Membrane curvature governs the distribution of Piezo1 in live cells
Curation statements for this article:-
Curated by Biophysics Colab
Endorsement statement (6 December 2022)
The preprint by Yang et al. asks how the shape of the membrane influences the localization of mechanosensitive Piezo channels. The authors use a creative approach involving methods that distort the plasma membrane by generating blebs and artificial filopodia. They convincingly show that curvature of the lipid environment influences Piezo1 localization, such that increased curvature causes channel depletion, and that application of the chemical modulator Yoda1 is sufficient to allow channels to enter filopodia. The study provides support for a provocative “flattening model” of Yoda1 action, and should inspire future studies by researchers interested in mechanosensitive channels and membrane curvature.
(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)
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Abstract
Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvaturedependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of Ca 2+ , suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.
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Endorsement statement (6 December 2022)
The preprint by Yang et al. asks how the shape of the membrane influences the localization of mechanosensitive Piezo channels. The authors use a creative approach involving methods that distort the plasma membrane by generating blebs and artificial filopodia. They convincingly show that curvature of the lipid environment influences Piezo1 localization, such that increased curvature causes channel depletion, and that application of the chemical modulator Yoda1 is sufficient to allow channels to enter filopodia. The study provides support for a provocative “flattening model” of Yoda1 action, and should inspire future studies by researchers interested in mechanosensitive channels and membrane curvature.
(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised …
Endorsement statement (6 December 2022)
The preprint by Yang et al. asks how the shape of the membrane influences the localization of mechanosensitive Piezo channels. The authors use a creative approach involving methods that distort the plasma membrane by generating blebs and artificial filopodia. They convincingly show that curvature of the lipid environment influences Piezo1 localization, such that increased curvature causes channel depletion, and that application of the chemical modulator Yoda1 is sufficient to allow channels to enter filopodia. The study provides support for a provocative “flattening model” of Yoda1 action, and should inspire future studies by researchers interested in mechanosensitive channels and membrane curvature.
(This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)
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Authors’ response (5 November 2022)
GENERAL ASSESSMENT
Piezo1 and Piezo2 are stretch-gated ion channels that are critically important in a wide range of physiological processes, including vascular development, touch sensation and wound repair. These remarkably large molecules span the plasma membrane almost 40 times. Cryo-EM and reconstitution experiments have shown that Piezos adopt a cup-like structure and, by doing so, curve the local membrane in which they are embedded. Importantly, membrane tension is a key mediator of Piezo function and gating, an idea well-supported several independent studies. Cells have varied three-dimensional shapes and are dynamic assemblies surrounded by plasma membranes with complex topologies and biochemical landscapes. How these microenvironments influence mechanosensation and Piezo function are unknown.
Authors’ response (5 November 2022)
GENERAL ASSESSMENT
Piezo1 and Piezo2 are stretch-gated ion channels that are critically important in a wide range of physiological processes, including vascular development, touch sensation and wound repair. These remarkably large molecules span the plasma membrane almost 40 times. Cryo-EM and reconstitution experiments have shown that Piezos adopt a cup-like structure and, by doing so, curve the local membrane in which they are embedded. Importantly, membrane tension is a key mediator of Piezo function and gating, an idea well-supported several independent studies. Cells have varied three-dimensional shapes and are dynamic assemblies surrounded by plasma membranes with complex topologies and biochemical landscapes. How these microenvironments influence mechanosensation and Piezo function are unknown.
The current preprint by Zheng Shi and colleagues asks how the shape of the membrane influences Piezo location. The authors use creative approach involving methods to distort the plasma membrane by generating “blebs” and artificial “filopodia”. Overall, the work convincingly shows that the curvature of the lipid environment influences Piezo localization. Specifically, they show that Piezo1 molecules are excluded from filopodia and other highly curved membranes. These experiments are well controlled and the results fully consistent with previous structural and biochemical work. Furthermore, the work explores the hypothesis that a chemical modulator of Piezo1 channels called Yoda1 functions by “flattening” the channels, a movement previously proposed to be linked to mechanical gating. Consistent with this model, the authors show that Yoda1 application is sufficient to allow Piezo1 channels to enter filopodia. While the flattening model is provocative hypothesis, hard evidence awaits structural verification.
