Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts

  1. Kojiro Suda
  2. Yohsuke Moriyama
  3. Nurhanani Razali
  4. Yatzu Chiu
  5. Yumiko Masukagami
  6. Koutarou Nishimura
  7. Hunter Barbee
  8. Hiroshi Takase
  9. Shinju Sugiyama
  10. Yuta Yamazaki
  11. Yoshikatsu Sato
  12. Tetsuya Higashiyama
  13. Yoshikazu Johmura
  14. Makoto Nakanishi
  15. Keiko Kono

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Abstract

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence—stable cell cycle arrest contributing to organismal aging—is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III ( SNF7 ) and AAA-ATPase ( VPS4 ) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca 2+ –p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

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  1. Version published to 10.1038/s43587-024-00575-6
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    RC-2021-00739

    “Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts”

    Kono et al.

    __Point-by-point response __

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this article, Kono et al worked on cellular outcomes induced by plasma membrane damage (PMD) in yeast and in human cells. Plasma membrane damage is induced by some stresses and alteration of its repair can lead to some diseases. Globally little is known about PMD. Authors observed that PMD-induced by low concentration of SDS in yeast and in human cells can limit their replicative lifespan. A genetic screen in yeast has identified the endosomal sorting complexes required for transport (ESCRT) genes as required for PMD response. In human cells, the authors observed that PMD-induced premature senescence is dependent of p53 activity but independent of DNA damage. This work sounds novel and interesting in the context of senescence on human cells. Nevertheless, they are some limits and questions that should be addressed to strongly improve this interesting work.*

    Thank you very much for reviewing our manuscript. We are delighted to know that reviewer #1 thinks our work is novel and interesting.

    **Major comments:**

    *- can the authors describe and explain what are common and divergent betweenreplicative lifespan in yeast and human cells, for instance on telomere biology? It is particularly important as the authors jumped from replicative lifespan in yeast to replicative senescence in human cells.

    Thank you for raising this point. The telomere biology in yeast and human cells share at least three central mechanisms but obviously there are limitations of using yeast as a model. We included this point in discussion (page 12, line 10-22).

    - a better characterization of premature senescence induced by SDS is required to delineate this new type of senescence: for instance, SASP content characterization and EdU incorporation assays to properly demonstrate the proliferation arrest.

    According to the reviewer’s suggestion, we added SASP qPCR results (Fig. 3I and J). We also performed EdU incorporation assays and included in the revised manuscript (Fig. 3F).

    *- the authors claimed that PMD-induced senescence is DNA damage-independent and that PMD could occur during replicative senescence. As mentioned in some references cited by the authors, replicative senescence normally occurs in response to telomere shortening and this shortening results in a DNA damage response which initiates senescence (ref 23). So authors should formulate their conclusions and discussion in the light of these well described results and tone down some of their conclusions. *

    We agree with the reviewer’s point that the best-studied mechanism underlying replicative senescence is telomere shortening (Blackburn, 2001; Shay and Wrightas, 2001) and telomere-dependent replicative senescence is mediated by the DNA repair pathway (d'Adda di Fagagna et al., 2003). We changed the title, abstract, and introduction (title, “Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts”, abstract page 2 line 12-13, introduction page 4 line 1-2). We hope new sentences describe our findings more precisely.

    In that context it will be also interesting to investigate whether PMD occurs in other types of cellular senescence (different inducers and different cell types).

    Thank you very much for the suggestion. We performed the experiment. The results indicate that PMD does not occur in DNA damage (doxorubicine)-dependent premature senescence (Fig. S8A and B).

    - this story will be strongly improved if the authors provide some mechanistic insights. In particular if they can link their observations in yeast to their observation in human cells. For instance, does ESCRT impact SDS-induced senescence in human cells? Can this be linked to p53 activity?

    Thank you very much for the suggestion. According to the comment, we tested whether VPS4A/B overexpression extends replicative lifespan in human cells analogous to what we observed in yeast. Unfortunately, VPS4A/B overexpression from CMV promoter gradually decreased cell viability within several days. Therefore, we could not conclude their functions on lifespan extension.

