The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins

Abstract

The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.

Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins

“…Such autocatalysis is also suggested by the finding that addition of pre-formed filaments to actin polymerization mixture containing Arp2/3 and WASP family members enhances the polymerization [Machesky et al, 1999]. The observations of filament structures and kinetic analyses of polymerization have led to the proposal of two models of branched filament formation: the side branching model, in which branch occurs at the side of filaments, and the barbed end branching model, in which branch occurs at the barbed ends of filaments Pantaloni et al, 2000;Pollard et al, 2000].…”

“…Therefore, when the Arp2/3 complex is associated with pre-existing filaments, new filaments can form on preexisting filaments and generate an actin cytoskeleton immediately after the initiation of polymerization. Such filaments appear as branched filaments with branch angles of 70° Pantaloni et al, 2000]. …”

“…2A) activates the Arp2/3 complex Pantaloni et al, 2000;Rohatgi et al, 1999], suggesting that the VCA domain is a constitutive activator of the Arp2/3 complex at least in vitro. The V domain associates directly with actin monomers ], and the CA region interacts with the Arp2/3 complex, forming a nucleation core for actin polymerization [Machesky et al, 1999;Rohatgi et al, 1999].…”

“…Amann and Pollard [2001] observed hydrolyzed actin filaments directly by light microscopy, but they added phalloidin to the branch-forming solution, which might affect branching. Boujemaa-Paterski et al [2001] and Pantaloni et al [2000] used light microscopy as well as electron microscopy, which involves the washing of filaments with heavy metals, and this also might affect filament length. They did not add phalloidin in branchforming reaction.…”

“…Indeed, Aman and Pollard [2001] used 300 nM WAVE1/SCAR VCA domain to activate 10 nM Arp2/3 complex. In contrast, Pantaloni et al [2000] used 500 nM N-WASP VCA to activate 17 nM Arp2/3 complex and BoujemaaPaterski et al [2001] used 600 nM ActA to activate 10.6 nM Arp2/3 complex. In both conditions, the Arp2/3 complex is thought to be fully activated.…”