VICTORIA ASKEW
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 spontaneous initiation of actin-filament assembly requires the formation of a nucleus of three actin monomers. Spontaneous nucleation is kinetically unfavourable and is the rate-limiting step in polymerization; therefore, factors that can accelerate or bypass this step are important for efficient actin assembly in the cell. The long-term search for the actin nucleator of the cell came to fruition in 1994 with the discovery of the actin-related protein-2/3 (Arp2/3) complex.
Laura Machesky in Tom Pollard's laboratory purified the intact Arp2/3 complex from Acanthamoeba castellanii based on its affinity for the actin-binding protein profilin, and showed that it consists of a stable assembly of seven polypeptides. Two of the subunits were actin-related proteins of the Arp2 and Arp3 subfamilies, giving the complex its name. The authors localized the complex at the leading edge of motile cells and presciently suggested that the unconventional actins dimerize to "form a cryptic nucleus for actin filament formation". Matt Welch in Tim Mitchison's laboratory confirmed this hypothesis by showing that the Arp2/3 complex was the key factor involved in nucleating Listeria monocytogenes actin tails in in vitro motility assays.
This work paved the way for exploring the regulators of the Arp2/3 complex, many of which turned out to be effectors of Rho GTPases. In 1998, Machesky and Insall showed that a domain shared by Wiskott–Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE; also known as suppressor of cyclic AMP repressor (SCAR)) binds to the Arp2/3 complex. In parallel, Ma et al. provided a direct link between signalling pathways and actin nucleation, by showing that the Rho GTPase CDC42 binds and activates proteins that stimulate actin polymerization by the Arp2/3 complex. Machesky and collaborators, followed by several other laboratories, found that WASP/WAVE proteins are nucleation-promoting factors (NPFs) for the Arp2/3 complex.
At the same time, Mullins, Heuser and Pollard showed that the purified Arp2/3 complex binds to the side of actin filaments, caps the slow-growing pointed ends of actin filaments with nanomolar affinity and promotes the formation of actin-filament branches that elongate only at their barbed ends; these findings led to the side-branching (or dendritic-nucleation) model for the protrusion of the lamellipodium at the leading edge of migrating cells.
Studies over the past decade have 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. In addition, since the discovery of the Arp2/3 complex, other actin nucleators (including formins and spire) have also been identified.
