Abstract
Dynamic formation and turnover of branched actin networks is essential for neuronal processes, such as membrane remodeling and synaptic vesicle trafficking, that occur during clathrin-mediated endocytosis (CME), the major pathway for receptor-mediated endocytosis in eukaryotic cells. During CME, the polymerization of branched actin networks generates the force necessary to drive endocytic membrane invagination and the inward movement of clathrin-coated pits (CCPs), which internalize important cellular factors that must be recycled. These branched actin networks are nucleated by the actin-related protein (Arp2/3) complex, a complex molecular machine that upon activation by a nucleation promoting factor (NPF) binds to the side of an existing ‘mother’ filament and nucleates the formation of a new ‘daughter’ filament, creating a 70 degree branch junction. Actin branches are inherently stable structures and in the absence of other cellular factors, they persist for 30-60 minutes in vitro before spontaneously dissociating. However, the lifetimes of actin branches in vivo are more than 100 times shorter (2-30 seconds), indicating that additional cellular factors are required to drive rapid pruning, or ‘debranching’. Daughter branch dissociation is critical for rapid actin network remodeling and turnover, yet the molecular mechanisms that enable cells to regulate debranching with temporal and spatial precision are only beginning to be understood. The goal of my thesis work has been to clarify our understanding of how cells control branched actin network turnover and how these mechanisms influence endocytosis and neuronal function. In Chapter 2, I describe a novel mechanism by which the actin branch stabilizer, cortactin, potently blocks/protects Arp2/3-actin branch junctions from debranching mediated by the debrancher, glia maturation factor (GMF). In Chapter 3, I discover that cortactin does not block GMF association with branch junctions but instead recruits GMF to branch junctions while simultaneously blocking its debranching activity. Further, I show that dynamin (shibire in Drosophila), which binds to cortactin and F-actin, synergizes with GMF in promoting debranching and is able to overcome the branch-stabilizing effects of cortactin. In Chapter 4, I explore the physiological effects of cortactin mutants on presynaptic actin dynamics and endocytosis at the Drosophila neuromuscular junction (NMJ). Finally, in Chapter 5, I show that the robust debranching activity previously described for budding yeast and fission yeast ADF/cofilins extends to all three human cofilin proteins (Cof1, Cof2, and ADF), demonstrating that it is a conserved activity in the cofilin family. Further, I find that cortactin, which completely blocks GMF-mediated debranching, has no effect on cofilin-mediated debranching, suggesting that the GMF- and cofilin-dependent debranching mechanisms are distinct and differentially regulated.