The Septin Cytoskeleton is Required for Plasma Membrane Repair

  1. M. Isabella Prislusky
  2. Jonathan GT Lam
  3. Viviana Ruiz Contreras
  4. Marilynn Ng
  5. Madeline Chamberlain
  6. Sarika Pathak-Sharma
  7. Madalyn Fields
  8. Xiaoli Zhang
  9. Amal O. Amer
  10. Stephanie Seveau

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Abstract

Mammalian cells are frequently exposed to mechanical and biochemical stressors resulting in plasma membrane injuries. Repair mechanisms reseal the plasma membrane to restore homeostasis and prevent cell death. In the present work, a silencing RNA screen was performed to uncover plasma membrane repair mechanisms of cells exposed to a pore-forming toxin (listeriolysin O). This screen identified molecules previously known to repair the injured plasma membrane such as annexin A2 (ANXA2) as well as novel plasma membrane repair candidate proteins. Of the novel candidates, we focused on septin 7 (SEPT7) because the septins are an important family of conserved eukaryotic cytoskeletal proteins. Using diverse experimental approaches, we established for the first time that SEPT7 plays a general role in plasma membrane repair of cells perforated by pore-forming toxins and mechanical wounding. Remarkably, upon cell injury, the septin cytoskeleton is extensively redistributed in a Ca 2+ -dependent fashion, a hallmark of plasma membrane repair machineries. The septins reorganize into subplasmalemmal domains arranged as knob and loop (or ring) structures containing F-actin, myosin II, and annexin A2 (ANXA2) and protrude from the cell surface. Importantly, the formation of these domains correlates with the plasma membrane repair efficiency. Super-resolution microscopy shows that septins and actin are arranged in intertwined filaments associated with ANXA2. Silencing SEPT7 expression prevented the formation of the F-actin/myosin II/ANXA2 domains, however, silencing expression of ANXA2 had no observable effect on their formation. These results highlight the key structural role of the septins in remodeling the plasma membrane and in the recruitment of the repair molecule ANXA2. Collectively, our data support a novel model in which the septin cytoskeleton acts as a scaffold to promote the formation of plasma membrane repair domains containing contractile F-actin and annexin A2.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This study highlights the role of the septin cytoskeleton in plasma membrane repair in HeLa cells perforated by the pore-forming toxin listeriolysin O (LLO). The authors performed a silencing RNA screen targeting protein-coding genes involved in endocytosis, exocytosis and intracellular trafficking. Besides the recovery of proteins that were previously identified to be part of the membrane repair machinery, they uncovered novel plasma membrane repair candidates, including septin 7 (SEPT7).

    They found that upon LLO treatment, septins redistribute from actin stress fibers to the cell surface where they form knobs and loops together with F-actin, Myosin-IIA and Annexin A2 (ANXA2). Using super resolution microscopy and 3D reconstruction, they showed that these structures often protruding from the cell surface are formed by septins and F-actin that are organized in intertwined filaments associated with Annexin A2, and that they are functionally correlated with plasma membrane repair efficiency. Silencing SEPT7 further revealed that the remodeling of the repair protein ANXA2 at the cell surface is greatly decreased in LLO-injured cells, whereas the down regulation of ANXA2 had no impact on the arrangement of septins and F-actin into knobs and loops. Altogether, their results evidenced that the septin cytoskeleton triggers the organization of membrane domains containing the actomyosin cytoskeleton and ANXA2, that are essential for the repair to occur.

    Major comments:

