Fig. 1: Single-molecule imaging and super-particle fusion of labelled PIEZO1.
From: Direct observation of the conformational states of PIEZO1
a, Structural models of PIEZO1, viewed extracellularly. The most complete cryo-EM structure of PIEZO1 with the missing distal approximately one third of the blade highlighted (left) and the AlphaFold II structure prediction of PIEZO1 (right) are shown. The C-terminal extracellular domain (CED) is removed from the AlphaFold II model due to poor prediction. TCO*K tags at position 103 are shown as magenta stars. Each protomer is separately coloured. b, Segmentation of candidate PIEZO1 particles from iPALM localizations. A representative ×100 differential interference contrast image (from n = 5 cells) of a HEK293 cell expressing TCO*K 103 PIEZO1 labelled with tetrazine–Alexa Fluor 647 (AF647) (top left; scale bar, 10 µm). A 3 nm per pixel rendering of plasma membrane localizations from the magenta inset on the top left (scale bar, 3 µm) (top middle). Binary AF647 localizations (magenta points) with candidate PIEZO1 molecules meeting nearest neighbour requirements highlighted with cyan boxes (scale bar, 3 µm) (top right). Representative 3 nm per pixel renderings of candidate triple-labelled PIEZO1 molecules that meet minimum interlocalization separation requirements are also shown (scale bars, 30 nm) (bottom). c, Fused super-particle of identified PIEZO1 trimeric localizations with threefold symmetry promotion, viewed top down (n = 5 imaged cells, n = 726 molecules and n = 8,500 localizations). Localizations were visualized with size and colour proportional to local density (see Methods). d, Super-particle thresholded for a local density of more than 0.5 × 10−5, the minimum that encompassed localizations within a 60-nm sphere (left). Localizations associated with each blade isolated with k-means clustering with k = 3 clusters (right). e, Scatter plot of average per-localization interblade distances between each localization within a blade cluster from the super-particle in part c and all localizations in a neighbouring blade cluster, coloured by local density. At position 103, the most probable interblade distance is 25.4 ± 5.9 nm (mean ± s.d.), compared with 19.2 nm calculated from the AlphaFold II model.