    • in the discussion section, the authors discuss calcium signaling as a possible actor of PMD-induced p53 activation, can they show some data in that direction at least by measuring cytosolic calcium levels during PMD-induced senescence.*

    According to the reviewer’s suggestion, we measured cytosolic calcium levels and included them in our revised manuscript (Fig. 5A-C). Our new results indicate that the cytosolic Ca2+ is increased after SDS treatment. We also added new figures confirming the previously reported result that KCl-dependent Ca2+-influx is sufficient for senescence induction (Fig. 5D-F). To test whether Ca2+ is required for PMDS, we treated the cells with both SDS and Ca2+ chelators but the cells ruptured immediately due to the failure of membrane resealing. Therefore, although it is likely that Ca2+ is required for PMDS, we could not dissect Ca2+’s function in membrane resealing and premature senescence. We will intensively analyze this point in our next paper.

    - ESCRT is involved in nuclear envelope repair. Can the authors ruled out any effects of SDS on nuclear envelopes as nuclear envelope alterations can be involved in cellular senescence?*

    We appreciate reviewer #1 for raising an important point. We can rule out the possibility based on the following evidence. Nuclear deformation and subsequent upregulation of DNA damage signaling is a striking feature of nuclear envelope damage as observed in premature aging diseases Laminopathies (Eriksson et al., 2003; De Sandre-Giovannoli et al, 2003; Earle et al., 2019). We found that SDS treatment did not induce nuclear envelope deformation (Fig. 1F and Fig. S2A). Moreover, ESCRT did not accumulate at the nuclear membrane after SDS treatment (Fig. S3D, green). These results suggest that the SDS-dependent cellular senescence cannot be attributed to the nuclear envelope damage. We added sentences in discussion of the revised manuscript (page 12, line 1-5).

    **Minor comments:***　

    • images are used twice between Figure 1F and S2A, please replaced images to avoid this.*

    According to the reviewer’s comment, we replaced the images.

    - in Figure 3 it will be better to present cumulative population doublings which is a more classical way to present these results.

    According to the reviewer’s comment, we replaced the graphs.

    - several human cell lines are used but in most of time for different experiments. It will be good to show that at least one of them display the expected results with the different assays.*

    According to the reviewer’s comment, we added Fig. S7 to show that WI-38 cells also show PMDS. Thank you again for reviewing our manuscript despite your hectic schedule.

    Reviewer #1 (Significance (Required)):

    see above.*

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):*

    **Summary:**

    Makoto Nakanishi and co-workers use SDS (and EGTA) to induce plasma membrane damage (PMD) on budding yeast cells and human fibroblast. Their results correlate SDS induced PMD with reduced the replicative lifespan of budding yeast and p53 mediated senescence in human fibroblast.

    Using genetic screens in budding yeast, 48 SDS sensitive mutants were identified, including a large set of ESCRT mutants, V-ATPase mutants, and several mutants deficient in metabolic enzymes (amino acid metabolism and lipid metabolism). Three of the SDS sensitive yeast mutants showed a reduced replicative lifespan.

    SDS induced PMD on human fibroblast triggered p53 induction (without concomitant DNA damage) and subsequent p53 mediated senescence. SDS induced PMD also induced phosphatidyl-serine (PS) externalization of PM projections that co-localized with the ESCRT-III subunit CHMP4a.

    These results describe a potentially interesting and novel pathophysiological effect of PMD.

    Thank you very much for serving as a reviewer. We are delighted that the reviewer #2 considers our work to be novel and interesting.

    **Major points.**

    While the description of the PMD induced phenotypes in yeast and fibroblast are interesting, mechanistic insight is not provided. Perhaps the phenotypic description could be solidified by addressing the following points:

    1. Quantification of PMD using state-of-the-art FACS analysis in yeast cells and human fibroblasts e.g. using PI together with Annexin V.*

    Thank you so much for the valuable suggestion. According to the comment, we performed these experiments. We could successfully quantify the DAPI penetration in normal human fibroblasts by FACS (added to the revised manuscript as Fig. S2D). In contrast, we failed to detect the increase of Annexin V (PS externalization signals) by FACS, probably due to the detection limit of the FACS machine we used (please see below). Let me remind you that the signal at the PS externalizing spots after PMD are extremely weak; the signals cannot be compared with massive PS externalization during apoptosis. Instead, we quantified the Annexin V signals of entire cells using Zeiss inverted confocal microscope (LSM780) and Zen blue software and included them in Fig. S3B. We hope these new data serve as objective evidence supporting our conclusion.

