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

Most eukaryotic cells rely on localized actin Results Although the nucleation activity of the Arp2/3 complex polymerization to generate and sustain the protrusion activity necessary for cell movement has been extensively explored [5-7, 9-11, 30, 31], the branching activity of the complex, an essential step in the [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dendritic nucleation model [5], is much less well characterized, particularly in terms of its relevance in vivo. While dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] it seems clear now that the branching occurs during polymerization rather than after [6, 7], branching has been and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. modeled in two ways, either as a separate activity of the Arp2/3 complex [5, 6, 30] or as an integral part of the The complex has thus been proposed to generate the actin network at the leading edge of crawling nucleation mechanism [7]. Furthermore, the complex does indeed localize at Y branches of actin filaments in cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to vivo [3, 4], but multiple other actin cross-linking proteins are present at the leading edge that could also cause protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 branching [12, 13], and there is no direct evidence that the branching activity of the Arp2/3 complex is required complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side for the extension of the lamellipod. binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, Structural studies of the Arp2/3 complex from Acanthamicroinjection of these antibodies into cells moeba using chemical cross-linking have shown that of specifically inhibits lamellipod extension without the seven subunits of the complex, only two, Arp3 and affecting the EGF-stimulated appearance of free p35, seem to bind actin directly [14]. The atomic model barbed ends in situ. These results indicate that of Arp3, based on its sequence homology to actin, predicts while the side binding activity of the Arp2/3 complex that this subunit is likely to be involved in the nucleation is required for nucleation in vitro and for protrusive function of the complex by forming a heterodimer with force in vivo, it is not required for EGF-stimulated Arp2 that could serve as a pointed end nucleus for actin increases in free barbed ends in vivo. This suggests polymerization [14, 15]. The p35 subunit, on the other that the branching activity of the Arp2/3 complex is hand, is a potential candidate for binding to the side of essential for lamellipod extension, while the the actin filament, since it binds actin but is not obviously generation of nucleation sites for actin involved in the nucleation activity [14]. We have generpolymerization is not sufficient. ated a peptide antibody against p34, the human homolog Addresses: * Albert Einstein College of Medicine, Department of of the Acanthamoeba p35 subunit [16, 17]. This antibody Anatomy and Structural Biology,