Abstract
Cells generate highly dynamic branched F-actin structures which are found in different locations to facilitate specific cellular processes. For example, they are found at sites of endocytosis, phagocytosis, and at the leading edge of migrating cells. These densely arborized actin networks are assembled by the Arp2/3 complex, which is conserved across the animal, plant, and fungal kingdoms of life. Upon activation by a nucleation promoting factor (NPF), the Arp2/3 complex binds to the side of an existing mother actin filament and nucleates the polymerization of a new daughter filament at a 70° angle, creating a branched actin filament structure. The Arp2/3 complex remains at the branch junction as an integral structural component holding the mother and daughter filaments together. These branches are inherently stable structures and spontaneously dissociate in vitro only after about ~30-60 minutes. In contrast, branches in living cells exist for only 3-30 seconds before they are ‘pruned’. This process of dissociating the branches shortly after they are formed is thought to help drive rapid actin network remodeling and turnover. However, the mechanisms by which cells catalyze debranching with precise timing are still not well understood. In Chapter 2 of this thesis, I describe a novel mechanism by which two distinct binding partners of the Arp2/3 complex, yeast coronin and GMF, directly interact, form a ternary Crn1-Gmf1-Arp2/3 complex in solution, and synergize in inducing filament debranching. In Chapter 3, I explore the in vitro biochemical relationships and combined effects of mammalian GMFβ with Arpin, coronin-1B, and coronin-7 on Arp2/3 complex-mediated actin assembly and branch turnover