Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks

Background: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wi skott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overa ll reactions and allows visualization of actin filaments, we showed how the se proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks, Results: Actin filament branches formed by polymerization of ATP-actin in t he presence of activated Arp2/3 complex were found to be metastable, dissoc iating from the mother filament with a half time of 500 seconds, The ADF/co filin protein actophorin reduced the half time for both dissociation of gam ma -phosphate from ADP-P-i-actin filaments and debranching to 30 seconds. B ranches were stabilized by phalloidin, which inhibits phosphate dissociatio n from ADP-P-i-filaments, and by BeF3, which forms a stable complex with AD P and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (K-d similar to 40 nM) than those of ADP-actin filaments ( K-d similar to1 muM), explaining why phosphate dissociation from ADP-P-i-fi laments liberates branches. Capping protein prevented annealing of short fi laments after debranching and, with profilin, allowed filaments to depolyme rize at the pointed ends. Conclusions: The low affinity of Arp2/3 complex for the pointed ends of ADP -actin makes actin filament branches transient. By accelerating phosphate d issociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free poi nted ends.