    • The results from the yeast screens should be better characterized and explained. *

    Thank you very much for the suggestion. According to the reviewer’s comment, we performed characterization of the screening hits and identified four novel mechanisms involved in PMD response in budding yeast (Fig. S5, S6, and Supplementary texts).

    *Why do the authors focus on 'replicative lifespan' rather than on e.g. 'nutrient-utilization'. *

    Thank you for the comment. Indeed, we are also interested in the relation of PMD and other cellular processes, including nutrient utilization. The project is on-going. In this manuscript, we would like to focus on the point that the PMD response and the replicative lifespan regulation share some key regulators.

    In principle, this is fine with me, given that there are only 48 hits, but then the authors could rather argue e.g.: that they look into ESCRT mutants because the ESCRTs have been already implicated in resealing the PM in a Ca2+ dependent manner.

    Thank you for the comment. In the revised manuscript, we edited the text and emphasized that ESCRT was known to be involved in membrane repair in higher eukaryotes (page 6 line 25-page 7 line 2). Here, we looked into ESCRT to test our working hypothesis that the PMD responses and the replicative lifespan regulation could share part of the fundamental mechanisms.

    To drive home the point the ESCRTs (but also Vps34 and Erg2) limit the replicative life span of budding yeast due to the accumulation of PMD, this should be experimentally tested (e.g. compare replicative life span of the mutants +/- SDS to WT cells +/- SDS). Snf7, Vps34 and Erg2 mutants could affect the replicative life-span in a number of ways that is independent from PMD.*

    Thank you very much for raising this point. We performed the experiment. The result was that all mutants (snf7, vps34, and erg2) did not divide at all in the presence of SDS (replicative lifespan=0), consistent with the screening strategy that we isolated the mutants with absolutely no growth on SDS plates (Fig. S4). These results were added to the result section of the revised manuscript (page 7, line 11-13).

    • The rational for over-expressing Vps4 is not clear to me? Vps4 is most likely not the rate limiting factor for the ESCRT machinery under these conditions.*

    Thank you for asking this question. Vps4 is a AAA-ATPase promoting disassembly of the structural components (ESCRT-III filaments) and thus critical for pinching off the membrane. The most straightforward rate-limiting factor could be ATP but obviously it is nonspecific, having too many downstream consequences. Therefore, we decided to mildly overexpress VPS4 from TEF1 promoter and luckily the strategy worked well.

    Perhaps it would be more telling to overexpress Vps4 in a snf7 mutant and test if it still improves the replicative life-span?

    Thank you for the comment. According to the comment, we constructed pTEF1-VPS4 in a snf7 mutant and found that the strain is lethal. Thus, the lifespan extension by *pTEF1-VPS4 *is at least partly mediated by SNF7. In addition, the synthetic lethality suggests that pTEF1-VPS4 also does some deleterious function to a part of the ESCRT functions. That makes sense because ESCRT is involved in many cellular processes including nuclear membrane repair, lysosome repair, multivesicular body formation, cytokinesis, and exosome production.

    • The finding that PMD induces p53 mediated senescence in fibroblast is an important initial finding, as is the observation of the formation of PM extrusion that contain ESCRTs and externalize PS. Unfortunately, also these experiments remain rather descriptive. Many questions remain open:** a. How is p53 activated? b. Are these 'protrusion' formed by the ESCRTs? c. Are the protrusions essential for entry into senescence or a consequence?**

    We cannot thank more for these fascinating suggestions. We are thrilled to tackle these questions. Using mRNA seq and pathway analysis, we identified upstream regulators of p53 during PMDS. We are ready to submit it as an independent manuscript because it involves large datasets.

