Fig. 3: Talin-1, but not kindlin, tensin, or septin, is the primary mechanosensitive module in curved adhesions.

A Representative images of GFP-talin-1, GFP-talin-2, mCherry-tensin-3, anti-kindlin-2, septin-2-GFP, and GFP-septin-7 on nanobar substrates. The averaged nanobar images are shown on the top-left corners. B Quantifications of the normalized nanobar end-to-side intensity ratios of five cytoskeletal adapters and septins. C SEM images and schematic illustration of vertical nanopillars inducing membrane curvature by their cylindrical shape. Scale bar: 1 µm. D, E Anti-ITGβ5, FCHo2-GFP, and mCherry-talin-1 accumulate and colocalize in curved adhesions formed at nanopillar locations (D), while mCherry-tensin-3 is not involved in curved adhesions (E). F, G Neither septin-2-GFP (F) nor GFP-septin-7 (G) is spatially correlated with anti-ITGβ5 and mCherry-FCHo2. H, I Quantifications of the Pearson’s correlation coefficients between five cytoskeletal adapters or septins and ITGβ5 (H), or FCHo2 (I), at nanopillars. J Cartoon illustration of talin F3 subdomain interacting with ITGβ5 intracellular tail and the plasma membrane. K Domain structures of five mCherry-talin-1 variants. F: FERM domains; R: rod domains; DD: dimerization domain. L ITGβ5-GFP forms abundant focal adhesions on flat substrates when co-expressed with the wild-type mCherry-talin-1, mCherry-talin-1(CAM), or mCherry-talin-1(THD). However, it forms fewer and smaller ones when either mCherry-talin-1(LBM) or mCherry-talin-1(K316E) is co-expressed. M Quantifications of the focal adhesion area percentage (left) and the number of focal adhesions per cell (right) of ITGβ5-GFP, when co-expressed with mCherry-talin-1 variants. N Anti-ITGβ5, FCHo2-GFP, and mCherry-talin-1(LBM) all accumulate and colocalize in curved adhesions formed at nanopillars. O mCherry-talin-1(THD) displays low spatial correlations with anti-ITGβ5 and FCHo2-GFP at nanopillars. P, Q Quantifications of Pearson’s correlation coefficients between mCherry-talin-1 variants and anti-ITGβ5 (P) or FCHo2-GFP (Q), at nanopillars. R Quantifications of the normalized nanopillar-to-surrounding intensity ratios of ITGβ5 when co-expressed with mCherry-talin-1 variants. Scale bar: 10 µm for all the cell images. White arrows indicate enrichments at nanobar ends, white empty arrowheads indicate no preferential enrichment at nanobar ends, while yellow arrowheads indicate focal adhesions. In (D–G) and (N–Q), white arrows indicate high-intensity correlations, while white triangles indicate low-intensity correlations at nanopillars. Welch’s t-tests (unpaired, two-tailed, not assuming equal variance) are applied for statistical analyses for (B, H, I, P–R), while Mann Mann-Whitney test (unpaired, two-tailed) was used for statistical analyses for (M). For (B, H, I, P–R), data are presented as mean ± SD; for (M), the solid black lines indicate the median.