Overall, the preprint by Shi and colleagues will be of interest to scientists studying how mechanical forces are detected at the molecular level. The work introduces important concepts regarding how the shape of cellular membranes affects the movement and function of proteins within it. The technical advance for changing the shape of a plasma membrane is of note.
We thank the reviewers for the accurate summary and positive assessments of our manuscript. We address each of the concerns below.
RECOMMENDATIONS
Revisions essential for endorsement:
As is evident from the comments below, our endorsement of the study is not dependent on additional experiments. However, we feel more experimental clarification is needed, that providing clearer images would be helpful, and, most importantly, we would like alternative conclusions and caveats to be mentioned.
1. Can the authors comment on the link between the conclusions that (1) the presence of filopodia prohibits Piezo1 localization (Fig 1) and (2) Piezo1 expression prohibits the formation of filopodia (Fig 3). As it stands, it is hard to understand if there is a cause and effect relationship here or if these are separate, unrelated observations? We recommend revising the discussion to clarify.
We now clarify the link between Piezo1’s curvature sensing (depletion from filopodia) and its inhibition effect on filopodia formation before presenting the current Fig. 5: “Curvature sensing proteins often have a modulating effect on membrane geometry. For example, N-BAR proteins, which strongly enrich to positive membrane curvature, can mechanically promote endocytosis by making it easier to form membrane invaginations (Shi and Baumgart, 2015; Sorre et al., 2012). Thus, we hypothesize that Piezo1, which strongly depletes from negative membrane curvature (Fig. 1, Fig. 2), can have an inhibitory effect on the formation of membrane protrusions such as filopodia.”
2. When comparing the images of Fig. 2A, B to those of Fig. 2C, D, it appears that bleb formation induces a drastic enrichment of Piezo1 in the bleb membrane. Is this due to low membrane tension in the bleb? If this is the case, it indicates that the level of membrane tension has a prominent role in determining the localization of Piezo1.
We apologize for this confusion due to our poor wording and figure presentation in the manuscript. By “Piezo1 clearly locates to bleb membranes” we didn’t mean to indicate that Piezo1 is enriched on bleb membranes as compared to the cell body. Rather, we meant to emphasize Piezo1’s localization to the *membrane* of the blebs rather than in the cytosolic space.
Cells in 2C, 2D are different from that in 2A and 2B and were presented with different image contrasts. We now include the images of the full cell for Fig. 2C and 2D as the current Figure S8. To focus on the equator of the bleb, the cell body was out of focus. However, there is no indication that Piezo1 density is significantly different between the bleb membrane and the intact parts of the plasma membrane.
We changed the main text to: “Similar to previous reports (Cox et al., 2016), bleb membranes clearly contain Piezo1 signal, but not significantly enriched relative to the cell body (Fig. 2C, 2D; Fig. S8).”
In line with this, it appears more Piezo1 proteins are localized in less tensed tethers. Thus, might your observations be equally consistent with tension rather than curvature as a key regulator of Piezo1 localization? We recommend adding this to your discussion.
We now explain the deconvolution between tension and curvature effects in detail. We also performed additional experiments to quantify the membrane tension in cells and blebs (current Fig. S9).
In the Results section, we add: “Tethers are typically imaged > 1 min after pulling, whereas membrane tension equilibrates within 1 s across cellular scale free membranes (e.g., bleb, tether) (Shi et al., 2018). Therefore, the sorting of Piezo1 within individual tension-equilibrated tether-bleb systems (Fig. 2C – 2G) suggests that membrane curvature can directly modulate Piezo1 distribution beyond potential confounding tension effects.”
