Arp2/3 complex is bound and activated by two WASP proteins

Abstract

Actin related protein 2/actin related protein 3 (Arp2/3) complex nucleates new actin filaments in eukaryotic cells in response to signals from proteins in the Wiskott-Aldrich syndrome protein (WASP) family. The conserved VCA domain of WASP proteins activates Arp2/3 complex by inducing conformational changes and delivering the first actin monomer of the daughter filament. Previous models of activation have invoked a single VCA acting at a single site on Arp2/3 complex. Here we show that activation most likely involves engagement of two distinct sites on Arp2/3 complex by two VCA molecules, each delivering an actin monomer. One site is on Arp3 and the second is on ARPC1 and Arp2. The VCAs at these sites have distinct roles in activation. Our findings reconcile apparently conflicting literature on VCA activation of Arp2/3 complex and lead to a new model for this process.

Cellular functions of WASP family proteins at a glance

“…This is accomplished by two WCA domains interacting with two unique sites on the Arp2/3 complex simultaneously to promote actin nucleation (Padrick et al, 2011). binding WASH homology domain 2 (WHD2), a proline-rich region and C-terminal WCA domain. WASH is intrinsically inactive and exists in a macromolecular pentameric complex named the WASH regulatory complex (SHRC) that also includes FAM21A or FAM21C (WASHC2A and WASHC2C), strumpellin (WASHC5), KIAA1033 (WASHC4) and CCDC53 (WASHC3) (Derivery et al, 2009;Gomez and Billadeau, 2009;Jia et al, 2010) (see poster).…”

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

“…The formation of the initial closed Arp2/3-filament complex (A 2 F) is reversible, but the Arp-filament interaction is stabilized by subsequent activation processes (13,17), which may include binding of actin monomers (13,15), changes in the nucleotide state of Arp2 and/or Arp3 (16,25), additional Arp2/3 complex conformational changes (6,10,17), or alteration of the mother filament structure or curvature (6,28,31). Because the experiments were conducted at either zero or maximally activating concentrations A B actin-AF488 Arp2/3 binding heat map merge of VCA, the scheme does not explicitly show VCA binding steps or define the occupancies of the multiple VCA-binding sites (10,14,15). Instead, we make the simplifying assumption that the arrangement of states is the same in both cases and, consequently, the effect of VCA can be incorporated by changing the apparent rate or equilibrium constants.…”

“…Nucleation of actin filament branches involves large-scale conformational changes in the Arp2/3 complex (6-10), binding of one or more verprolin homology, central, and acidic (VCA) domains (10)(11)(12)(13)(14)(15), recruitment of one or more actin monomers (2,16), and formation of a stable association of the complex with a mother filament (6). The ordering, dynamics, and regulation of these individual reaction steps in the overall process of branch nucleation have been subjects of intense interest (13,15,17,18).…”

Actin filament curvature biases branching direction

“…Upon activation by two molecules of nucleation-promoting factor (NPF) localized at or near a membrane, the Arp2/3 complex nucleates a new, "daughter" filament from the side of a preexisting "mother" filament, forming a Y-shaped branch that serves as the basic structural unit of these networks (26)(27)(28) (Fig. S1).…”

“…S1) is understood to involve conformational changes in the Arp2/3 complex induced by the binding of NPFs (26,27,28,30,31). Additional conformational changes in both the Arp2/3 complex and several monomers in the mother filament probably occur upon the binding of the ternary complex of NPFs, Arp2/3, and G-actin to the mother filament or during a subsequent activation step that is necessary to allow branch nucleation (26,31), because the bound NPFs appear to partially overlap the F-actin binding surface of the Arp2/3 complex (32).…”