For more than a decade the Arp2/3 complex, a handful of nucleation-promoting factors, and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have brought a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASH, WHAMM, and JMY stimulate Arp2/3 complex activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, Cordon-bleu, and Leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization.

The cellular functions of the actin cytoskeleton require precise regulation of both the initiation of actin polymerization and the organization of the resulting filaments. The actin-related protein-2/3 (ARP2/3) complex is a central player in this regulation. A decade of study has begun to shed light on the molecular mechanisms by which this powerful machine controls the polymerization, organization and recycling of actin-filament networks, both in vitro and in the living cell.

The pathogenic bacterium Listeria monocytogenes is capable of directed movement within the cytoplasm of infected host cells. Propulsion is thought to be driven by actin polymerization at the bacterial cell surface, and moving bacteria leave in their wake a tail of actin filaments. Determining the mechanism by which L. monocytogenes polymerizes actin may aid the understanding of how actin polymerization is controlled in the cell. Actin assembly by L. monocytogenes requires the bacterial surface protein ActA and protein components present in host cell cytoplasm. We have purified an eight-polypeptide complex that possesses the properties of the host-cell actin polymerization factor. The pure complex is sufficient to initiate ActA-dependent actin polymerization at the surface of L. monocytogenes, and is required to mediate actin tail formation and motility. Two subunits of this protein complex are actin-related proteins (ARPs) belonging to the Arp2 and Arp3 subfamilies. The Arp3 subunit localizes to the surface of stationary bacteria and the tails of motile bacteria in tissue culture cells infected with L. monocytogenes; this is consistent with a role for the complex in promoting actin assembly in vivo. The activity and subunit composition of the Arp2/3 complex suggests that it forms a template that nucleates actin polymerization.

Actin filament assembly at the cell surface of the pathogenic bacterium Listeria monocytogenes requires the bacterial ActA surface protein and the host cell Arp2/3 complex. Purified Arp2/3 complex accelerated the nucleation of actin polymerization in vitro, but pure ActA had no effect. However, when combined, the Arp2/3 complex and ActA synergistically stimulated the nucleation of actin filaments. This mechanism of activating the host Arp2/3 complex at the L. monocytogenes surface may be similar to the strategy used by cells to control Arp2/3 complex activity and hence the spatial and temporal distribution of actin polymerization.

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat–containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

Eukaryotic cells use actin polymerization to change shape, move, and internalize extracellular materials by phagocytosis and endocytosis, and to form contractile structures. In addition, several pathogens have evolved to use host cell actin assembly for attachment, internalization, and cell-to-cell spread. Although cells possess multiple mechanisms for initiating actin polymerization, attention in the past five years has focused on the regulation of actin nucleation-the formation of new actin filaments from actin monomers. The Arp2/3 complex and the multiple nucleation-promoting factors (NPFs) that regulate its activity comprise the only known cellular actin-nucleating factors and may represent a universal machine, conserved across eukaryotic phyla, that nucleates new actin filaments for various cellular structures with numerous functions. This review focuses on our current understanding of the mechanism of actin nucleation by the Arp2/3 complex and NPFs and how these factors work with other cytoskeletal proteins to generate structurally and functionally diverse actin arrays in cells.

Summary The Arp2/3 complex is an actin nucleator that plays a critical role in many cellular processes. Its activities are regulated by nucleation-promoting factors (NPFs) which function primarily during plasma membrane dynamics. Here we identify a new mammalian NPF called WHAMM (WASP Homologue associated with Actin, Membranes, and Microtubules) that localizes to the cis-Golgi apparatus and tubulo-vesicular membrane transport intermediates. The modular organization of WHAMM includes an N-terminal domain that mediates Golgi membrane association, a coiled-coil region that binds microtubules, and a WCA segment that stimulates Arp2/3-mediated actin polymerization. Overexpression and depletion studies indicate that WHAMM is important for maintaining Golgi structure and facilitating anterograde membrane transport. The ability of WHAMM to interact with microtubules plays a role in membrane tubulation, while its capacity to induce actin assembly promotes tubule elongation. Thus, WHAMM is an important regulator of membrane dynamics functioning at the interface of the microtubule and actin cytoskeletons.

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