Visualizing PIEZO1 localization and activity in hiPSC-derived single cells and organoids with HaloTag technology

Abstract

PIEZO1 is critical to numerous physiological processes, transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging, our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore, the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca²⁺-sensitive HaloTag ligands, achieving temporal resolution approaching that of patch clamp electrophysiology. Finally, we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems, with potential for elucidating disease mechanisms and targeted drug screening.

Fig. 1. Generation and validation of the PIEZO1-HaloTag hiPSC line.

A Flowchart illustrating PIEZO1-HaloTag CRISPR knock-in in WTC-11 hiPSCs; multiple human cell types differentiated from the PIEZO1-HaloTag hiPSC line; and subsequent HaloTag-ligand probe labeling and imaging. B Structural schematic of the trimeric PIEZO1 channel (Dark blue, PDB: 5Z10) with HaloTag (Cyan, PDB: 5UY1) attached to the cytosolic C-terminus. Dashed gray lines highlight the linker sequence (G-S-G-A-G-A) between PIEZO1 and HaloTag. C Representative traces of cell-attached patch clamp measurements with mechanical stimulation imparted through negative suction pulses for ECs derived from WTC-11, PIEZO1 KO, and PIEZO1-HaloTag hiPSCs. Blue color gradient indicates strength of negative pressure steps associated with suction pulses (light blue: lowest pressure, darkest blue: highest pressure). D Maximal suction-evoked current amplitudes recorded in each condition from 5 independent experiments. All values are expressed as mean ± SEM (WTC-11 mean: − 71 ± 12.1 pA, n = 21; PIEZO1 KO mean: − 10.6 ± 7.5 pA, n = 17; PIEZO1-HaloTag mean: − 60.8 ± 13.4 pA, n = 24) ***p-value < 0.005, Mann-Whitney. WTC-11 compared to PIEZO1 KO p = 8 × 10⁻⁶; PIEZO1-HaloTag compared to PIEZO1 KO p = 2 × 10⁻⁴. Cohen’s d effect sizes are − 1.31 for PIEZO1 KO as compared to WTC-11. WTC-11 and PIEZO1-HaloTag did not show a statistically significant difference (p-value = 0.58) E TIRF images, representative of 3 independent experiments, showing unmodified WTC-11 hiPSCs (left), PIEZO1-HaloTag hiPSCs (middle), and PIEZO1-HaloTag KO hiPSCs (right), all treated with JF646 HTL. F TIRF images, representative of 3 independent experiments, of differentiated PIEZO1-HaloTag EC, keratinocyte, and NSC labeled with JF646 HTL. See also Supplementary Figs. 1, 2, and 3 and Supplementary Movies 1, 2, and 3. Data points are shown as mean ± SEM unless otherwise noted.

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 τ_HT = 38.1 ± 0.15 s and τ_tdT = 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.

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.

Fig. 4. PIEZO1 activity monitored in endothelial and neural stem cells.

A Representative single frame TIRF images of PIEZO1-HaloTag ECs and NSCs labeled with JF646-BAPTA HTL. B JF646-BAPTA HTL puncta density in ECs (mean = 0.23 ± 0.02 puncta per µm², n = 18) and NSCs (mean = 0.13 ± 0.02 puncta per µm², n = 16) from 3 independent experiments. Each dot indicates the puncta density in a cell (see Methods). Means are indicated by black lines. The two groups were significantly different from one another, ***p-value = 0.003, Mann-Whitney. Cohen’s d effect size − 1.20. C Same as (B), for JF646 HTL puncta density in ECs (mean = 0.45 ± 0.02 puncta per µm², n = 26 cells) and NSCs (mean = 0.30 ± 0.02 puncta per µm², n = 26 cells) from 3 independent experiments, *** p-value = 7.37 × 10⁻⁵, Mann-Whitney. The Cohen’s d effect size was − 1.21. D Distributions of puncta intensities from all detected (i.e., bright state) JF646-BAPTA puncta across every frame in EC videos (n = 15) and NSC videos (n = 16). Videos were recorded at 500 fps over 10 s, from 3 independent experiments. E Representative background-subtracted fluorescence intensity traces of immobile PIEZO1-HaloTag puncta from 500-fps TIRF imaging of an EC (top) and an NSC (bottom) labeled with JF646-BAPTA. Right, expanded traces from the sections marked with a red line on the left. F Corresponding all-points amplitude histograms from the 10-s traces shown in (E). Counts are shown on a log₁₀ scale. G Average of all-points amplitude histograms from 46 immobile puncta from 3 independent experiments of JF646-BAPTA labeled PIEZO1-HaloTag ECs (black) and NSCs (red). The two distributions were significantly different from each other, using a Two-Sample Kolmogorov-Smirnov test (p-value = 0.002). For individual fluorescence traces and all-points amplitude histogram of each punctum, see Supplementary Figs. 11, 12. Data points are shown as mean ± SEM unless otherwise noted.

Fig. 5. Visualizing the spatial distribution and activity of PIEZO1-HaloTag puncta in micropatterned neural rosettes (MNRs).

A Top and side view schematics of an MNR, showing cells organized radially around a central lumen. B Left schematic shows the confocal imaging plane (orange) in an MNR. Right: Confocal image (representative of 3 independent experiments) of a PIEZO1-HaloTag MNR labeled with JF646 HTL, fixed, and stained with phalloidin (magenta), anti-N-cadherin (yellow), and Hoechst (cyan). Zoomed-in sections show PIEZO1-HaloTag at cell-cell interfaces and actin-rich regions at the lumen and outer edges. A gamma of 0.5 was applied to the zoomed-in actin image. C Left schematic showing lattice light-sheet microscopy imaging in MNRs. Right: Representative volumetric rendering of an MNR labeled with actin-phalloidin and JF635 HTL showing top-down and side views. Middle: A 2 µm slab projection of actin (orange) and PIEZO1-HaloTag detections (cyan) with zoomed-in insets on far right. D Representative maximum intensity projection (MIP) of 3 optical planes acquired 215 nm apart in an MNR. Left, actin channel, middle, lumen and outer edge masks, right, PIEZO1-HaloTag. Representative images in C-D are from 4 independent experiments. E Density scatter plot of distances of PIEZO1-HaloTag puncta localizations to the lumen edge mask and outer edge mask (n = 103 videos from 21 MNRs, 4 independent experiments). The color scale indicates relative density of puncta at each position, scaled to the total number of puncta in the plot. The red cluster indicates PIEZO1 localization near the lumen edge. F Representative MIPs from 3-plane stacks of actin and Ca²⁺-sensitive JF646-BAPTA-labeled PIEZO1-HaloTag puncta in an MNR (n = 3 independent experiments). Bottom, kymograph generated from blue line (upper panel) showing fluorescence intensity flickering corresponding to PIEZO1 activity. G Density scatter plot of distances of active (bright) PIEZO1-HaloTag puncta localizations to the lumen edge mask and outer edge mask (n = 39 videos from 12 MNRs, 3 independent experiments). The color scale indicates relative density of puncta at each position, scaled to the total number of puncta in the plot. See also Supplementary Figs. 16−21, Supplementary Results, and Supplementary Movie 10.