Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation.
Cells constantly sense, and react to, their environments. They can monitor or alter their surroundings by taking up or secreting various substances, and may also migrate toward food supplies, or toward signaling molecules—for example, to clot blood or heal wounds. These actions depend on the cytoskeleton, a protein meshwork that gives cells their shape; allows them to transport materials into, out of, or across their cytoplasms; and enables them to move.
The filaments of the cytoskeleton are constructed from several different types of proteins, one of which is called actin. In response to signals, actin can assemble into linear filaments, or can form branches with one end anchored on an existing filament. Branch formation requires the Arp2/3 complex, which initiates and anchors branches on existing filaments, and also various ‘nucleation-promoting factors’ (NPFs), which turn on the branching activity of the Arp2/3 complex.
Two types of NPFs have been identified: type I interact with individual actin molecules, while type II bind to actin filaments. Previous work has shown that type I NPFs—including the N-WASP protein—have a specialized domain called VCA that binds to both the Arp2/3 complex and to actin molecules. VCA brings actin molecules to the branch site, which initiates branch formation, but how N-WASP collaborates with type II NPFs to build branches is not well understood.
Helgeson and Nolen now examine how a type II NPF called cortactin works with the Arp2/3 complex and N-WASP to construct new branches on actin filaments in vitro. Cortactin appears to displace the VCA domain of N-WASP to stimulate branch formation, and then to remain associated with—and stabilize—the growing branch. Helgeson and Nolen suggest that these NPFs work together to create branches using an “obligatory displacement” model. According to this scheme, N-WASP (or another type I NPF), the Arp2/3 complex and two actin molecules are bound at the site of a future branch on an actin filament, poised for branch formation. However, before more actin molecules can be added, N-WASP must be released, either slowly on its own—as Smith et al. also report in findings published concurrently in eLife—or rapidly with the help of cortactin or other type II NPFs.
Although the rationale for N-WASP removal is not yet understood, type I NPFs are generally attached to the plasma membrane. When N-WASP releases the mother filament, the membrane should no longer be able to block the addition of actin molecules to a growing branch.