    • The authors show that silencing SEPT6 or SEPT7, but not SEPT2 or SEPT9, perturbed plasma membrane repair of LLO-injured cells. The authors explain this result by indicating that the reduced expression of SEPT7 and SEPT6 (according to the siRNA), but not that of SEPT2 results in a reduced expression of septins from other groups. This could have been an explanation but, in Fig. S2B, downregulating SEPT2 clearly seem to impact the expression of SEPT6 and SEPT7 (except for siRNA#3) once normalized with the loading control tubulin. Moreover, it is well accepted in the literature and has been observed in many cell types, including HeLa cells, that knocking down a septin from one group (with sometimes the exception of septins of Group 3) induces the downregulation of septins of the other groups, and that it consistently results in the loss of septin filaments. Therefore, the fact that silencing SEPT2 does not perturb plasma membrane repair is quite surprising. This could suggest that SEPT6 and SEPT7, independently of their filament organization, play a role in membrane repair after LLO treatment. Nevertheless, the SEPT2 staining to study the fate of septin filaments following LLO exposure indicated that it is the redistribution of septin filaments that is crucial in this repair process. Interestingly, BORG proteins which are involved in the association of septin filaments to the actin cytoskeleton in interphase cells bind to the SEPT6/SEPT7 coiled-coil region of septin polymers. Could these proteins be involved, knowing that they are Cdc42 effector proteins, and that links exist between Cdc42 activation and Ca2+ entry? OPTIONAL: silencing BORG proteins (BORG2 for example) and studying septin and F-actin remodeling following LLO exposure could help the authors to understand the reason of such a redistribution.
    • What about the terms "knob" and "loop": Are they structurally related to the "specks" described in other papers? Or are they new structures that no one observed before? Nobody has never looked at septins in this repair process before, but actin has long been described to be involved.
    • It seems that knobs are formed before loops take over. This would deserve further investigation. Is that a reality? Or are they two independent structures? OPTIONAL: it would be interesting to do time-lapse video microscopy to follow the fate of a knob. Related to the previous point: why to show 3 sets of images in the LLO condition in Fig. 2A? Does the top b-panel represent the knob stage? Where there are still many stress fibers indicating that septins have not yet fully redistributed? And when septins are fully dissociated from actin cables, which are then lost, loops are forming (middle c-panel) and then increase in size (bottom d-panel)?
    • Even though, some information is given in the discussion section, it would be helpful to mention in the introduction section the different pathways that cells activate to repair plasma membrane defects, and to precise which one(s) has(ve) already been described in the literature to be switched on in response to the LLO toxin.
    • Some experiments are not rigorous enough: Sometimes, they have not been repeated, as exemplified in Fig. 7B. Count less cells but repeat the experiment at least three times. Sometimes, one condition is missing, as in Fig. 6C. Where is the DMSO condition? What about the statistics? Fig. 6A: In the calcium free condition, it seems that the two cells that are illustrated depict a telophase. The subcellular organization is obviously different at the end of cell division. Show only one interphase cell as in the top panels. Fig. 6B and C: It is mentioned in the figure legend: "Cells were treated as indicated in (A) and (D)". But the "C" condition is not mentioned anywhere: Does "C" stand for no DMSO or FCF treatment, in the presence of calcium, but under LLO treatment? Likewise, it would be very helpful to indicate in each figure panel whether cells have been treated with LLO or FCF. Please help the reader. Fig. 7A: Whatever SEPT7 is expressed or downregulated, the actin stress fibers are still present. If these cells were not well transfected, replace the images.
    • Concerning the FCF experiments: The FCF cytokinin has been used by many authors to perturb septin dynamics. It induces the stabilization of septin polymers, thus promoting the formation of thick ectopic fibers. It is a potent inducer of septin polymerization and acts as a stabilizer. Fig. 6D: In the Ctr condition, a DMSO condition is needed to visualize the impact of FCF on septin filaments. Does FCF stabilize septins and induce the formation of thick filaments? The SEPT2 image in the FCF condition without LLO is of bad quality (see above remark). Also in Fig. 6D (FCF condition without LLO), the F-actin staining revealed that there are no stress fibers!!??? Usually, the more septins are associated with actin, the thicker stress fibers you get, since septins stabilize actin cables. FCF treatment often induces thick ectopic septin filaments that are not associated with stress fibers (which are therefore lost). Was it the case in all FCF-treated cells? Does FCF treatment really mimic what happens physiologically in the cell? Many off-target effects have been observed with this molecule in non-plant cells.
    • The image quality in Figs 3A and B, and 6A and D needs to be improved regarding the septin staining. In control conditions, septin filaments cannot be clearly distinguished.
    • Fig. 3B: It seems that ANXA2 is overexpressed in LLO-injured cells. Its accumulation level between both conditions should be compared by immunoblot. ANXA2 is indeed recovered on loops, but it is difficult to consider whether it is a redistribution.
    • Fig. 7D: Compared to the control condition (we have to refer to Fig. S5D), ANXA2 again seems to be overexpressed under LLO treatment. To affirm that ANXA2 remodeling in LLO-injured cells requires the formation of septin/F-actin knobs and loops, data in Fig. 7D must be quantified.
    • Fig. 6 (B-D): In panel B, there is a significant difference between the "C" and "FCF" conditions regarding the number of knobs + loops per cell. Where are the images corresponding to the "C" condition?

