Zyxin-mediated regulation of traction force and actin assembly at adhesive contacts

Cytoskeletal regulation of cell adhesion and traction force is vital to the organization of multi-cellular structures. The assembly, disassembly, and size of adhesion plaques have been shown to alter traction force exerted by migrating cells. However, the mechanisms by which adhesive complexes regulate the transmission of traction force remain unclear. At focal and cell-cell adhesion sites, actin bundles also rapidly polymerize, which suggests that highly dynamic protein interactions and rapid actin assembly may be tightly coupled to the regulation of traction force at adhesive contacts. The focal adhesion protein zyxin emerged as a key regulator of traction force since zyxin has the ability to sense mechanical force and recruit Ena/VASP proteins to promote actin assembly. Using elastic micro-pillars to analyze traction force, the intensity of GFP-tagged zyxin in epithelial cells was found to directly correlate with increasing traction force at cell-matrix adhesion sites. Suppression of zyxin using siRNA prevented VASP from localizing to focal adhesion plaques, which were sites of poor actin assembly. Surprisingly, zyxin deficient cells migrated normally and retained the ability to transmit traction force to the substrate. Since zyxin also localize to cell-cell contacts, the effect of zyxin suppression on actin assembly was analyzed for cadherin-mediated adhesion. In contrast to focal adhesion sites, VASP accumulated at cell-cell contacts despite the absence of zyxin. Surprisingly, actin assembly was reduced at VASP positive cell-cell contacts. Cell spreading on E-cadherin coated surface and the formation of cell clusters were slower for zyxin deficient cells than wildtype cells. While actin assembly at both focal adhesion and cell-cell adhesion was limited in zyxin deficient cells, only actin assembly at focal adhesion was VASP dependent. These results suggest that zyxin regulates actin assembly by different mechanisms at the sites of focal adhesion and cell- cell adhesion.