Effects of villin on the polymerization and subunit exchange of actin.

We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low concentrations of villin (villin/actin approximately 1:400) but is stimulated at higher concentrations (villin/actin greater than 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or upon the addition of phalloidin. The exchange of actin subunits at steady state is inhibited at low concentrations of villin (villin/actin approximately 1:200) but is stimulated at higher concentrations (villin/actin approximately 1:50). None of the above effects is observed at less than 10(-8) M free [Ca2+].