    **Minor points:***

    I understand that the author can use FIB-SEM as a very powerful technique for volumetric ultrastructural analysis. I'm wondering why it was used in Figure 5c? Would 'simple' SEM not yield exactly the same results but given the relative ease of SEM, many more cells could be quantified...? FIB-SEM would actually be great for the analysis of PMD more directly, right after SDS treatment in both yeast cells (were the entire volume of the cell could be analyzed) and in human cells.

    Thank you very much for a valuable advice. As reviewer #2 may know very well, SEM requires dehydration of cells, and the data acquisition is performed under high-vacuum condition. These two treatments significantly alter the structure of the plasma membrane of human normal fibroblasts. In contrast, for FIB-SEM observation, the cells in a culture dish can be directly fixed and embedded in resin, which preserves fine structures of the plasma membrane including soft and tiny projections (280-2500 nm). Based on these reasons, we decided to utilize FIB-SEM in Fig. 5C (now Fig. 6C in the revised manuscript).

    Reviewer #2 (Significance (Required)):

    The authors report very exciting observations that describe novel effects of plasma membrane damage (PMD) on cell (patho)physiology. Unfortunately, I find it difficult to connect the yeast part to the studies using human fibroblast (expect that SDS is used to cause PMD). While the description of the PMD induced phenotypes in yeast and fibroblast are interesting, mechanistic insight (e.g. the role of the ESCRTs in PMD and induction of p53 mediated senescence) is largely lacking at the moment. Provided that a more through phenotypic description (see major points) and perhaps some mechanistic insight can be provided, this work will be of interest to a wide audience in molecular cell biology.*

    Thank you very much for the encouraging comment. We are delighted to know that reviewer #2 highly evaluates the potential impact of this work. Here, we would like to report that 1) the PMD limits replicative lifespan in two independent eukaryotic cell types, and 2) the PMD response and the replicative lifespan regulations partly share their fundamental mechanisms, especially the mechanisms underlying cell cycle checkpoint activation. This work opens up many exciting future directions and we are extensively following them up. We hope we will be able to report detailed mechanisms very soon. Thank you again for reviewing our manuscript despite your hectic schedule.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Makoto Nakanishi and co-workers use SDS (and EGTA) to induce plasma membrane damage (PMD) on budding yeast cells and human fibroblast. Their results correlate SDS induced PMD with reduced the replicative lifespan of budding yeast and p53 mediated senescence in human fibroblast.

    Using genetic screens in budding yeast, 48 SDS sensitive mutants were identified, including a large set of ESCRT mutants, V-ATPase mutants, and several mutants deficient in metabolic enzymes (amino acid metabolism and lipid metabolism). Three of the SDS sensitive yeast mutants showed a reduced replicative lifespan.

    SDS induced PMD on human fibroblast triggered p53 induction (without concomitant DNA damage) and subsequent p53 mediated senescence. SDS induced PMD also induced phosphatidyl-serine (PS) externalization of PM projections that co-localized with the ESCRT-III subunit CHMP4a.

    These results describe a potentially interesting and novel pathophysiological effect of PMD.

    Major points.

    While the description of the PMD induced phenotypes in yeast and fibroblast are interesting, mechanistic insight is not provided. Perhaps the phenotypic description could be solidified by addressing the following points:

    1. Quantification of PMD using state-of-the-art FACS analysis in yeast cells and human fibroblasts e.g. using PI together with Annexin V.
    2. The results from the yeast screens should be better characterized and explained. Why do the authors focus on 'replicative lifespan' rather than on e.g. 'nutrient-utilization'. In principle, this is fine with me, given that there are only 48 hits, but then the authors could rather argue e.g.: that they look into ESCRT mutants because the ESCRTs have been already implicated in resealing the PM in a Ca2+ dependent manner.
    3. To drive home the point the ESCRTs (but also Vps34 and Erg2) limit the replicative life span of budding yeast due to the accumulation of PMD, this should be experimentally tested (e.g. compare replicative life span of the mutants +/- SDS to WT cells +/- SDS). Snf7, Vps34 and Erg2 mutants could affect the replicative life-span in a number of ways that is independent from PMD.
    4. The rational for over-expressing Vps4 is not clear to me? Vps4 is most likely not the rate limiting factor for the ESCRT machinery under these conditions. Perhaps it would be more telling to overexpress Vps4 in a snf7 mutant and test if it still improves the replicative life-span?
    5. The finding that PMD induces p53 mediated senescence in fibroblast is an important initial finding, as is the observation of the formation of PM extrusion that contain ESCRTs and externalize PS. Unfortunately, also these experiments remain rather descriptive. Many questions remain open: a. How is p53 activated?
      b. Are these 'protrusion' formed by the ESCRTs? c. Are the protrusions essential for entry into senescence or a consequence?

