Formation of talin-vinculin pre-complexes dictates maturation of nascent adhesions by accelerated force transmission and vinculin recruitment

Talin, vinculin, and paxillin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). Using machine learning, traction microscopy, single-particle-tracking, and fluorescence fluctuation analysis, we find that talin, vinculin, and paxillin are recruited in near-synchrony to NAs maturing to focal adhesions. After initial recruitment of all three proteins under minimal load, vinculin accumulates in these NAs at a ~5 fold higher rate than in non-maturing NAs and with faster growth in traction. We identify a domain in talin, R8, which exposes a vinculin-binding-site (VBS) without requiring load. Stabilizing this domain via mutation lowers load-free vinculin binding to talin, impairs maturation of NAs, and reduces the rate of additional vinculin recruitment. Taken together, our data show that talins concurrent localization with vinculin, before engagement with integrins, is essential for NA maturation, which entails traction-mediated unfolding of talin and exposure of additional VBSs triggering further vinculin binding.