Vinculin transmits high-level integrin tensions that are dispensable for focal adhesion formation

Focal adhesions (FAs) transmit force and mediate mechanotransduction between cells and the matrix. Previous studies revealed that integrin-transmitted force is critical to regulate FA formation. As vinculin is a prominent FA protein implicated in integrin tension transmission, this work studies the relation among integrin tensions (force), vinculin (protein), and FA formation (structure) by integrin tension manipulation, force visualization and vinculin knockout (KO). Two DNA-based integrin tension tools are adopted: tension gauge tether (TGT) and integrative tension sensor (ITS), with TGT restricting integrin tensions under a designed T_tol (tension tolerance) value and ITS visualizing integrin tensions above the T_tol value by fluorescence. Results show that large FAs (area >1 μm²) were formed on the TGT surface with T_tol of 54 pN but not on those with lower T_tol values. Time-series analysis of FA formation shows that focal complexes (area <0.5 μm²) appeared on all TGT surfaces 20 min after cell plating, but only matured to large FAs on TGT with T_tol of 54 pN. Next, we tested FA formation in vinculin KO cells on TGT surfaces. Surprisingly, the T_tol value of TGT required for large FA formation is drastically decreased to 23 pN. To explore the cause, we visualized integrin tensions in both wild-type and vinculin KO cells using ITS. The results showed that integrin tensions in FAs of wild-type cells frequently activate ITS with T_tol of 54 pN. With vinculin KO, however, integrin tensions in FAs became lower and unable to activate 54 pN ITS. Force signal intensities of integrin tensions reported by 33 and 43 pN ITS were also significantly reduced with vinculin KO, suggesting that vinculin is essential to transmit high-level integrin tensions and involved in transmitting intermediate-level integrin tensions in FAs. However, the high-level integrin tensions transmitted by vinculin are not required by FA formation.