Figures and data
Piezo1 is depleted from filopodia.
(A) Fluorescence images of HeLa cells co-expressing hPiezo1-eGFP (green) and GPI-mCherry (magenta). The two boxed regions (A1, A2) are merged and contrast-adjusted on the right. (B) Fluorescence images of HeLa cells co-expressing GPI-eGFP (green) and mOrange2-CaaX (magenta). The two boxed regions (B1, B2) are merged and contrast-adjusted on the right. All scale bars are 10 µm. (C) Fluorescence intensity profiles along the marked yellow lines in A1 (up) and B2 (down). Green: hPiezo1 (up) and GPI (down). Magenta: GPI (up) and CaaX (down). (D) Filopodia sorting of eGFP fused hPiezo1 (T22: at 22 °C, n.f. = 129, n.c. = 12; T37: at 37 °C: n.f. = 113, n.c. = 8), CaaX (n.f. = 87, n.c. = 9), and D2R (n.f. = 222, n.c. = 15) relative to GPI-mCherry. C.S.: color swap, indicating the molecule of interest was fused with mCherry (or mOrange2 for CaaX) while the reference was GPI-eGFP. hPiezo1-C.S.: n.f. = 24, n.c. = 4; mP1-C.S. (mouse Piezo1): n.f. = 47, n.c. = 8; CaaX-C.S.: n.f. = 123, n.c. = 9; TREK1-C.S.: n.f. = 73, n.c. = 12. n.f.: number of filopodia, n.c.: number of cells. (E) Filopodia radii of cells co-expressing GPI and hPiezo1 (n = 266), DRD2 (n = 222), or CaaX (n = 210). All radii were determined from the GPI channel. All quantifications were done in HeLa cells except the sorting of TREK-1 was measured in HEK293T cells. p values given by one-way ANOVA with post hoc Tukey’s test. ***p < 10−7.
Sorting of Piezo1 on membrane tethers.
(A) Fluorescence images of a HeLa cell co-expressing hPiezo1-eGFP (left) and GPI-mCherry (middle). The transmitted light image (right) shows the position of a motorized micropipette (fused) in contact with the cell before tether pulling. (B) Fluorescence images of the HeLa cell in (A) after a 20 µm tether was pulled out (arrow). The tether region is merged and contrast-adjusted on the right. (C, D) Fluorescence images of tethers pulled from membrane blebs on HeLa cells co-expressing hPiezo1-eGFP (left) and GPI-mCherry (middle). Merged images shown on the right. Significantly less Piezo1 signals were observed on the tether from tense bleb (C) compared to the tether from floppy bleb (D). All fluorescence images here are shown in log-scale to highlight the dim tether. All scale bars are 5 µm. (E) Sorting of Piezo1 on tethers pulled from cell membranes (Cell, n = 31) and membrane blebs (Bleb, n = 28) relative to GPI. Filopodia sorting of Piezo1 (n = 313) and CaaX (n = 210) from Fig. 1D are shown here for comparison. (F) Radii of tethers pulled from cell membranes (Cell) and membrane blebs (Bleb, converted to absolute tether radii) co-expressing GPI and hPiezo1. Radii of filopodia on cells co-expressing GPI and hPiezo1 or CaaX (Fig. 1E) are shown here for comparison. All radii were determined from the GPI channel. (G) Sorting of hPiezo1 on tethers pulled from blebs (black) plotted as a function of the apparent (lower axis) and absolute (upper axis) radii of the tethers. Sorting of hPiezo1 on tethers pulled from cells are shown in gray. The fraction of ‘outside-out’ Piezo1 when reconstituted into small liposomes (according to ref10) is shown in blue. The red circle shows the cluster of tense bleb data used to calculate the conversion factor for tether radius (361 ± 61 nm/A.U.). The radius of the thickest tether was converted to 242 nm, consistent with the directly measured upper limit of tether radii (264 nm; Fig. S6). The solid line is a two-parameter fit (R2 = 0.85) with the shaded area representing the 95% confidence interval. Insect: Sorting of Piezo1 as a function of bleb radius, where the line represents a linear fit with slope = 0.005 ± 0.017 µm−1. Error bars are SEM. p values given by one-way ANOVA with post hoc Tukey’s test. ***p < 10−7.
