Piezo1 as a force-through-membrane sensor in red blood cells

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Piezo1 is the stretch activated Ca 2+ channel in red blood cells that mediates homeostatic volume control. Here we study the organization of Piezo1 in red blood cells using a combination of super resolution microscopy techniques and electron microscopy. Piezo1 adopts a non- uniform distribution on the red blood cell surface, with a bias towards the biconcave “dimple”. Trajectories of diffusing Piezo1 molecules, which exhibit confined Brownian diffusion on short timescales and hopping on long timescales, also reflect a bias towards the dimple. This bias can be explained by “curvature coupling” between the intrinsic curvature of the Piezo dome and the curvature of the red blood cell membrane. Piezo1 does not form clusters with itself, nor does it co-localize with F-actin, Spectrin or the Gardos channel. Thus, Piezo1 exhibits the properties of a force-through-membrane sensor of curvature and lateral tension in the red blood cell.

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  1. eLife assessment

    This important paper uses advanced imaging approaches to explore how Piezo1 distributes on surface red blood cells. The study provides compelling evidence that this molecule 'reads' the membrane curvature and clear support for the force-through-membrane model of mechanosensation.

  2. Reviewer #1 (Public Review):

    This paper has many strengths that support its conclusions. Specifically, the use of natively expressed Piezo1 engineered to carry the HA tag allowed the authors to explore the distribution of the protein from primary cells isolated from a mouse at native expression levels. Thus, over-expression effects could be avoided. The super-resolution imaging is nicely controlled and convicting in its analysis of the distribution of the channel in 3D. The supporting EM data also supports the findings from fluorescence. Likewise, the theory is convincing in proving a mechanistic reason why the channel distributes into this region of the cell. While the data are quite nice and well analyzed, the paper is lacking in an exploration of what function this distribution of the channel would provide to the cell. Likewise, if this distribution was disturbed, would the red blood cell's behavior change? For example, would calcium signals in response to an osmotic challenge or squeezing change if the channel was not concentrated in the dimple? As it stands now, the paper presents a structural view of the distribution of piezo1 in a primary cell plasma membrane but lacks direct experimental evidence for the mechanism of this concentration or mechanistic insight into the effects of this spatial distribution on red blood cell physiology.

  3. Reviewer #2 (Public Review):

    The manuscript by Vaisey et al investigates the organization of Piezo1 on the surface of mouse red blood cells. The authors found that Piezo1 prefers to distribute within the concave dimple as compared to the convex rim regions of the RBC. Additionally, Piezo1s form individual trimers that do not show an apparent tendency to cluster or interact with cytoskeletal components.

    The manuscript addresses a timely topic regarding the mechanisms underlying the subcellular distribution of Piezo1, a major mammalian mechanosensitive ion channel. The findings regarding the behavior (curvature sensing, lack of clustering) of Piezo1 in live cells potentially have broad implications in biophysics, mechanobiology, and physiology. Overall, I found the manuscript well written. The experimental data collected with super-resolution microscopy and electron microscopy are compelling and of high quality. However, important details of the modeling aspects are unclear and several key control experiments are missing.

  4. Reviewer #3 (Public Review):

    Vaisey et al., 2022 utilize super-resolution and electron microscopy techniques to characterize the distribution of Piezo1 ion channels in red blood cells. Prior theoretical research has proposed that the highly curved Piezo1 conformation may bias the channel localization in cell membranes through a mechanism of curvature coupling (Haselwandter and Mackinnon, 2018). Vaisey et al., 2022 find that Piezo1 channels diffuse in the membrane, are not clustered and that their localization is biased to the highly curved RBC dimple, thus matching the hypothesis of curvature coupling. The findings in this paper advance our understanding of how Piezo1 channel conformation affects its localization. With some exceptions the experiments and analyses are performed carefully and rigorously, and the numbers of biological replicates are sufficient. I find this manuscript exciting.