A mechanosensing mechanism controls plasma membrane shape homeostasis at the nanoscale
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Evaluation Summary:
When a cell undergoes rapid shrinking, excess plasma membrane becomes available. The authors show that excess plasma membrane forms very small bleb-like evaginations that disappear after a few minutes. They show a new role for the I-BAR protein IRSp53 and Arp2/3-dependent actin polymerization which surprisingly leads to the flattening of the bud instead of its growth, as it is the case in filopodial protrusions. This manuscript will be of general interest to cell biologists working on membrane-cortex interactions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.
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Evaluation Summary:
When a cell undergoes rapid shrinking, excess plasma membrane becomes available. The authors show that excess plasma membrane forms very small bleb-like evaginations that disappear after a few minutes. They show a new role for the I-BAR protein IRSp53 and Arp2/3-dependent actin polymerization which surprisingly leads to the flattening of the bud instead of its growth, as it is the case in filopodial protrusions. This manuscript will be of general interest to cell biologists working on membrane-cortex interactions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors have shown in a previous paper (ref 20) that relaxation of the plasma membrane after mechanical stretching induces the formation of different membrane structures that transiently accommodate the excess area, among them small membrane evaginations of typically 100 nm diameter on the apical side of the cells. The stretching technique was already described in (20). They focus here on the mechanism of the active re-absorption process of these small buds. Using fluorescence microscopy, correlative fluorescence/SEM microscopy and TEM, they show here that the evaginations are actively re-adsorbed in an actin-dependent manner. They evidence that the I-BAR domain protein, which was previously shown to be a curvature sensor is very important for the flattening of these evaginations by locally triggering …
Reviewer #1 (Public Review):
The authors have shown in a previous paper (ref 20) that relaxation of the plasma membrane after mechanical stretching induces the formation of different membrane structures that transiently accommodate the excess area, among them small membrane evaginations of typically 100 nm diameter on the apical side of the cells. The stretching technique was already described in (20). They focus here on the mechanism of the active re-absorption process of these small buds. Using fluorescence microscopy, correlative fluorescence/SEM microscopy and TEM, they show here that the evaginations are actively re-adsorbed in an actin-dependent manner. They evidence that the I-BAR domain protein, which was previously shown to be a curvature sensor is very important for the flattening of these evaginations by locally triggering actin polymerization in a Rac1/Arp2/3-dependent manner. By using different constructs, they show that the I-BAR and SH3 domains are key, for the recruitment in the curved structures and for the downstream actin polymerization, respectively. They also show that the re-absorption process is independent of Myo2, formins and N-WASP.
Concluding that IRSp53 is a mechanosensor is not so original: it was already proposed for the mechanism of filopodia generation by G. Scita (A. Disanza et al., EMBO J. 32, 2735 (2013)). It is rather a "curvature-sensor" to be more precise, as already shown in vitro as mentioned by the authors. However, the originality of this work is to show that IRSp53 is involved in the resorption of a protrusion rather than on its growth as observed in filopodia for instance. I think this result brings novel insights on the already-rich range of functions in which this protein is involved. It also shows that this process involves branched actin and not linear actin bundles like in filopodia.
To explain how resorption happens, the authors develop a theoretical model based on a composite membrane made of an active gel layer (the cortex) with a frictional coupling to the lipid bilayer. They propose that IRSp53 is recruited to the membrane buds due to its affinity for curved membranes. It locally nucleates branched actin polymerization, with induces a gradient of actin density, and thus a lateral actin flow away from the bud. Due to the frictional coupling between the membrane and the cortex, the evagination eventually vanishes. This is an interesting mechanism, but so far, the authors do not have considered other possibilities, neither ways to test the model, beyond the fact that this mechanism does not require Myo2 in agreement with the experiments.
On the same line, the authors do not discuss why resorption still occurs, although less efficiently, when IRSp53 is silenced or absent.
