Fig. 2: PIEZO1-HaloTag localization and tracking reveal populations of PIEZO1 with different mobilities.
A Data from TIRF videos of PIEZO1-HaloTag ECs labeled with JF549 HTL and of PIEZO1-tdTomato mLSECs, acquired for 2 min with identical settings. Red trace indicates the average fluorescence intensity of PIEZO1-HaloTag puncta (n = 19 videos from 3 experiments), and gray trace for PIEZO1-tdTomato puncta (n = 20 videos from 3 experiments). Black dashed curves represent exponential fits, with τHT = 38.1 ± 0.15 s and τtdT = 21.5 ± 0.13 s (Mann-Whitney p-value = 5.29 × 10−7, Cohen’s d = − 2.73). B Puncta localization error distributions for JF549-HTL-labeled PIEZO1-HaloTag ECs and PIEZO1-tdTomato mouse fibroblasts (see Methods), imaged with identical settings. C TIRF images of a migrating JF646-HTL-labeled PIEZO1-HaloTag NSC, representative of 3 independent experiments, imaged at 10 fps. The top row shows the full cell, bottom row a zoomed-in region. D Schematic of super-resolution single particle tracking of PIEZO1-HaloTag puncta, with two mobility behaviors: mobile (magenta) and immobile (cyan). E Representative trajectories from 11 immobile and 11 mobile puncta tracked for 10 s, with starting positions normalized to the origin; color indicates different trajectories. F Cumulative distribution functions (CDF) of Single Lag Displacements (SLD). The black dotted curve is experimental data; gray and red curves represent single- and two-component exponential fits, respectively. The inset shows residuals for both fits. G Path length distribution at 5 s from PIEZO1-HaloTag ECs after fixation (14,943 trajectories, 13 videos). The dashed red line indicates the 3-µm cutoff for immobile trajectories. H Path length distribution for live JF646-HTL-labeled PIEZO1-HaloTag ECs (889,971 trajectories, 40 videos). The gray curve represents a fit to a sum of two Gaussian curves; individual curves shown in cyan and magenta. The dashed red line indicates the cutoff discriminating mobile and immobile trajectories. I Mean squared displacement (MSD) for immobile (cyan) and mobile (magenta) puncta. Gray traces represent mean MSD for each video; solid curves represent mean MSD across all videos; black lines are linear fits to data at t < 2 s, dashed black lines show linear extrapolations. Data for panels E-I are from 4 independent experiments. See also Supplementary Movie 4. Data points are shown as mean ± SEM.
