Peer review process
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.)