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.