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 Ca2+, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In the revised version, we carried out extensive new experiments: 1) We added a new main figure (current Fig. 3), quantitatively demonstrating that Piezo1 enriches to ∽100 nm cellular membrane invaginations, in agreement with the prediction from our model (current Fig. S10). 2) We carried out new experiments (current Fig. 5E) to evaluate the effect of Yoda1 on Piezo1 knockout cells. We show that the number of filopodia per (Piezo1 knockout) cell does not significantly change with adding Yoda1. This confirms that Yoda1 molecules alone (without activating Piezo1) do not directly alter the formation of filopodia. 3) We did new optical trap measurements (current Fig. 5F - 5H), comparing the mechanical forces needed to hold a tether for wild-type vs. hPiezo1 overexpressing HEK293T cells. The hPiezo1-overexpressing cells require significantly higher force to pull/hold a tether. This supports our claim that Piezo1 can mechanically inhibit membrane protrusions. 4) We did new experiments (current Fig. 5I - 5K) and found filopodia formation is inhibited at endogenous Piezo1 expression level. Both WT and Het. MEFs showed significantly less filopodia compared to their Piezo1-KO counterparts. This result suggests that endogenous Piezo1 can already inhibit filopodia formation. We also performed additional control experiments. 1) We measured the filopodia radii of TREK1-expressing cells (in current Fig. 1D, 1E, S6C). 2) We separated the channels in the current Fig. 1A and 1B. 3) We updated the current Fig. S10. 4) We quantified the stiffness of bleb membranes (current Fig. S9). 5) We collected and analyzed more data points for the current Fig. 2E - 2G, Fig. 4B - 4D and clarified the data selection criteria for Fig. 4D. 6) We did negative control (current Fig. S12D), confirming that shear stress applied during washing steps alone does not change the sorting of Piezo1 on filopodia. 7) We carried out confocal and TIRF imaging (current Fig. S1B, S1C) to confirm the plasma membrane localization of hPiezo1-eGFP. 8) We showed Fig. 1A in regular contrast (current Fig. S1A). 9) We measured the sorting of Piezo1 and filopodia radius in MEFs where tdTomato-labelled Piezo1 is expressed at endogenous levels. The results (in current Fig. 1D, 1E, S5) confirm that the depletion of Piezo1 from membrane protrusions also happens at physiological condition. We estimated that the amount of overexpressed Piezo1 in HeLa cells is about 2.5-fold of the endogenous level (Fig. S5C). 10) We modified Fig. 4H.