In the Discussion section, we add: “In addition to membrane curvature, tension in the membrane may affect the subcellular distribution of Piezo1 (Dumitru et al., 2021). Particularly, membrane tension can activate the channel and potentially change Piezo1’s nano-geometries. This tension effect is unlikely to play a significant role in our interpretation of the curvature sorting of Piezo1 (Fig .2): (1) HeLa cell membrane tension as probed by short tethers (Fig. S9F; 45 ± 29 pN/ µm on blebs and 270 ± 29 pN/ µm on cells, with the highest recorded tension at 426 pN/ µm) are significantly lower than the activation tension for Piezo1 (> 1000 pN/µm (Cox et al., 2016; Lewis and Grandl, 2015; Shi et al., 2018; Syeda et al., 2016)). (2) With more activated (and potentially flatten) channels under high membrane tension, one would expect a higher density of Piezo1 on tethers pulled from tenser blebs. This is the opposite to our observations in Fig. 2C - 2G, where Piezo1 density on tethers was found to decrease with the absolute curvature, thus tension (eq. S6), of membrane tethers.”
3. Given the intrinsically curved structure of Piezo1, it is difficult to understand the model’s prediction that curved Piezo1 is not enriched in 25-75 nm invaginations. Where will Piezo1 normally reside in the plasma membrane? It would be helpful if this could be discussed.
The spontaneous curvature from our model _C_0 (_C_0-1 = 83 ± 17 nm, the value is updated after refitting to more data points collected for Fig. 2G) represents a balance between the intrinsic curvature of Piezo1 trimers (0.04 ~ 0.2 nm-1 as suggested by CryoEM studies(Haselwandter et al., 2022; Lin et al., 2019; Yang et al., 2022)) and that of the associated membrane (0 nm-1, assuming lipid bilayers alone do not have an intrinsic curvature). We now refer to _C_0 as the “spontaneous curvature of the Piezo1-membrane complex” throughout the manuscript, rather than the “spontaneous curvature of Piezo1”.
Our model, when extrapolated to membrane invaginations, predicts a weak enrichment of Piezo1 on ~100 nm invaginations (peak at 83 nm), but a depletion of Piezo1 on more highly curved invaginations. This is simply because it would be energetically costly to fit a protein-membrane complex to a curvature that is different from what the complex prefers (in the case of 25-75 nm membrane invaginations, the membrane curvature would be too high for the Piezo1-memrbane complex).
However, it is worth pointing out that Piezo1-membrane complex may not present the same spontaneous curvature on positively and negatively curved membranes. More importantly, we do not yet have direct evidence to show that this depletion indeed happens in the exact range of invagination curvature we predicted. We now acknowledge this limitation in the Discussion section: “However, it is worth noting that we assumed a zero spontaneous curvature for membranes associated with Piezo1 and that the spontaneous curvature of Piezo1-membrane complex is independent of the shape of surrounding membranes. These assumptions may no longer hold when studying Piezo1 in highly curved invaginations or liposomes (Lin et al., 2019).”
We also took this opportunity to verify the key prediction from the extrapolated model - that Piezo1 would enrich towards ~ 100 nm radius cell membrane invaginations. To achieve this, we utilized a recent development in nanotechnology, pioneered by Wenting Zhao and Bianxiao Cui’s labs (Lou et al., 2019; Zhao et al., 2017). An illustration of the experimental design and detailed findings are summarized in the current Fig. 3 and briefly discussed below.
In collaboration with Wenting Zhao’s lab, we cultured cells on precisely engineered nanobars with curved ends and flat central regions. For a labelled membrane protein of interest, the end-to-center fluorescence ratio would report the protein’s curvature sorting ability. We find that Piezo1 enriches to the curved ends of nanobars, whereas membrane marker signals are homogeneous across the entire nanobar (Fig. 3). The finding achieved strong statistical significance via hundreds of repeats on nanobars of the exact same geometry, a major technical strength of our chosen system. Furthermore, the enrichment of Piezo1 was observed on nanobars with 3 different curvatures (corresponding to diffraction-limited radii between 100 to 200 nm) and qualitatively agrees with our model (current Fig. S10). While further investigations on a wider range of membrane curvature are required to fully map out the sorting of Piezo1 on membrane invaginations, our data in the current Fig. 3 clearly verifies the prediction that membrane curvature can lead to enrichment of Piezo1 on cellular invaginations.
