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 Ttol (tension tolerance) value and ITS visualizing integrin tensions above the Ttol value by fluorescence. Results show that large FAs (area >1 µm2) were formed on the TGT surface with Ttol of 54 pN but not on those with lower Ttol 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 Ttol of 54 pN. Next, we tested FA formation in vinculin KO cells on TGT surfaces. Surprisingly, the Ttol 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 Ttol 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.

Filopodial adhesive force in discrete nodes revealed by integrin molecular tension imaging.

Filopodia are narrow cell extensions involved in various physiological processes. Integrins mediate filopodia adhesion and likely transmit adhesive force to regulate filopodia formation and functions, but the force is extremely weak to study and remains poorly understood. Using integrative tension sensor (ITS), we imaged filopodia adhesive force at the single molecular tension level and investigated the force dynamics and sources. Results show that filopodia integrin tension (FIT) is generated in discrete foci (force nodes) along single filopodia with a spacing of ∼1 µm. Inhibitions of actin polymerization or myosin II activity markedly reduced FIT signals in force nodes at filopodia tips and at filopodia bases, respectively, suggesting differential force sources of FIT in the distal force nodes and proximal ones in filopodia. Using two ITS constructs with different force thresholds for activation, we showed that the molecular force level of FIT is greater at filopodia bases than that at filopodia tips. We also tested the role of vinculin and myosin X in the FIT transmission. With vinculin knockout in cells, filopodia and associated force nodes were still formed normally, suggesting that vinculin is dispensable for the formation of filopodia and force nodes. However, vinculin is indeed required for the transmission of strong FIT (capable of rupturing DNA in a shear conformation), as the strong FIT vanished in filopodia with vinculin knockout. Co-imaging of FIT and myosin X shows no apparent co-localization, demonstrating that myosin X is not directly responsible for generating FIT, despite its prominent role in filopodium elongation.