Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts

Background: Actin filaments polymerize in vivo primarily from their fast-gr owing barbed ends. In cells and extracts, GTP gamma S and Rho-family GTPase s, including Cdc42, stimulate barbed-end actin polymerization; however, the mechanism responsible for the initiation of polymerization is unknown. The re are three formal possibilities for how free barbed ends may be generated in response to cellular signals: uncapping of existing filaments; severing of existing filaments; or de novo nucleation. The Arp2/3 complex localizes to regions of dynamic actin polymerization, including the leading edges of motile cells and motile actin patches in yeast, and in vitro it nucleates the formation of actin filaments with free barbed ends. Here, we investigat ed actin polymerization in soluble extracts of Acanthamoeba. Results: Addition of actin filaments with free barbed ends to Acanthamoeba extracts is sufficient to induce polymerization of endogenous actin. Additi on of activated Cdc42 or activation of Rho-family GTPases in these extracts by the non-hydrolyzable GTP analog GTP gamma S stimulated barbed-end polym erization, whereas immunodepletion of Arp2 or sequestration of Arp2 using s olution-binding antibodies blocked Rho-family GTPase-induced actin polymeri zation. Conclusions: For this system, we conclude that the accessibility of free ba rbed ends regulates actin polymerization, that Rho-family GTPases stimulate polymerization catalytically by de novo nucleation of free barbed ends and that the primary nucleation factor in this pathway is the Arp2/3 complex.