Vinculin-dependent actin bundling regulates cell migration and traction forces

Abstract

Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited . In this study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further , mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading, and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites. Lastly, we herein discuss the viability of models detailing the F-actin-binding surface on vinculin in context of our mutational analysis.

The Structural Basis of Actin Organization by Vinculin and Metavinculin

“…2b), suggesting that both regions of the protein contribute to the density and engage the actin surface. This conformation of the C-terminus is consistent with the recent report that R1049 is important for actin binding 29 , bringing this residue close to the negatively charged surface of subdomain 2 (Fig. 2c).…”

“…Both activities are required for vinculin function in vivo, as lesions that selectively disrupt them in the context of the full-length molecule lead to defects in cell spreading, adhesion formation and maturation, and mechanotransduction 6; 27; 28; 29 . Cross-linking and tomographic studies indicate that actin bundling occurs through oligomerization of the Vt domain 26; 27; 30 .…”

Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior

“…The different effects of actin binding on the cell areas at physiological levels of stiffness and on extremely rigid substrates have also been reported [35]. One possibility to explain the discrepancy between the effects on the physiological levels of stiffness (gel substrates) and extremely rigid (glass) substrates is as follows: two tensile forces transmitted from two actin-binding surfaces are required for activation and CSB resistance of vinculin on the physiological level of stiffness because of the weak intracellular tension, while a high tensile force transmitted from one actin-binding surface on extremely rigid substrates is enough for them.…”

Vinculin in cell–cell and cell–matrix adhesions

“…They predicted F-actin binds two distinct surfaces in the vinculin tail: an upper (amino acids 925–952) and lower monomer site (amino acids 1050–1056) [15]. Experimental data from four groups, including our laboratory, support residues or regions identified by Janssen are important for the interaction between vinculin and actin [13, 15, 59, 60]. However, other studies revealed that mutation of residues outside the upper and lower monomer perturb actin binding to vinculin [46, 58].…”