We now refer to this new finding in the Abstract, along with the previously observed depletion of Piezo1 on filopodia. We present a detailed description of the experiment and associated findings in the Results and the Method sections.
4. It is currently unknown whether and how long Yoda1 might keep Piezo1 in a flattened state. Given that Yoda1 is highly hydrophobic, it might affect membrane properties instead of the curvature of Piezo1. These caveats should be discussed.
We thank the reviewers for pointing out the potential effect of Yoda1. We did additional experiments to confirm that on Piezo1-KO cells, Yoda1 molecules alone do not significantly alter the formation of filopodia, in contrast to observations in WT cells. This data suggests Yoda1 (at the concentration we use) is unlikely to significantly alter the mechanical properties of the plasma membrane. The data is now presented as Fig. 5E in the updated manuscript. We added: “In Piezo1 knockout (Piezo1-KO) cells, adding Yoda1 to the culture medium does not significantly change the number of filopodia (Fig. 5E), suggesting the agonist does not directly regulate filopodia formation without acting on Piezo1.”
5. The authors state that “Yoda1 leads to a Ca2+ independent increase of Piezo1 on tethers”. It has not been determined yet that Yoda1 leads to Piezo1 flattening (or even opening). In Electrophysiology experiments, unless there is pressure applied, Yoda1 does not lead to substantial currents. Therefore, the cartoon of Yoda1 flattening Piezo1(3H) is misleading. We recommend revising this. So far, the best experimental evidence on flattening is via purified channels reconstituted in various sizes of liposomes. However, it is plausible that the flattened shape is closed or open inactivated. Because most of the claims of this paper depend on the curved vs flattened shape of Piezo1, the authors should address these caveats carefully.
We thank the reviewers for pointing out the limitations in our current understanding of Yoda1. We agree that our data do not directly show the flattening of Piezo1 by Yoda1, rather it is consistent with the flattening hypotheses. We lowered the tone of our conclusion to Fig. 4 to: “Our study suggests this conformational change of Piezo1 may also happen in live cells (Fig. 4H).” We also added arrows in Fig. 4H to suggest that membrane tension helps the proposed flattening of Piezo1 by Yoda1.
We think our experiment may also provide new insights on the action of Yoda1: First, we note that only a small fraction of filopodia responded to Yoda1, and pre-stressing of the cell membrane was required to amplify the Yoda1 effect (current Fig. 4E). This observation is consistent with the reviewers’ notion that membrane tension is likely required to flatten Piezo1, even in the presence of Yoda1. Secondly, highly curved liposome or detergents can confine the shape of Piezo1 trimers. Therefore, the inability to observe Yoda1-induced flattening of Piezo1 in small liposomes is not necessarily in contradiction with our observation in the mostly flat cell membranes.
We add to the Discussion section: “Yoda1 induced flattening of Piezo1 has not been directly observed via CryoEM. Our results (Fig. 4) point to two challenges in determining this potential structural change: (1) Yoda1 induced changes in Piezo1 sorting is greatly amplified after pre-stretching the membrane (Fig. 4E), pointing to the possibility that a significant tension in the membrane is required for the flattening of Yoda1-bound Piezo1. (2) Piezo1 is often incorporated in small (< 20 nm radius) liposomes for CryoEM studies. The shape of liposomes can confine the nano-geometry of Piezo1 (Lin et al., 2019; Yang et al., 2022), rendering it significantly more challenging to respond to potential Yoda1 effects. This potential effect of membrane curvature on the activation of Piezo1 would be an interesting direction for future studies.”
6. Page 9: "Our study shows this conformational change of Piezo1 in live cells (Fig. 3H)." We recommend that this claim be removed as it seems too strong for the provided data.
