Figures and data
Piezo1 is depleted from filopodia.
(A) Fluorescence images of HeLa cells co-expressing hPiezo1-eGFP (left) and GPI-mCherry (middle). The two boxed regions (A1, A2) are zoomed-in on the right. Images are contrast-adjusted to show the dim filopodia, see Fig. S1A for images under regular contrast. (B) Fluorescence images of HeLa cells co-expressing GPI-eGFP (left) and mOrange2-CaaX (middle). The two boxed regions (B1, B2) are zoomed-in 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). Each fluorescence trace was normalized to the mean intensity on the corresponding flat cell body. (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 red or orange fluorescent proteins (tdTomato for MEF, mOrange2 for CaaX, mCherry for the rest) while the reference was GPI-eGFP. hPiezo1-C.S.: n.f. = 24, n.c. = 4; mPiezo1-C.S.: n.f. = 47, n.c. = 8; mPiezo1-MEF-C.S.: n.f. = 24, n.c. = 5; CaaX-C.S.: n.f. = 123, n.c. = 9; TREK1-C.S.: n.f. = 77, n.c. = 12. n.f.: number of filopodia, n.c.: number of cells. (E) Filopodia radii of cells co-expressing GPI and hPiezo1 (n.f. = 266), mPiezo1 (n.f. = 71), CaaX (n.f. = 210), D2R (n.f. = 222), or TREK1 (n.f. = 65). All radii were determined from the GPI channel. Data labelled with ‘MEF’ were done in mouse embryonic fibroblasts with Piezo1-tdTomato at endogenous expression level and ‘TREK1’ were measured in HEK293T cells due to TREK1’s poor expression in HeLa cells, all other quantifications were done in HeLa cells. In the box (25% ~ 75%) plots, the mid-lines represent the median and the whiskers represent the 1.5 inter-quartile range (same below). p values given by one-way ANOVA with post hoc Tukey’s test. ***p < 10-7. **p < 10-3.
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-tip) 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. Full images of the cell in (C) and (D) are shown in Fig. S8. (E, F) Sorting of hPiezo1 (E) and radii of tethers (F) pulled from cell membranes (Cell, n = 31 cells) and membrane blebs (Bleb, n = 38 blebs) relative to GPI. Corresponding data of hPiezo1 (n = 266 filopodia) and CaaX (n = 210 filopodia) from Fig. 1D and 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 yellow circle shows the cluster of tense bleb data used to calculate the conversion factor for tether radius. The solid line is a two-parameter fit (R2 = 0.85, n = 69 tethers) with the shaded area representing the 95% confidence interval. Inset: Sorting of Piezo1 as a function of bleb radius (n = 38 blebs), where the line represents a linear fit with slope = −0.007 ± 0.015 μm-1. Error bars are SEM. p values given by one-way ANOVA with post hoc Tukey’s test. ***p < 10-7.
Enrichment of Piezo1 towards the ends of nanobars.
(A) Illustration of a nanobar. (B)l Scanning electron microscopy image of the cell culture substrate with 200, 300, and 400 nm wide nanobars. (C) A representative image of cells cultured on nanobar substrate, expressing hPiezo1-eGFP (left) and stained with CellMask Deep Red (middle). The transmitted light image is shown on the right with the width of nanobars labelled on top. (D) Averaged fluorescence images (up: hPiezo1; down: CellMask Deep Red) of nanobars with widths of 200 nm (left; n = 141 nanobars); 300 nm (middle; n = 167 nanobars); 400 nm (right; n = 155 nanobars). Colormap: blue: low; green: medium; red: high. In the upper right image, the dashed circles and square regions are used to calculate the ‘end’ and ‘center’ signals, respectively, for all nanobar images. (E) Mean intensity profile along the dashed line in (D) for all three nanobar widths. Fluorescence traces are normalized to the intensity at the center of the nanobar. Green: hPiezo1; Magenta: CellMask Deep Red. Error bars are SEM. (F) Scattered plot of the ‘end’ to ‘center’ ratio for all nanobars in both the hPiezo1 channel and the CellMask channel (Mem.). Estimated radius of curvature for the curved ends are labelled on the x-axis. p values given by one-way ANOVA with post hoc Tukey’s test. ***p < 10-7, **p < 10-3. All scale bars are 2 μm.
Yoda1 leads to increased sorting of Piezo1 on filopodia, independent of Ca2+.
(A) Left: fluorescence images of a HeLa cell (see Fig. S11A 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), n = 66 filopodia. p values given by paired Student’s t test, ***p <10-7. (D) Sfilo plotted as a function of filopodia radius before (red triangle) and after (black triangle) adding Yoda1. The black line is a one-parameter fit of the +Yoda1 data to equation (1) with 
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 cells). Dash line represents the average number of filopodia per cell. Solid line represents a linear fit (Pearson’s r = −0.13). (C) Fluorescence images of HEK293T cells coexpressing hPiezo1-eGFP (up) and GPI-mCherry (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. (D) Relation between the number of filopodia and hPiezo1-eGFP expression level in HEK293T cells cultured in regular (black, n = 52 cells) and 5 μM Yoda1 containing (red, n = 50 cells) 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. (E) The number of filopodia per Piezo1-KO MEF incubated with 5 μM Yoda1 (n = 31 cells) or 0.1% DMSO (n = 34 cells). (F) A HEK293T cell co-expressing hPiezo1-eGFP (left) and GPI-mCherry (middle), with a tether (arrow) pulled by an optically-trapped bead (right). (G) Time dependent pulling force for the tether in (F). The tether was stretched at t = 3 s. The gray area was used to calculate the equilibrated pulling force. (H) Equilibrium tether pulling force for HEK293T cells only expressing GPI-eGFP (-Piezo1) or co-expressing hPiezo1-mCherry and GPI-eGFP (+Piezo1). (I) Representative (of the median number of filopodia per cell) fluorescence images of WT (left) and Piezo1-KO (right) MEF, both stained with CellMask Deep Red. MEFs were from littermate embryos that were dissected on the same day. (J) The number of filopodia per cell for WT (n = 38 cells) and Piezo1-KO (P1-KO; n = 36 cells) MEFs from a pair of littermates. Cells were imaged 30 days after dissection (3 passages). (K) The number of filopodia per cell for Het. (n = 32 cells) and P1-KO (n = 89 cells) MEFs from a pair of littermates. Cells were imaged 52 days after dissection (6 passages). All fluorescence images here are shown in log-scale to highlight the filopodia. All scale bars are 10 μm. p values given by two-tailed Student’s ttest after checking for normality. ***p < 10-7 **p < 10-3.