Activation via Yoda1 leads to increased sorting of Piezo1 on filopodia, independent of Ca2+.
(A) Left: fluorescence images of a HeLa cell (see Fig. S9A for the full cell) co-expressing GPI-mCherry (up) and hPiezo1-eGFP (down). Right: 10 min after adding 10 µM Yoda1 to the cell on the left. (B, C) Quantifications of hPiezo1 sorting on filopodia (B) and filopodia radii (C) for the cell in A. p values given by paired Student’s t test, **p <10−3. (D) Sfilo plotted as a function of filopodia radius before (red) and after (black) adding Yoda1. The black line is a one-parameter fit of the +Yoda1 data to equation (1) with fixed 
Piezo1 inhibits filopodia formation.
(A) Fluorescence images of HeLa cells co-expressing GPI-mCherry (up) and hPiezo1-eGFP (down). (B) Relation between the number of filopodia and hPiezo1-eGFP expression level in HeLa cells (n = 129). Dash line represents the average number (118) of filopodia per cell. Solid line represents a linear fit (Pearson’s r = -0.13). p value given by Student’s t test. (C) Relation between the number of filopodia and hPiezo1-eGFP expression level in HEK293T cells cultured in regular (black, n = 52) and 5 µM Yoda1 containing (red, n = 50) media. Lines are linear fits between y and log(x). Without Yoda1 (black): slope = -21.5 ± 4.0, Pearson’s r = -0.61. With Yoda1 (red): slope = -2.0 ± 5.0, Pearson’s r = -0.06. (D) Fluorescence images of HEK293T cell co-expressing GPI-mCherry (up) and hPiezo1-eGFP (down). Cells are arranged so that the expression level of hPiezo1-eGFP increases from left to right, the number of filopodia per cell decreases correspondingly. All fluorescence images here are shown in log-scale to highlight the filopodia. All scale bars are 10 µm.
Fluorescence images corresponding to Fig. 1D
Fluorescence images of HeLa cells co-expressing: (A) hPiezo1-mCherry (magenta) and GPI-eGFP (green); (B) mPiezo1-mCherry (magenta) and GPI-eGFP (green); (C) eGFP-CaaX (green) and GPI-mCherry (magenta); (D) D2R-eGFP (green) and GPI-mCherry (magenta). (E) Fluorescence images of HEK-293T cells co-expressing mTREK1-mCherry (magenta) and GPI-eGFP (green). The boxed regions are merged and contrast-adjusted on the right. All scale bars are 10 µm.
Calculation of membrane curvature sorting and filopodia/tether radii
(A) Fluorescence images of a HeLa cell in the GPI-mCherry channel (intensity in log scale), with the boxed region enlarged in (B). (B) Left: ROIs for each tether/filopodium boxed in yellow, flat regions on the cell body boxed in blue. Right: two background regions for each ROI boxed in yellow. (C) Left: ROIs for a tether (yellow) and for a bleb (blue). Right: two background regions corresponding to each ROI on the left. (D) Illustration to show the imaged regions of a cylindrical tether/filopodium (left) and a flat cell membrane (right). Illumination profile shown in blue.
Depletion of Piezo1 from filopodia of HEK293T cells
(A-C): Transmitted light (left), hPeizo1-eGFP fluorescence (middle), and GPI-mCherry (right) images of HEK293 cells. (D) Fluorescent intensity along the two yellow lines shown in (A). (E) log-fluorescence of (A). All scale bars are 10 μm.
Curvature sensitivity of Pieoz1 and D2R on filopodia
(A) Sorting of hPiezo1 does not change with filopodia curvature (black line: linear fit with slope 0.05 ± 0.51 nm), Person’s r value = 0.009. (B) Sorting of DRD2 increases with filopodia curvature (black line: linear fit with slope 4.83 ± 0.86 nm), Person’s r value = 0.35.
Sorting of Piezo1 does not change with the relaxation of tether radius.
