Calcium dependence of villin-induced actin depolymerization.

"Cutting" of actin filaments by villin was evaluated from the time course of filament depolymerization. Depolymerization was initiated by diluting polymerized actin, labeled with a fluorescent probe on either lysine-374 or cysteine-375, to a concentration well below the critical into a medium containing free villin and various concentrations of calcium (in addition to potassium and magnesium). It was observed that at high calcium concentrations (200 microM) the time course of depolymerization could not be described by the single exponential that defines it at low calcium and low villin levels. Instead, at high calcium, the exponent increased with time and the rate of depolymerization became greater than that of controls in the absence of villin. This contrasts with the inhibition of depolymerization by villin at low calcium. The latter inhibition is a consequence of the capping of the barbed filament end by villin as are the inhibition of filament elongation and the elevation of the critical concentration. Evidence is presented that the effects of villin at high calcium are the result of cutting of the actin filaments by villin. It thus appears that different calcium binding sites control capping and cutting and that the calcium binding sites regulating cutting have a much lower affinity for calcium than the sites regulating capping of the barbed filament ends.