    Minor comments:

    • Fig. 2A: Report the white squares (selected enlarged areas) in all panels (SEPT2 and overlay). In panels b, do not place an arrowhead where we are supposed to observe an enlarged area. Also, from panels b, it would be worth showing an enlarged area including a knob. Show enlarged areas also from panels d.
    • Fig. 3B'i: Septins are not on stress fibers. Select a transfected cell where septins still coalign with actin fibers, not a cell that was impaired by the transfection.
    • Fig. 3C: Add the time point "0min". What was the % of colocalization before LLO treatment? and in DMSO condition? What about ALIX at 5 and 10min? Again, it's only one experiment.
    • Figs 4 and 5: Very nice images but obtained following FCF exposure. Hopefully FCF would not have induced an aberrant organization!
    • The "Ctr" abbreviation is often used, in different conditions, and may be confusing. Precise in the figure (not in the figure legend) whether it is siRNA ("Ctr siRNA"). Mention "DMSO" for the controls of your drugs (like in Figs S4 and S5).
    • Fig. S4C: How is this figure different or does it provide additional information compared to Fig. 2C?
    • Fig. S5D: It is hard to know that the ANXA2 siRNA worked, since no difference of staining between the Ctr and the transfected cells can be observed. Were these cells really transfected? It would have been helpful to use fluorescent siRNAs. The same applies to Fig. S5C: Silencing SEPT7 supposedly greatly reduces the level of expression of all septins. The SEPT2 staining is still high, and many actin stress fibers are still observable (whereas the loss of septin filaments results in the loss of actin stress fibers, as observed by many authors, including in HeLa cells). Same remark for Fig. S6, regarding the SEPT7 silencing in the Ctr condition (no LLO). No impact on stress fibers! Are these cells transfected? The authors themselves mention that sometimes cells are less effectively silenced (like in Fig. 7A, B). Why not to show cells effectively silenced!!
    • In the abstract, it is specified that SEPT7 also plays a role in membrane repair after mechanical wounding. Based only on one type of experiment (SEPT7 silencing, Fig. 1H), this statement should only be mentioned in the text or used to discuss the putative repair mechanisms that septins are involved in, but not stated in the abstract as a main conclusion.

    Significance

    Strengths:

    Despite septins have been involved in endocytosis, exocytosis, membrane protrusions, cell junction integrity or actomyosin constriction at cytokinesis, the involvement of the septin cytoskeleton in the plasma membrane repair machinery has, to my knowledge, never been reported before. The authors not only showed that septins are present in specific membrane protrusions (knobs and loops) but also evidenced that septin filaments trigger the formation of these plasma membrane repair domains by recruiting F-actin and ANXA2, essential for the repair to occur. The novelty of this study has therefore to be acknowledged, and these data will benefit the scientific community, and the septin community in particular.

    This is a descriptive paper that nevertheless clearly shows, by different means, the reorganization of the septin cytoskeleton in LLO-injured cells. The use of high-resolution microscopy coupled to 3D reconstruction which enables to easily appreciate the organization of septins, F-actin and ANXA2 in the knobs and loops is a true strength of the paper.

    Limitations:

    The authors mention in the abstract that septins act as scaffolds to recruit contractile actin fibers and ANXA2. Biochemical experiments such as co-immunoprecipitations could strengthen this notion. The molecular mechanism by which septins are involved in this repair process has not been addressed at all in the paper. Even though the silencing RNA screen highlighted several proteins involved in known membrane repair mechanisms, the authors just presented a few data concerning ALIX, a component of the ESCRT-III machinery. A % of colocalization of SEPT2 and SEPT7 with ALIX is reported in Fig. 3C but this experiment has only been done once (n=1) and only following 15-min exposure to LLO. Is that too late? Immunofluorescence images of SEPT2 and ALIX with or without LLO (15min) are also provided in Fig. S5A but no quantification is reported. Is it sufficient to say that the ESCRT machinery is not involved?

    My field of expertise:

    Cytoskeleton, Septin, Actin, Microtubule, Signaling pathways

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    Referee #1

    Evidence, reproducibility and clarity

    Review of "The septin cytoskeleton is required for plasma membrane repair" by Prislusky et al.

    Eukaryotic cells rapidly repair damage to their plasma membrane and underlying cortical cytoskeleton. Such repair is increasingly recognized as being of major importance to human health (PMID: 33849525). Two broadly conserved cell damage responses have been described: a very rapid membrane resealing response which commences within a second or so following damage, and a cortical cytoskeletal response which commences within ~15-30s and which is based on activation of the Rho GTPases. However, our understanding of either of these responses is extremely limited, a situation which has engendered considerable debate about not only the mechanistic bases of these responses but also their relative roles and the extent to which they may be interdependent.

