ABSTRACT
The cyclic peptide phalloidin binds and stabilizes actin filaments. It is widely used in studies of actin filament assembly, including analysis of branch formation by Arp2/3 complex, but its influence on the branching reaction has not been considered. Here we show that rhodamine-phalloidin binds both Arp2/3 complex and the VCA domain of Arp2/3 complex activator, hWASp, with dissociation equilibrium constants of about 100 nM. Not only does phalloidin promote nucleation of pure actin monomers but it also dramatically stimulates branch formation by actin, Arp2/3 complex, and hWASp-VCA more than 10-fold and inhibits dissociation of branches. Therefore, the appearance of more branches in samples treated with rhodamine-phalloidin arises from multiple influences of the peptide on both the formation and dissociation of branches.
Subject(s)
Actin-Related Protein 2-3 Complex/chemistry , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Phalloidine/chemistry , Phalloidine/metabolism , Actin-Related Protein 2/chemistry , Actin-Related Protein 2/metabolism , Actin-Related Protein 3/chemistry , Actin-Related Protein 3/metabolism , Actins/chemistry , Amino Acid Sequence , Animals , Cattle , Chickens , Fluorescence Polarization , Microscopy, Fluorescence , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Wiskott-Aldrich Syndrome Protein/biosynthesis , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
The actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother" filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (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 filament 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 filaments 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 filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-P(i)-bound filaments. 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 filaments. We conclude that the nucleotide on the mother filament 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-P(i) filaments than on ADP-actin filaments.
