Fig 1: Force transduction in FAs is vinculin isoform-dependent.a Representative images of vinculin-deficient (vinc(-/-)) cells expressing vinculin tension sensor (V-TS), metavinculin tension sensor (M-TS), and the no-force control (Con-TS) 4 h after spreading on FN-coated glass coverslips show localization of all constructs to FAs (YPet), which are visualized by paxillin staining. Scale bar: 20 µm, in zoom: 5 µm. b Expression of V-TS or M-TS rescues the spreading defect of vinc(-/-) cells; data of the parental (vinc(f/f)) and vinc(-/-) cells are the same as in Fig. 1b. (n = 23, 32, 21, 14 cells). The bar chart shows the mean values ± SD. c Live-cell FLIM measurements of vinc(-/-) cells expressing FL-based tension sensors demonstrate FRET efficiency differences between V-TS and M-TS. Impairing actin binding by inserting the I997A mutation into vinculin (V-TS-I997A) and I1065A into metavinculin (M-TS-I1065A) strongly reduces tension and eliminates vinculin isoform-specific differences. (n = 73, 73, 74, 72, 73 cells). d Live-cell FLIM measurements of FL-based Con-TS, V-TS, and M-TS expressed in talin-deficient cells (tln1-/-tln2-/-), seeded on pLL-coated dishes and treated with Y-27632, confirmed that FRET differences are force-specific. (n = 80, 80, 82 cells). e Highly similar FRET efficiencies of force-insensitive vinculin (V-F7-TS) and metavinculin (M-F7-TS) tension sensor controls expressed in vinc(-/-) cells demonstrate that vinculin isoform-specific effects are conformation-independent (n = 86, 85 cells). f Talin-2 tension sensor (T2-TS) measurements in vinc(-/-) cells expressing TagBFP-tagged vinculin (V-B) and metavinculin (M-B) show that vinculin isoform-specific force transduction propagates across talin-2. T2-Con: talin-2 no-force control. (n = 30, 36, 31, 31 cells). g, h The A50I point mutation, which reduces the binding affinity of (meta)vinculin to talin, caused a FRET efficiency decrease in FL- and F40-based vinculin (V-TS-A50I) and metavinculin (M-TS-A50I) samples. (g: n = 84, 83, 78, 85, 86 cells; h: n = 60, 59, 77, 57, 78 cells). i Examination of stretched sensor molecules in V-TS-expressing cells, using four different TS modules, shows that vinculin is exposed to a wide range of forces; in average, 20–30% of molecules experience mechanical tension (n = 77, 73, 81, 77 cells). j Analogous analysis of M-TS-expressing cells indicates that the fraction of mechanically engaged metavinculin molecules is <20%. Note the equal amounts of stretched molecules in samples containing sensors sensitive to 1–?6 pN, 3–?5 pN, and 6–?8 pN indicating comparably high force per molecule across metavinculin. (n = 80, 74, 80, 77 cells). k Analyzing the differences of medians shown in (i) and (j) indicates that cells expressing M-TS instead of V-TS have less mechanically-engaged linkages that experience higher tension per molecule. Boxplots show median, 25th and 75th percentile with whiskers reaching to the last data point within 1.5× interquartile range. Two-sided Kolmogorov–Smirnov test: ***p < 0.001, **p < 0.01, *p < 0.05, n.s. (not significant) p = 0.05. Source data and exact p values are provided in the Source Data file.