Fig. 2: The plasma membrane expands the blades of PIEZO1.
From: Direct observation of the conformational states of PIEZO1
a, MINFLUX imaging and PIEZO1 trimer identification. Representative ×100 confocal scan (from n = 5 cells) of a HEK293 cell membrane expressing labelled TCO*K 103 PIEZO1 (scale bar, 2 µm) (left). Colour represents scaled intensity in each detector (650–685 nm (green) and 685–720 nm (red)). Clusters of raw localizations (traces) rendered as 15-nm spheres from the same region (scale bar, 2 µm) (middle). Zoomed-in rendering (3-nm spheres) showing single-labelled (red), double-labelled (yellow) and triple-labelled (green) PIEZO1 identified by the algorithm (scale bar, 20 nm) (right). b, Representative PIEZO1 trimer. Raw localizations (3-nm spheres) coloured by DBSCAN cluster. The centre positions were determined by a 3D GMM fit (white circles). c, Ostu-thresholded super-particle with threefold symmetry enforcement (n = 5 cells and n = 41 molecules). d, Histograms of GMM fit error for each trimer fluorophore position (n = 41 molecules and n = 123 fluorophore positions). Bin width = 1 nm. The black dashed line indicates median fit error. e, Interblade distances of PIEZO1 in a membrane (grey circles; n = 41 molecules and n = 5 cells), the PIEZO2 structure (PDB ID: 6KG7) at the nearest aligned position and the AlphaFold II prediction (E2JF22) (top). A schematic of labelled PIEZO1 in a membrane is also shown (bottom). f, Interblade distances per detergent-solubilized PIEZO1 (17.1 ± 4.0 nm; orange circles; n = 7 molecules), AlphaFold II prediction (black circle) and PIEZO1 in a membrane (grey circles) (top). Kolmogorov–Smirnov test: *P = 0.0106 and D = 0.662. A schematic of the protein immobilization method and blade compaction is also shown (bottom). g, Interblade distances in a cell exposed to 20 µM GsMTx-4 (20.7 ± 6.6 nm; yellow circles; n = 20 molecules and n = 3 cells), AlphaFold II prediction (black circle) and PIEZO1 in a membrane (grey circles) (top). Kolmogorov–Smirnov test: *P = 0.0126 and D = 0.4341. A schematic of blade compaction from GsMTx-4 is also shown (bottom). All statistical tests are two-tailed. Values in e–g are mean ± s.d.
