Fig. 3: PIEZO1-HaloTag enables imaging of endogenous PIEZO1 activity with temporal resolution approaching that of patch-clamp electrophysiology.
A TIRF images of ECs labeled with either JF646-BAPTA HTL or JF646 (non-Ca2+-sensitive HTL). B Puncta densities (per µm2) for JF646-BAPTA HTL (blue) and JF646 (black) from images as in A. JF646-BAPTA HTL: Basal, 0.07 ± 0.004 (n = 12); PIEZO1-HaloTag KO, 0.02 ± 0.002 (n = 14); 2 µM Yoda1, 0.22 ± 0.02 (n = 11); JF646: 0.34 ± 0.01 (n = 12). Data from 4 independent experiments. Statistical comparisons: JF646-BAPTA HTL Basal vs. PIEZO1-HaloTag KO (two-sample t test p = 4.01 × 10−11, Cohen’s d = − 4.46), JF646-BAPTA HTL Basal vs. 2 µM Yoda1 (two-sample t-test p = 1.25 × 10−6, Cohen’s d = 2.28) and JF646-BAPTA HTL Basal vs. JF646 (Mann-Whitney p = 3.62 × 10−5, Cohen’s d = 7.72); ***p < 0.005 for all conditions with. C Distributions of JF646-BAPTA puncta intensities for all puncta from 32 untreated, 12 DMSO, and 13 2 µM Yoda1 videos. D Representative background-subtracted fluorescence intensity traces of immobile PIEZO1-HaloTag puncta in TIRF imaging of JF646-BAPTA-labeled PIEZO1-HaloTag ECs treated with DMSO. Right, expanded traces corresponding to the red lines. E All-points amplitude histograms from 30 s traces in (C), shown on a log10 scale. F Representative background-subtracted fluorescence intensity traces of immobile puncta from JF646-BAPTA-labeled PIEZO1-HaloTag ECs treated with 2 µM Yoda1. G All-points amplitude histogram of puncta intensities from the 30-s traces in (F). H Average of all-points amplitude histograms from 21 immobile puncta for untreated, DMSO, or 2 µM Yoda1-treated JF646-BAPTA-labeled PIEZO1-HaloTag ECs. Yoda1 distribution was significantly different with: Yoda1 vs. untreated p = 4.12 × 10−7, Yoda1 vs. DMSO p = 1.61 × 10−7, untreated vs. DMSO p = 0.05 (3 independent experiments, Two-Sample Kolmogorov-Smirnov test). I Representative fluorescence intensity trace of an immobile JF646-BAPTA-labeled PIEZO1-HaloTag punctum imaged at 500 fps. Expanded trace shown on the right. J Representative trajectory and fluorescence intensity trace of a mobile JF646-BAPTA-labeled PIEZO1-HaloTag punctum. Data for panels C-G are from 3 independent experiments. See Supplementary Figs. 8, 9, and 1,0 and Supplementary Movies 7, 8, and 9. Data points are shown as mean ± SEM unless otherwise noted.