We changed the sentence to: “Our study suggests this conformational change of Piezo1 may also happen in live cells (Fig. 4H).”
Additional suggestions for the authors to consider:
1. Based on the calculated spontaneous curvature of Piezo1-membrane C0 of 87 nm, is it possible to derive the curvature of Piezo1 protein itself and the associated membrane footprint? This would be a nice addition.
It is possible to do such an estimation, however, many (unverified) assumptions must be made, in addition to the ones already in our model. First, we need to assume a size of the Piezo1 trimers and of the Piezo1-membrane complex. If we assume Piezo1 trimers are ~170 nm2 in the plane of lipid bilayers (based on estimates from PDB) and that the complex takes on the shape of a 10 -20 nm radius half-sphere. Effectively, Piezo1 occupies an area fraction of 6.7%~27% in the Piezo1-membrane complex. Next, we assume that the membrane and the Piezo1 trimer have the same bending rigidity. Finally, we assume that the membrane itself does not have an intrinsic curvature.
With those assumptions, the intrinsic curvature of Piezo1 trimers (_C_p) would relate to the spontaneous curvature of membrane-Piezo1 complex (_C_0) following: _C_p-1 = _C_0-1 * (6.7%~27%). Knowing _C_0-1 = 83 ± 17 nm, we get _C_p-1 = 5.6 nm ~ 22.4 nm.
2. It is hard to see the filopodia and their localization in the figures. It would be better for readers and more convincing if clearer/higher resolution example images could be provided.
We now provide high resolution figures.
3. Can the authors better explain how the calculations done in panel 1C and S3D are done and their importance?
Each fluorescence trace along the drawn yellow line was normalized to the mean intensity on the corresponding flat cell body, so that the average fluorescence of the cell body has a y-axis value of 1. We think the intensity traces are important because image contrast can be adjusted, therefore Fig. 1A alone would not convincingly show that there are no Piezo1 on filopodia.
4. In Figure 2E, are these data from hPiezo1 or mPiezo1? In other cases, hPiezo1 is specified, this this may be a typo?
Corrected.
5. Figure 3 F&G: We assume these cells are the same in all panels, just visualized with either mCherry or eGFP in each condition. Accordingly, we would have expected more swelling in hypotonic conditions, and wonder if further evaluation may resolve this apparent discrepancy? If not, please provide more clarification.
This is a good point. Indeed, we do observe a significant swelling of the cell right after the hypotonic shock.
However, this effect is expected to be transient (volume of the cell would recover after ~ 1 min), see Figure. 1C here: https://www.pnas.org/doi/10.1073/pnas.2103228118. Our images in Fig. 3F and 3G were taken ~10 min after the hypotonic shock.
6. On a lighter note, we’d recommend not using in cellulo.
We changed in cellulo to “in live cells”
Reference List
Cox, C.D., Bae, C., Ziegler, L., Hartley, S., Nikolova-Krstevski, V., Rohde, P.R., Ng, C., Sachs, F., Gottlieb, P.A., and Martinac, B. (2016). Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension. Nature Communications 7, 1-13.
Dumitru, A.C., Stommen, A., Koehler, M., Cloos, A., Yang, J., Leclercqz, A., Tyteca, D., and Alsteens, D. (2021). Probing PIEZO1 Localization upon Activation Using High-Resolution Atomic Force and Confocal Microscopy. Nano Letters 21, 4950-4958.
Haselwandter, C.A., MacKinnon, R., Guo, Y., and Fu, Z. (2022). Quantitative prediction and measurement of Piezo's membrane footprint. bioRxiv
Lewis, A.H., and Grandl, J. (2015). Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension. Elife 4, e12088.
Lin, Y., Guo, Y.R., Miyagi, A., Levring, J., MacKinnon, R., and Scheuring, S. (2019). Force-induced conformational changes in PIEZO1. Nature 573, 230-234.
