clear whether this is causal of any of the NE abnormalities. It should be better explained how these data add to the story.
    • Inducing actin polymerization and depolarization impact nuclear morphological abnormalities and nuclear blebbing. Do these treatments impact nuclear fragility and cGAS accumulation at NE break sites?
    • Depletion of SUN1 in CS-A cells increased nuclear circularity, decreased blebbing, and phosphorylation of TBK1. The impact of SUN1 depletion in cGAS foci formation at NE break sites and phosphorylation of STING is not shown. Such experiments will provide stronger evidence that CS-A activates the cGAS-STING pathway in a SUN1 (mechanical stress)-dependent manner.

    Referees cross-commenting

    I am more positive than the other reviewers. I agree with reviewer 1 that another patient line would be great, and that some of the westerns need improvement. However, I still find that the phenotypes found in this cell line, which are rescued by the wild-type protein, are interesting and worth reporting. I do not fully agree with technical recommendations from reviewer 3, or suggesting that that the authors address questions outside the focus of their study.

    Significance

    The significance of the study is that shows for the first time the nuclear envelop defects and fragility in cells from Cockayne Syndrome patients (mutations in ERCC8) are due to mechanical stress that is alleviated by depletion of the LINC complex. In addition, the study shows activation of the cGAS-STING pathway in these cells, although the evidence about this phenotype could be strengthen.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Cockayne syndrome (CS), an autosomal recessive premature ageing/progeria disease, is caused by mutations in the genes coding for CSA and CSB. These play a well-studied role in transcription-coupled nucleotide excision repair. In this manuscript, CSA but not CSB dysfunction is associated with nuclear envelope defects, a typical phenotype of cells from progeria diseases caused by mutations in NE proteins. HPA model cells lacking CSA show an irregular nuclear envelope not observed in cells lacking CSB. Patient cells with defective CSA, compared to isogenic cells expressing the wild-type CSA protein, show more severe nuclear envelope deformation and blebbing and activation of the cGAS/STING signaling pathway, indicating nuclear envelope damage, a slight reduction in the expression of LEM2, a protein of the inner nuclear membrane, and a reduced interaction of LEM2 with the lamina component lamin A/C. Patient cells with defective CSA show aberrant acThe proposed link between CSA and nuclear envelope integrity is interesting. CSA would in this case a factor involved in DNA repair as well as nuclear envelope integrity, two processes linked to progeria. However, the work relies on a single patient derived cell line which is compared to the same cell line expressing the wild type protein. To consolidate, more cells form different CSA patients need to be included. If worked out this idea will be interesting all cell biologists interested in DAN damage repair and nuclear envelope structure and function as well as researchers interested in the molecular mechanisms of progeria syndromes. My expertise: nuclear envelope structure and function, nuclear transport tin stress fiber and their nuclei show different effects toward actin polymerization inhibitors or stabilizers. Downregulation of SUN1, one of the nuclear envelope proteins linking to the cytoskeleton, improves nuclear envelope blebbing phenotypes in CSA patient cells

    Major comments:

    Albeit the link between CSA and NE integrity the work is in my eyes too preliminary. Although the data presented are well done and carefully evaluated they mostly (except Fig 1A) rely on direct comparisons of one patient cell line (CS-A or CS-B) to the same cells expression the wildtype protein. It remains thus open whether the effects seen on LEM2 expression, LEM2-LaimA/C interaction, stress fibre formation, cGAS/STING signaling pathway activation in the CS-A cells are representative for a number of different CS patient derived cells. This is especially important given the small changes observed. Please note that there are alos clear differences between the CSA-wt and CSB-wt cells. Would the HPA CSA KO cells show in addition to NE irregularities (not even quantified) the same phenotypes and can they be reverted by re-expression of the wildtype CSA protein? The link between CSA and SUN1 is not well worked out. What is the effect of SUN2 downregulation and that of nespirins? It remains unclear whether the observed effects are indeed LINC mediated.

    Minor comments:

    Fig 1B: Why is HA-Tagged CSA not shown on the CSA western? This would be helpful to compare to the endogenous levels at least in CSB cells. A western showing an housekeeping marker would allow better comparison. Judging from the proteins markers HA-tagged CSA seems much larger as endogenous CSA (first versus second row). Again, less cropped western blots would help.

    Fig 3A: Is CSA FLAG or HA-tagged? Or both? If both are expressed the question raises of why the CSA-LEM 2 interaction is only seen in an overexpression situation.

    Fig 5: Inconsistency between figure and figure legend: 20 vs 25 nM Jasplakinolide. I assume Latrunculin A should read Cytochalasin D?

    Fig5B: Not clear why in "CSA-wT cells" Cytochalasin D and Jasplakinolide have the same effect on nuclear envelope shape yet only Jasplakinolide increases the number of blebs.

    Page 10: Method for IF: 4% (v/v) paraformaldehyde and 2% /v/v) should likely read (w/v).

    Page 19: replace "withl" by "with".

    Significance

    The proposed link between CSA and nuclear envelope integrity is interesting. CSA would in this case a factor involved in DNA repair as well as nuclear envelope integrity, two processes linked to progeria. However, the work relies on a single patient derived cell line which is compared to the same cell line expressing the wild type protein. To consolidate, more cells form different CSA patients need to be included. If worked out this idea will be interesting all cell biologists interested in DAN damage repair and nuclear envelope structure and function as well as researchers interested in the molecular mechanisms of progeria syndromes.

    My expertise: nuclear envelope structure and function, nuclear transport

  5. Version published to 10.1101/2023.12.14.571633v1 on bioRxiv