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Reviewer #2 (Public Review):
Quiroga et al investigate what happens with the excess plasma membrane that becomes available when a cell undergoes rapid shrinkage / compression after a phase of stretching. Using fluorescence and electron microscopy they find that apically small bottle-like evaginations form and disappear again after a few minutes. They find that, in analogy to much larger membrane blebs, ezrin and actin get recruited to the evaginations before they disappear. As a possible mechanism, they investigate the involvement of IRSP53 as a putative sensor of membrane curvature. They use IRSP53 deficient fibroblasts, which show a delayed resolution of evaginations, as a platform and rescue with different established deletion constructs and results suggest that Rac1 and is downstream of curvature sensing. This is confirmed by …
Reviewer #2 (Public Review):
Quiroga et al investigate what happens with the excess plasma membrane that becomes available when a cell undergoes rapid shrinkage / compression after a phase of stretching. Using fluorescence and electron microscopy they find that apically small bottle-like evaginations form and disappear again after a few minutes. They find that, in analogy to much larger membrane blebs, ezrin and actin get recruited to the evaginations before they disappear. As a possible mechanism, they investigate the involvement of IRSP53 as a putative sensor of membrane curvature. They use IRSP53 deficient fibroblasts, which show a delayed resolution of evaginations, as a platform and rescue with different established deletion constructs and results suggest that Rac1 and is downstream of curvature sensing. This is confirmed by dominant negative Rac1 constructs. Quantitative imaging and APEX based electron microscopy provide evidence that the IRSP53 acts locally at the evaginations, not globally, e.g. at lamellipodia or filopodia.
Finally the authors demonstrate that Arp2/3 mediated polymerisation of actin is required for the effect and suggest a model where in plane polymerisation of actin generates friction with the membrane that flattens out the local curvature.
This is an interesting, well executed study that might be relevant in many different physiological settings. For example, when cells get passively deformed in motile metazoans or when they actively deform upon contraction and migration. Membrane evaginations did get less attention in this context than invaginations and the suggested mechanism is plausible and will trigger future studies that sort out the biophysics and physiological relevance in more detail. At this stage the local action of actin in the context is completely hypothetical.
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Reviewer #3 (Public Review):
The study by Quiroga et al. explores an interesting new mechanism used by cells to repair nanoscale outward membrane deformations (i.e. evaginations) that form upon rapid drop in membrane area. Using a combination of fluorescence and electron microscopy, the authors show that formation of these membrane evaginations, which are approximately 150nm in height and 100nm in diameter, cause recruitment of IRSP53 that locally augments Rac and Arp2/3 dependent actin polymerization to flatten and reconnect these membrane folds to the cell cortex.
To quantify the proposed mechanism, the authors use wild-type cells upon knockdown of IRSP53 as well as on IRSP53-/- cells. Reabsorption dynamics of membrane evagination is being measured for several proteins involved in the mechanism, as well as for a set of mutant and …
Reviewer #3 (Public Review):
The study by Quiroga et al. explores an interesting new mechanism used by cells to repair nanoscale outward membrane deformations (i.e. evaginations) that form upon rapid drop in membrane area. Using a combination of fluorescence and electron microscopy, the authors show that formation of these membrane evaginations, which are approximately 150nm in height and 100nm in diameter, cause recruitment of IRSP53 that locally augments Rac and Arp2/3 dependent actin polymerization to flatten and reconnect these membrane folds to the cell cortex.
To quantify the proposed mechanism, the authors use wild-type cells upon knockdown of IRSP53 as well as on IRSP53-/- cells. Reabsorption dynamics of membrane evagination is being measured for several proteins involved in the mechanism, as well as for a set of mutant and deletion constructs of IRSP53. The membrane structure and protein localization at these membrane folds is visualized using SEM, CLEM and APEX. The link from IRSP53 to its actin-regulatory binding partners is being explored using IRSP53 mutants, Rac mutants and chemical perturbations. These experimental data is complemented by a nice mathematical mode that further strengthens the feasibility of the proposed mechanism.
The manuscript is well structured and clearly written. The systematic analysis presents a compelling set of experiments that support the main findings of the manuscript.
Following aspect could benefit from some revisions: The authors show that membrane evagination are still reabsorbed upon knockdown of IRSP53 and in IRSP53-/- cells. If the proposed homeostatic mechanism is mediated solely by IRSP53, as the title implies, this should not be the case. Considering their redundant function, additional I-BAR domain proteins may contribute to this mechanism as well. To address this possibility, the authors would need to monitor the localization of other candidate proteins (e.g. MTSS1, MTSS2, IRTKS) at membrane evagination. Optionally, protein function could be tested via knockdown to delineate the contribution of individual I-BAR domain proteins.
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