    Minor points:

    I understand that the author can use FIB-SEM as a very powerful technique for volumetric ultrastructural analysis. I'm wondering why it was used in Figure 5c? Would 'simple' SEM not yield exactly the same results but given the relative ease of SEM, many more cells could be quantified...? FIB-SEM would actually be great for the analysis of PMD more directly, right after SDS treatment in both yeast cells (were the entire volume of the cell could be analyzed) and in human cells.

    Significance

    The authors report very exciting observations that describe novel effects of plasma membrane damage (PMD) on cell (patho)physiology. Unfortunately, I find it difficult to connect the yeast part to the studies using human fibroblast (expect that SDS is used to cause PMD). While the description of the PMD induced phenotypes in yeast and fibroblast are interesting, mechanistic insight (e.g. the role of the ESCRTs in PMD and induction of p53 mediated senescence) is largely lacking at the moment. Provided that a more through phenotypic description (see major points) and perhaps some mechanistic insight can be provided, this work will be of interest to a wide audience in molecular cell biology.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this article, Kono et al worked on cellular outcomes induced by plasma membrane damage (PMD) in yeast and in human cells. Plasma membrane damage is induced by some stresses and alteration of its repair can lead to some diseases. Globally little is known about PMD. Authors observed that PMD-induced by low concentration of SDS in yeast and in human cells can limit their replicative lifespan. A genetic screen in yeast has identified the endosomal sorting complexes required for transport (ESCRT) genes as required for PMD response. In human cells, the authors observed that PMD-induced premature senescence is dependent of p53 activity but independent of DNA damage. This work sounds novel and interesting in the context of senescence on human cells. Nevertheless, they are some limits and questions that should be addressed to strongly improve this interesting work.

    Major comments:

    • can the authors describe and explain what are common and divergent between replicative lifespan in yeast and human cells, for instance on telomere biology? It is particularly important as the authors jumped from replicative lifespan in yeast to replicative senescence in human cells.
    • a better characterization of premature senescence induced by SDS is required to delineate this new type of senescence: for instance, SASP content characterization and EdU incorporation assays to properly demonstrate the proliferation arrest.
    • the authors claimed that PMD-induced senescence is DNA damage-independent and that PMD could occur during replicative senescence. As mentioned in some references cited by the authors, replicative senescence normally occurs in response to telomere shortening and this shortening results in a DNA damage response which initiates senescence (ref 23). So authors should formulate their conclusions and discussion in the light of these well described results and tone down some of their conclusions. In that context it will be also interesting to investigate whether PMD occurs in other types of cellular senescence (different inducers and different cell types).
    • this story will be strongly improved if the authors provide some mechanistic insights. In particular if they can link their observations in yeast to their observation in human cells. For instance, does ESCRT impact SDS-induced senescence in human cells? Can this be linked to p53 activity?
    • in the discussion section, the authors discuss calcium signaling as a possible actor of PMD-induced p53 activation, can they show some data in that direction at least by measuring cytosolic calcium levels during PMD-induced senescence.
    • ESCRT is involved in nuclear envelope repair. Can the authors ruled out any effects of SDS on nuclear envelopes as nuclear envelope alterations can be involved in cellular senescence?

    Minor comments:

    • images are used twice between Figure 1F and S2A, please replaced images to avoid this.
    • in Figure 3 it will be better to present cumulative population doublings which is a more classical way to present these results.
    • several human cell lines are used but in most of time for different experiments. It will be good to show that at least one of them display the expected results with the different assays.

    Significance

    see above.

  5. Version published to 10.1101/2021.03.26.437120 on bioRxiv