Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex

The actin. lament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother''. laments growing near the plasma membrane. The nucleotide bound to the mother. laments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother. lament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single. laments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother. laments, we observed fewer branches by microscopy on ADP-bound. laments than on ADP-Pi-bound. laments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother. laments. We conclude that the nucleotide on the mother. lament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-Pi. laments than on ADP-actin. laments.