To orchestrate complex processes like motility and endocytosis, cells rely on regulatory proteins that control the dynamics and architectures of the actin cytoskeleton. Actin filament nucleators are important actin regulators because they allow cells to control the initiation of new actin networks and dictate the architectures of these networks. Arp2/3 complex is a seven subunit actin filament nucleating machine that specifically generates branched actin filaments. Regulated branching by Arp2/3 complex is important not only for processes required for normal cellular function, such as endocytosis and protrusion of lamellipodia, but also in pathogenic processes like host cell infection by bacteria or metastasis of tumor cells. The majority of studies on Arp2/3 complex regulation have focused on WASP proteins, an activator that mediates propagation of branched actin networks. These studies revealed the basic tenets of WASP mediated activation; that WASP binds to Arp2/3 complex, recruits actin monomers, and stimulates an activating conformational change. However, the molecular aspects of WASP mediated-activation most important for understanding the biological function of the complex are still unknown. For instance, it is unknown why WASP proteins require actin filaments to activate Arp2/3 complex, a requirement that allows Arp2/3 complex to function specifically as a branched actin filament nucleator. Further, it is not known why WASP (but not other activators) must recruit actin monomers to Arp2/3 complex to trigger nucleation, despite the fact that this requirement regulates the density of branched filament ends in Arp2/3-assembled actin networks. In addition to WASP, several other regulatory proteins are now known to activate Arp2/3 complex. We recently discovered that the WISH/DIP/SPIN90 (WDS) proteins are distinct from WASP in that they can activate Arp2/3 complex without preformed filaments to induce it to nucleate linear actin filaments. These linear filaments serve as seeds to kickstart WASP-mediated branching thereby initiating actin network assembly. Despite the fact that initiation is perhaps the most important step in controlling branched network assembly, how WDS proteins activate the complex to create seed filaments is unknown. Furthermore, it is not understood how the activity of WDS proteins is coordinated with other activators like WASP, or how WDS proteins themselves are regulated. Here we propose a combination of biochemical, biophysical, structural, computational, and cell biological approaches to investigate these questions, with a long-term goal of understanding how these regulatory mechanisms allow Arp2/3 complex to control actin dynamics in complex cellular processes.
In this work, we are studying the cellular machinery that controls actin polymerization. Bacteria and viruses use this machinery to infect human cells and cancerous cells depend on it to spread. Therefore, improving our understanding of the molecules that constitute this machinery will contribute to our understanding of diseased states in humans and how to treat them.
