The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate an d sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex l ocalizes within that network in vivo [3, 4] and nucleates actin polymerizat ion and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leadin g edge of crawling cells in vivo [3, 4, 8]. However, the relative contribut ions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selective ly inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 c omplex in vitro. However, microinjection of these antibodies into cells spe cifically inhibits lamellipod extension without affecting the EGF-stimulate d appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stim ulated increases in free barbed ends in vivo. This suggests that the branch ing activity of the Arp2/3 complex is essential for lamellipod extension, w hile the generation of nucleation sites for actin polymerization is not suf ficient.