    In the current study, the authors use an siRNA screen to identify Septin7 (hereinafter SEPT7) as a critical participant in the cell repair response. They further demonstrate that cell damage, as induced either by bacterial pore-forming proteins or by mechanical abrasion results in accumumulation of septins (including SEPT7) in curious ring-like structures associated at the plasma membranes; these structures are often associated with plasma membrane protrusions, which are a common feature of damaged cells. Additionally, the authors show that the septins colocalize with F-actin, myosin-2 (an F-actin-based motor protein) and annexin-2A, a protein previously implicated in cell repair. Lastly, the authors show that depletion of septins reduces the recruitment of annexin-2A to the plasma membrane in wounded cells, implying that the septins are upstream of the annexin in the wound response.

    This is an exciting study that is also very well-documented. The excitement is provided by the following observations: first, septins have not previously been implicated in cell repair; second, the association of the septins with F-actin and myosin-2 in ring-like structures at the plasma membrane is suggestive of the possibility that local contraction may promote healing, a long-standing idea derived from studies of frog oocyte healing (PMID: 10359696; PMID: 11502762) which has proven controversial for healing of other cell types (see below); third, a link between septins and annexins in cell repair or, for that matter, any other process, is novel. With respect to the support for their claims, the authors go above and beyond to make their case-every point is supported by multiple approaches-for example the importance of septins is shown via siRNA, shRNA, and inducible depletion-and the imaging is very, very nice.

    The potential role for actomyosin-powered contraction in healing of wounds made in cultured mammalian cells has been largely discounted because of studies wherein cells are wounded after pharmacological treatment with actin poisons have shown that healing is actually improved. The problem with such studies, is that depolymerization of actin prior to cell damage will dramatically alter the response to damage due to loss of cortical tension (PMID: 19846787). Thus, besides being important in its own right, the current study opens new doors for experimental assessment of the possible roles for cortical actomyosin in cell repair.

    I have only minor concerns or questions:

    1. What is the spatial relationship between the septin rings and actual damage sites? This could be addressed by wounding in the presence of a lysine fixable dextran.
    2. The information in table 1 could be made more reader-friendly. In particular, it is not clear how the authors are getting their gene/protein names for their hits and what they correspond to. This was most noticeable for IQSEC1, ABI1, and GBF1 which the authors describe in the text as "genes that control the actin cytoskeleton" but in the table are listed as "Signaling proteins". I may have the abbreviations wrong (which is more reason for additional clarity) but GBF1 is the abbreviation for a protein involved in intracellular trafficking; IQSEC1 is a GEF for Arf proteins, and ABI1 is best known as a subunit of the WAVE complex.
    3. The statement that begins the abstract "Mammalian cells are frequently exposted to mechanical and biochemical stresses..." could just as easily be "Eukaryotic cells..." or even "Cells..." as the membrane repair response is apparently universal and, indeed, was first described in nonmammalian cells. Similarly, the introduction begins "The plasma membrane of mammalian cells forms a biophysical barrier that separates the cell from its external environment". As far as I know, this is not a specific feature of mammalian plasma membranes but rather all plasma membranes. I don't know if it is the author's intention to imply their work is only relevant to mammals, but that is certainly not the case and they end up reducing the impact of their work by making it sound like cell repair is a phenomenon specific to mammalian cells.
    4. The word "subplasmalemmal" is likely to be confusing for those who are not aware that plasmalemma is an antiquated term for the plasma membrane. It might be easier for the reader if the authors refer to "subdomains of the plasma membrane".

    Significance

    This is an exciting study that is also very well-documented. The excitement is provided by the following observations: first, septins have not previously been implicated in cell repair; second, the association of the septins with F-actin and myosin-2 in ring-like structures at the plasma membrane is suggestive of the possibility that local contraction may promote healing, a long-standing idea derived from studies of frog oocyte healing (PMID: 10359696; PMID: 11502762) which has proven controversial for healing of other cell types (see below); third, a link between septins and annexins in cell repair or, for that matter, any other process, is novel. With respect to the support for their claims, the authors go above and beyond to make their case-every point is supported by multiple approaches-for example the importance of septins is shown via siRNA, shRNA, and inducible depletion-and the imaging is very, very nice.

    The potential role for actomyosin-powered contraction in healing of wounds made in cultured mammalian cells has been largely discounted because of studies wherein cells are wounded after pharmacological treatment with actin poisons have shown that healing is actually improved. The problem with such studies, is that depolymerization of actin prior to cell damage will dramatically alter the response to damage due to loss of cortical tension (PMID: 19846787). Thus, besides being important in its own right, the current study opens new doors for experimental assessment of the possible roles for cortical actomyosin in cell repair.

  4. Version published to 10.1101/2023.07.12.548547v2 on bioRxiv
  5. Version published to 10.1101/2023.07.12.548547v1 on bioRxiv