The Arp2/3 complex was the first actin nucleator identified, and years of study have outlined its mechanism of action and its regulation. Alone, Arp2/3 complex nucleates actin inefficiently, and thus requires an activator (e.g. WASp). Arp2/3 complex associates with pre-existing filament sides, and then nucleates new filaments at a 70 degree angle, creating branched structures. When concentrations of existing actin filaments are low, Arp2/3 complex can be directly inhibited by coronin. While this general view of Arp2/3 mechanism and regulation has been established for several years, many important questions remain unanswered. What surfaces on Arp2/3 complex mediate filament side binding? Is association with the side of an existing filament a requisite step in nucleation? Is filament side binding and branching by Arp2/3 complex critical for its functions in vivo? What is the precise molecular mechanism by which coronin inhibits Arp2/3 complex?

The work presented in this thesis attempts to answer some of these questions by generating and using new mutants of the p35/ARPC2 subunit of the Arp2/3 complex. Previous observations suggested that p35/ARPC2, which is centrally located in the complex, is poised to accept signals from F-actin and coronin binding to change the conformation and activity of the Arp2/3 complex. Thus, it can be viewed as a structural and functional hub. In Chapter 2, I present a systematic mutagenesis of the conserved surfaces on p35/ARPC2, in which mutant alleles are analyzed genetically and biochemically. The data revealed that three separate sites on p35/ARPC2 are required for cell growth, endocytosis and in vitro Arp2/3-mediated actin nucleation. In Chapter 3, I focus on coronin and its mechanism of interaction with the p35/ARPC2 subunit by which it inhibits Arp2/3 complex. I also define sequences in the coronin coiled-coil domain important for inhibiting Arp2/3 and explore how mutations in this domain affect the ability of coronin to arrest cell growth upon overexpression.

Together, these new findings provide a more detailed view of the inner workings of the Arp2/3 complex, and how it performs its functions as a regulated biological machine.