Fig. 4: Vinculin recruitment modulates molecular-clutch stability and force-loading rate.

a, Numerical solution of the master equation describing NA maturation (Supplementary equation (I1)). The calculations show that the occupancy probability of sites available for molecular-clutch formation by molecular clutches with folded talin (blue curve) reaches a steady-state level within 10–20 s, whereas the amount of vinculin-bound molecular clutches (green curve) grows more slowly due to the limited pool of molecular clutches with force-unfolded talin (red curve). b, Total internal reflection fluorescence measurements of NA area (blue) and also talin (yellow) and vinculin (green) intensities in NAs in NIH-3T3 cells (mean ± s.e.m., N = 437 peripheral NAs pooled from four cells), fitted to the talin WT model. Talin accumulates first, because for vinculin to bind to it requires the unfolding of the mechanosensitive R3 domain of talin. c,d, Retrograde actin flow (d) and cell traction (c) data for WT (red circles), vinculin-KO (green triangles) and vinculin VD1-expressing MEF cells (blue triangles)^13,21 fitted to the WT-2 model, which accounts for vinculin-dependent changes in the dissociation rate and tension of molecular clutches. The fit shows that vinculin enhances the stability and mechanical load of molecular clutches. e, Force-loading rates of molecular clutches predicted by the model at their different tensions in mature FAs. Higher force-loading rates were predicted for molecular clutches under higher tension. f, Force-loading rates of molecular clutches in the presence of different vinculin mutants predicted by the talin WT model. KO of vinculin (blue) or vinculin with reduced talin binding affinity (vinA50I, red) markedly accelerates the force-loading rate of molecular clutches and increases the sensitivity to the density of sites available for molecular-clutch formation, whereas vinculin with reduced actin-binding affinity (vinI997A, yellow) has little effect compared to MEF WT cells (green). Experimental data were digitalized from ref. ²⁰ (mean ± 95% confidence interval for three independent experiments). g, Force–extension curves of talin alone (no VD1) and in the presence of vinculin VD1 (cycles 1–3)²². Binding of vinculin VD1 to force-unfolded talin domains prevents their refolding, increasing talin extension in the 5–10 pN range by almost threefold, thereby softening talin. Vcl, vinculin. Panel g adapted from ref. ²² under a Creative Commons license CC BY 4.0.