Date Published: April 13, 2004
Publisher: Public Library of Science
Author(s): Mark J Dayel, R. Dyche Mullins
Abstract: In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.
Partial Text: The actin cytoskeleton determines the shape, mechanical properties, and motility of most eukaryotic cells. To change shape and to move, cells precisely control the location and timing of actin filament assembly by regulating the number of fast-growing (barbed) filament ends (Pollard et al. 2000). The actin-related protein (Arp) 2/3 complex, a seven-subunit protein complex that contains two actin-related proteins, generates these new barbed ends in response to cellular signals (Welch et al. 1998; Machesky et al. 1999; Rohatgi et al. 1999). In a process called “dendritic nucleation,” the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments to produce a rigid and highly crosslinked filament array (Mullins et al. 1998; Machesky et al. 1999; Blanchoin et al. 2000a). Such crosslinked arrays form the core of many motile cellular structures, including the leading edges of amoeboid cells and the actin comet tails that propel endosomes and bacterial pathogens through eukaryotic cytoplasm. To understand the construction, function, and regulation of these structures, it is important to understand the molecular mechanism of Arp2/3 activation.
Conventional actin and all actin-related proteins share a conserved nucleotide binding pocket. Actin monomers bind ATP but do not hydrolyze it until they are induced to polymerize. Actin polymerization triggers rapid ATP hydrolysis, followed by a slow release of cleaved phosphate from the filament (Blanchoin and Pollard 2002). Arp2 also hydrolyzes its bound ATP, and we find that the conditions that promote ATP hydrolysis and the kinetics of the reaction are remarkably similar to those of conventional actin. In the presence of VCA and actin filaments, monomeric actin stimulates ATP hydrolysis on Arp2 (Table 1). We also find that binding of the Arp2/3 complex to the pointed end of a preformed actin filament is sufficient to trigger Arp2 ATP hydrolysis, even in the absence of VCA. The stimulation of Arp2 ATPase activity by both filament pointed ends and by actin monomers under nucleating conditions suggests that the geometry of the Arp2/3–actin interaction is the same in both cases.