Lou, H., Zhao, W., Li, X., Duan, L., Powers, A., Akamatsu, M., Santoro, F., McGuire, A.F., Cui, Y., and Drubin, D.G. (2019). Membrane curvature underlies actin reorganization in response to nanoscale surface topography. Proceedings of the National Academy of Sciences 116, 23143-23151.
Shi, Z., and Baumgart, T. (2015). Membrane tension and peripheral protein density mediate membrane shape transitions. Nature Communications 6, 1-8.
Shi, Z., Graber, Z.T., Baumgart, T., Stone, H.A., and Cohen, A.E. (2018). Cell membranes resist flow. Cell 175, 1769-1779. e13.
Sorre, B., Callan-Jones, A., Manzi, J., Goud, B., Prost, J., Bassereau, P., and Roux, A. (2012). Nature of curvature coupling of amphiphysin with membranes depends on its bound density. Proceedings of the National Academy of Sciences 109, 173-178.
Syeda, R., Florendo, M.N., Cox, C.D., Kefauver, J.M., Santos, J.S., Martinac, B., and Patapoutian, A. (2016). Piezo1 channels are inherently mechanosensitive. Cell Reports 17, 1739-1746.
Yang, X., Lin, C., Chen, X., Li, S., Li, X., and Xiao, B. (2022). Structure deformation and curvature sensing of PIEZO1 in lipid membranes. Nature 1-7.
Zhao, W., Hanson, L., Lou, H., Akamatsu, M., Chowdary, P.D., Santoro, F., Marks, J.R., Grassart, A., Drubin, D.G., and Cui, Y. (2017). Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nature Nanotechnology 12, 750-756.
(This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)
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Consolidated peer review report (26 August 2022)
GENERAL ASSESSMENT
Piezo1 and Piezo2 are stretch-gated ion channels that are critically important in a wide range of physiological processes, including vascular development, touch sensation and wound repair. These remarkably large molecules span the plasma membrane almost 40 times. Cryo-EM and reconstitution experiments have shown that Piezos adopt a cup-like structure and, by doing so, curve the local membrane in which they are embedded. Importantly, membrane tension is a key mediator of Piezo function and gating, an idea well-supported several independent studies. Cells have varied three-dimensional shapes and are dynamic assemblies surrounded by plasma membranes with complex topologies and biochemical landscapes. How these microenvironments influence mechanosensation and Piezo …
Consolidated peer review report (26 August 2022)
GENERAL ASSESSMENT
Piezo1 and Piezo2 are stretch-gated ion channels that are critically important in a wide range of physiological processes, including vascular development, touch sensation and wound repair. These remarkably large molecules span the plasma membrane almost 40 times. Cryo-EM and reconstitution experiments have shown that Piezos adopt a cup-like structure and, by doing so, curve the local membrane in which they are embedded. Importantly, membrane tension is a key mediator of Piezo function and gating, an idea well-supported several independent studies. Cells have varied three-dimensional shapes and are dynamic assemblies surrounded by plasma membranes with complex topologies and biochemical landscapes. How these microenvironments influence mechanosensation and Piezo function are unknown.
The current preprint by Zheng Shi and colleagues asks how the shape of the membrane influences Piezo location. The authors use creative approach involving methods to distort the plasma membrane by generating “blebs” and artificial “filopodia”. Overall, the work convincingly shows that the curvature of the lipid environment influences Piezo localization. Specifically, they show that Piezo1 molecules are excluded from filopodia and other highly curved membranes. These experiments are well controlled and the results fully consistent with previous structural and biochemical work. Furthermore, the work explores the hypothesis that a chemical modulator of Piezo1 channels called Yoda1 functions by “flattening” the channels, a movement previously proposed to be linked to mechanical gating. Consistent with this model, the authors show that Yoda1 application is sufficient to allow Piezo1 channels to enter filopodia. While the flattening model is provocative hypothesis, hard evidence awaits structural verification.
Overall, the preprint by Shi and colleagues will be of interest to scientists studying how mechanical forces are detected at the molecular level. The work introduces important concepts regarding how the shape of cellular membranes affects the movement and function of proteins within it. The technical advance for changing the shape of a plasma membrane is of note.
