Abstract
Actin filaments assemble into networks with distinct architectures to support different actin-dependent processes. Despite their different organizations, filament networks are interdependent and often transition from one type to another in response to shifts in the balance of competing regulatory activities. Although we know a lot about actin and its assembly into different structures, we still do not understand what drives the assembly of specific network architectures in different parts of the cell and why and how assembled networks transition from one type of architecture to another.
The overarching goal of my research is to answer the question: How do complex actin filament networks form and exist in the cell? In Chapter 1 of this dissertation, I will discuss what we know about actin networks, regulation of their assembly and stability and how studying the role of a novel actin regulator, the large GTPase dynamin2 dynamin2 (dyn2) in actin regulation can help us answer essential questions about how dynamic actin filament (F-actin) networks form and provide cellular function.
In Chapter 2, I will present my experimental research on the mechanism by which dyn2 specifies the architecture of branched F-actin networks. Previous data suggested that dyn2 regulates branched actin filament networks within lamellipodia of migrating U2-OS cells to organize assembly of contractile actomyosin bundles at the lamella. Other data demonstrated that dyn2, in complex with cortactin, bundles actin filaments in vitro. Based on these findings, I hypothesized that dyn2 and cortactin specify the architecture of transient bundled structures in lamellipodia to assemble actomyosin bundles at the lamella. Using biochemical and microscopic assays with reconstituted F-actin networks in vitro, I determined that dyn2, in complex with cortactin, bundles branched F-actin networks. Through the inhibition of F-actin nucleation, dyn2 promotes the formation of unbranched filaments that are subsequently bundled by a dyn2-cortactin complex. Dyn2/cortactin bundles selectively bind α-actinin, and GTP hydrolysis influences α-actinin binding to bundles. Together, these data provide a novel role for dyn2 and cortactin in promoting maturation of branched Arp2/3 complex-dependent networks into networks of filament bundles.
In Chapter 3, I propose a mechanism by which dyn2 regulates actin networks in lamellipodia to organize actomyosin assembly at the lamella and discuss future directions of this research. I speculate that transient dyn2/cortactin-dependent bundles in lamellipodia may co-assemble with myosin II filaments to promote the formation of actomyosin bundles at the lamella. The mechanism by which dyn2 bundles F-actin networks can be used in other cellular processes and structures where both Arp2/3 complex and dyn2 localize, such as endocytosis, exocytosis, phagocytosis, actin comets, filopodia, invadopodia, and cell-cell contacts.
