Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments (original) (raw)
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Proceedings of The National Academy of Sciences, 1998
The Arp2͞3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2͞3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2͞3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 M. The high affinity of Arp2͞3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2͞3 complex. Arp2͞3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2͞3 bound to sides and pointed ends of actin filaments and examples of Arp2͞3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ؎ 7°. From these in vitro biochemical properties, we propose a model for how Arp2͞3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells. 1 mM MgCl 2 , and 50 mM KCl.
Current Biology, 2001
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,
Pathway of Actin Filament Branch Formation by Arp2/3 Complex
Journal of Biological Chemistry, 2008
A spectroscopic assay using pyrene-labeled fission yeast Arp2/3 complex revealed that the complex binds to and dissociates from actin filaments extremely slowly with or without the nucleation-promoting factor fission yeast Wsp1-VCA. Wsp1-VCA binds both Arp2/3 complex and actin monomers with high affinity. These two ligands have only modest impacts on the interaction of the other ligand with VCA. Simulations of a mathematical model based on the kinetic parameters determined in this study and elsewhere account for the full time course of actin polymerization in the presence of Arp2/3 complex and Wsp1-VCA and show that an activation step, postulated to follow binding of a ternary complex of Arp2/3 complex, a bound nucleation-promoting factor, and an actin monomer to an actin filament, has a rate constant at least 0.15 s ؊1. Kinetic parameters determined in this study constrain the process of actin filament branch formation during cellular motility to one main pathway.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2013
Background-Arp2/3 complex is a key actin cytoskeletal regulator that creates branched actin filament networks in response to cellular signals. WASP-activated Arp2/3 complex assembles branched actin networks by nucleating new filaments from the sides of pre-existing ones. WASPmediated activation requires seed filaments, to which the WASP-bound Arp2/3 complex can bind to form branches, but the source of the first substrate filaments for branching is unknown. Results-Here we show that Dip1, a member of the WISH/DIP/SPIN90 family of actin regulators, potently activates Arp2/3 complex without preformed filaments. Unlike other Arp2/3 complex activators, Dip1 does not bind actin monomers or filaments, and interacts with the complex using a non-WASP-like binding mode. In addition, Dip1-activated Arp2/3 complex creates linear instead of branched actin filament networks. Conclusions-Our data show the mechanism by which Dip1 and other WISH/DIP/SPIN90 proteins can provide seed filaments to Arp2/3 complex to serve as master switches in initiating branched actin assembly. This mechanism is distinct from other known activators of Arp2/3 complex.