RECOMMENDATIONS
Revisions essential for endorsement:
As is evident from the comments below, our endorsement of the study is not dependent on additional experiments. However, we feel more experimental clarification is needed, that providing clearer images would be helpful, and, most importantly, we would like alternative conclusions and caveats to be mentioned.
1. Can the authors comment on the link between the conclusions that (1) the presence of filopodia prohibits Piezo1 localization (Fig 1) and (2) Piezo1 expression prohibits the formation of filopodia (Fig 3). As it stands, it is hard to understand if there is a cause and effect relationship here or if these are separate, unrelated observations? We recommend revising the discussion to clarify.
2. When comparing the images of Fig. 2A, B to those of Fig. 2C, D, it appears that bleb formation induces a drastic enrichment of Piezo1 in the bleb membrane. Is this due to low membrane tension in the bleb? If this is the case, it indicates that the level of membrane tension has a prominent role in determining the localization of Piezo1. In line with this, it appears more Piezo1 proteins are localized in less tensed tethers. Thus, might your observations be equally consistent with tension rather than curvature as a key regulator of Piezo1 localization? We recommend adding this to your discussion.
3. Given the intrinsically curved structure of Piezo1, it is difficult to understand the model’s prediction that curved Piezo1 is not enriched in 25-75 nm invaginations. Where will Piezo1 normally reside in the plasma membrane? It would be helpful if this could be discussed.
4. It is currently unknown whether and how long Yoda1 might keep Piezo1 in a flattened state. Given that Yoda1 is highly hydrophobic, it might affect membrane properties instead of the curvature of Piezo1. These caveats should be discussed.
5. The authors state that “Yoda1 leads to a Ca2+ independent increase of Piezo1 on tethers”. It has not been determined yet that Yoda1 leads to Piezo1 flattening (or even opening). In Electrophysiology experiments, unless there is pressure applied, Yoda1 does not lead to substantial currents. Therefore, the cartoon of Yoda1 flattening Piezo1(3H) is misleading. We recommend revising this. So far, the best experimental evidence on flattening is via purified channels reconstituted in various sizes of liposomes. However, it is plausible that the flattened shape is closed or open inactivated. Because most of the claims of this paper depend on the curved vs flattened shape of Piezo1, the authors should address these caveats carefully.
6. Page 9: "Our study shows this conformational change of Piezo1 in live cells (Fig. 3H)." We recommend that this claim be removed as it seems too strong for the provided data.
Additional suggestions for the authors to consider:
1. Based on the calculated spontaneous curvature of Piezo1-membrane C0 of 87 nm, is it possible to derive the curvature of Piezo1 protein itself and the associated membrane footprint? This would be a nice addition.
2. It is hard to see the filopodia and their localization in the figures. It would be better for readers and more convincing if clearer/higher resolution example images could be provided.
3. Can the authors better explain how the calculations done in panel 1C and S3D are done and their importance?
4. In Figure 2E, are these data from hPiezo1 or mPiezo1? In other cases, hPiezo1 is specified, this this may be a typo?
5. Figure 3 F&G: We assume these cells are the same in all panels, just visualized with either mCherry or eGFP in each condition. Accordingly, we would have expected more swelling in hypotonic conditions, and wonder if further evaluation may resolve this apparent discrepancy? If not, please provide more clarification.
6. On a lighter note, we’d recommend not using in cellulo.
REVIEWING TEAM
Reviewed by:
Alec Nickolls, Postdoctoral Fellow, NCCIH, USA: Piezo structure/function, iPSC cell technologies and disease genetics
Ruhma Syeda, Assistant Professor, University of Texas Southwestern Medical Center, USA: Piezo structure/function, lipids, biochemistry, biophysics
Bailong Xiao, Professor, Tsinghua University, China: Piezo structure/function, cryo-EM, ion channel biophysics, molecular genetics
Curated by:
Alex Chesler, Senior Investigator, NCCIH, USA
(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)
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