Nucleation of actin polymerization by villin and elongation at subcritical monomer concentration

Abstract

We have obtained a quantitative description of villin-nucleated actin polymerization in physiological salt by determining the concentrations of free villin (V), villin-actin monomer (VA), villin-actin dimer (VA2), and villin-actin oligomer (VAn). Over a range of actin-villin ratios from 0.1 to 20 we determined the concentration of actin-bound villin by measuring the low-intensity pyrenylactin fluorescence of the two terminal actins in each villin-actin polymer. (To this end we first showed that each villin-actin oligomer and polymer contains two low-intensity pyrenylactin molecules). We determined the concentration of free villin using a calibrated cutting activity assay. The pattern of increase in bound villin together with the pattern of increase in high-intensity pyrenylactin fluorescence with increasing G-actin concentration indicated, first, that villin-actin monomers were not formed at detectable levels even at a 12-fold villin excess over actin. Second, there was no stoichiometric villin-actin dimer formation at actin-villin ratios of 2. Instead there was an equilibrium between free villin, VA2, and VAn. Defining K1 = [VA]/[V][A] and K2 = [VA2]/[VA][A], a good fit of the data was obtained with K1 .mchlt. K2 and a value of K1K2 = KV = 1012-1013 M-2 = [VA2]/[V][A]2, i.e., 1/KV1/2 = (0.3-1) .times. 10-6 M. We have assumed here that the monomer binding constant of VA2 to form VA3 was equal to the monomer binding constant of pointed filament ends, K.infin. = 1/c.infin., obtained as described below. Extending polymerization measurements to actin-villin ratios greater than 100 showed that F-actin increased gradually with increasing G-actin concentrations until a limiting value for the G-actin concentration was obtained, c.infin., approaching 1/K.infin. for the pointed filament end. The data could be fitted with the same three constants, K1, K2, and K.infin., used for the data in the low range of actin-villin ratios. Retaining the term critical concentation for the limiting G-actin concentration during nucleated polymerization, F-actin is formed at subcritical actin concentrations. In other others, filament assembly no longer has the characteristics of condensation polymerization but instead is described by a series of equilibria that are very similar to those derived by Oosawa for the analogous case of "linear" polymerization.