Fluorescence images of a HeLa cell co-expressing GPI-mCherry (left) and hPiezo1-eGFP (right), 1 min after pulling tether (A) and 12 min after pulling tether (B). Arrows point to the tether. The sorting of hPiezo1 was (0.010 ± 0.010) 1 min after tether pulling and was (-0.004 ± 0.011) 12 min after tether pulling, while the tether radius changed from (17.19 ± 0.05) nm to (20.33 ± 0.06) nm. Representative of 5 tethers and cells. All fluorescence images here are shown in log-scale to highlight the dim tether. All scale bars are 10 µm. Change of tether radius (C) and Piezo1 sorting on tethers (D) (1min vs. 6∼12 min after tether pulling). Bar plot shows mean + S.D., p values given by paired Student’s t test.
Upper limit of the tether radii from blebs
Left: fluorescence image of the thickest tether pulled from a bleb (Fig. 2D). The shape of the tether gradually changes from cylindrical to catenoid-shape at the connections of the tether to the pulling handle and to the bleb, consistent with the behavior of low-tension membrane tubes 46. The measured apparent radius of the tether was (0.671 ± 0.003) A.U., which converts to an absolute diameter of 485 ± 82 nm. The conversion factor (361 ± 61 nm/A.U.) was determined by assuming that the average radius of filopodia and equilibrated tethers from cell body equal to the radii of tethers from tense blebs (Fig. 2G). Three yellow lines marked on the image: 1, a line across the tether-pipette junction; 2, a line across the majority of the tether (used for determining the apparent radius the tether); 3, a line across the tether-bleb junction.
Right: normalized fluorescence intensity profiles along the three lines marked on the left. The majority of the tether radius was within optical resolution (blue), while a peak-to-peak distance of 528 nm was measured from the fluorescence across the tether-pipette junction (black), serving as an upper limit of the absolute diameter for this tether. A peak-to-peak distance of 1047 nm was measured from the fluorescence across the tether-bleb junction (red).
Measurements of cell membrane bending stiffness
(A) A HeLa cell expressing hPiezo1-eGFP (up) and GPI-mCherry (down), with a 15 μm tether pulled by an optically trapped 4.5 μm diameter bead. Left: focus on the tether. Right: focus on the cell body. (B) Transmitted light image of the optically trapped bead that was used for calculating tether pulling force. All scale bars are 5 μm. (C) Time dependent tether pulling force after stretching the tether at t = 10 s. Images in (A) were taken during the period shaded in red, from which a 26.3 nm tether radius (based on GPI fluorescence) was determined. The gray area was used to calculate the equilibrated pulling force (30.5 pN). (D) Experiments of equilibrium tether pulling force vs. inverse tether radius repeated on 9 tethers pulled from 6 independent cells. The red line is a linear fit, with a positive intercept (14 ± 5 pN) potentially corresponding to contributions of cytoskeleton attachments and membrane asymmetry. The slope of the fit (351 ± 99 pN·nm) gave the cell membrane bending stiffness 13.6 ± 3.8 kBT.
Modeling the curvature sorting of Piezo1
Sorting of Piezo1 as a function of tube radius (eq. 1) plotted using parameters that correspond to closed Piezo1 (blue, based on data in Fig. 2G) and open/inactivated Piezo1 (orange, based on data in Fig. 3D). Negative Rt corresponds to membrane invaginations. The curvature sorting of a hypothetical ion channel with 1/10 of the area of Piezo1 are plotted in yellow (closed) and purple (open/inactive), showing that the opening of the channel has less significant effect on its curvature sorting if the area is small. The sorting of a hypothetical protein with 1/10 of the area of Piezo1 and 10 times higher spontaneous curvature is plotted in green, showing strong curvature sensitivity to invaginations, similar to that of N-BAR domain containing proteins.
Full images for Fig. 3A, 3F
Kinetics of hPiezo1 on plasma membranes
(A) Change of Piezo1 sorting on filopodia (left) and filopodia radii (right) after adding 100 µM Yoda1. (B) Fluorescence recovery after photobleaching (FRAP) of hPiezo1-eGFP in HeLa cells. Red line: fitting to eq. S8, τ0.5 = 342 ± 4 s, r2 = 0.994. Error bars are standard deviation. Scale bar, 5 μm. (C) Percentage of filopodia that showed strong (Sfilo > 0.3, dark), medium (0.1 < Sfilo < 0.3, light), and weak (Sfilo < 0.1, open) sorting of hPiezo1 10 min and 20 min after adding Yoda1. 10 µM Yoda1 was added after hypotonic shock.