Dissecting the Mechanism of Activation of Arp2/3 complex by WASP family proteins and other Nucleation Promoting Factor Proteins

Arp2/3 complex controls assembly of actin networks by nucleating branched actin filaments in response to cellular signaling pathways. These signaling pathways converge on activator proteins called NPFs (nucleation promoting factors) that bind to the complex and switch on its nucleation activity. Arp2/3 complex must also bind ATP, actin monomers, and (in most cases) actin filaments to nucleate a new filament. We are interested in understanding how all of these activating factors cooperate to influence the conformation the complex to turn on its activity. In one recent study, we used single molecule total internal reflectance microscopy to determine how two biochemically distinct classes of NPF proteins, cortactin and N-WASP, work together to synergistically nucleate branched actin filament networks. Cortactin and N-WASP are both required to assemble actin in invadopodia, cellular protrusions that form in metastasizing cells, so these studies are relevant to understanding regulation of actin dynamics in cancer cells.

Understanding how branched actin networks are initiated

In most contexts, Arp2/3 complex creates exclusively branched actin filaments. Biochemically, this property of Arp2/3 complex derives from the requirement that it bind to pre-existing filaments in order to nucleate new filaments. This sets up an apparent paradox: if the complex requires preformed filaments for branching nucleation, what is the source of the initial substrate filaments to create branched networks? We recently discovered a new class of Arp2/3 complex activators, the WISH/DIP/SPIN90 proteins (WDS) that can activate the complex without a preformed filament. Remarkably, these activators create linear filaments when they turn on the nucleation activity of the complex. Linear filaments seeded by Dip1 can then activate WASP-bound Arp2/3 complex. We are currently dissecting the mechanism of this new class of activators and exploring how they coordinate with WASP family NPFs in vitro and in vivo to assemble branched actin networks.

Small molecule inhibitors to control actin cytoskeletal dynamics

Small molecule inhibitors provide important tools for studying the protein function both in vitro and in vivo. In collaboration with Cytokinetics, we discovered two small molecule inhibitors of Arp2/3 complex. While these inhibitors have been widely used by cell biologists studying the actin cytoskeleton, how they inhibit activation of the complex was unknown. We recently showed that both classes of inhibitors block a key structural rearrangement of the complex required for activation. In collaboration with Zoe Cournia (Bioacademy, Athens), we have also developed more potent versions of these small molecule inhibitors that could serve as a starting point for creating high affinity compounds with clinical value.