Villin function in the organization of the actin cytoskeleton - Correlation of in vivo effects to its biochemical activities in vitro

Villin is an actin-binding protein of the intestinal brush border that bund les, nucleates, caps, and severs actin in a Ca2+-dependent manner in vitro, Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell t ransfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with it s importance for microfilament bundling in cells, the KKEK motif of the car boxyl-terminal F-actin-binding site is crucial for bundling in vitro, In ad dition, substitutions of basic residues in a second site, located in the am ino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca2+ as well as its bundling and s evering activities in vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border cor e bundle but does not initiate its assembly by nucleation of actin filament s.