Spatiotemporal Control of Actin Cytoskeletal Machinery in Cells: Multivalency Mediated Protein Clustering at the Plasma Membrane and Optogenetic Tool Development

Precise regulation of actin cytoskeleton dynamics is crucial for numerous cellular processes. My thesis work has demonstrated two different mechanisms that cells use to achieve spatiotemporal control of actin assembly -- multivalency mediated phase separation and subcellular activation of actin polymerization machinery. The plasma membrane of the living cell is believed to function as a dynamic platform for signal transduction, rather than existing simply as a static barrier between intracellular and extracellular environments. Moreover the membrane is organized into distinct lipid and protein microdomains. Recent work has shown the importance of protein assembly at the membrane in the formation of these domains. However, the mechanisms by which proteins organize at the membrane are largely unknown. Here I demonstrate that multivalent interactions among proteins in the Nephrin/Nck/N-WASP actin-regulatory pathway can drive clustering of the components at the plasma membrane in cells. Generation of the micronsized domains is dependent on the phosphorylation-mediated interaction between the transmembrane protein, Nephrin, and its effectors, Nck and N-WASP, in the cytoplasm. These structures are dynamic and associated with increased actin polymerization activity, suggesting the importance of clustering in local rearrangements of the cytoskeleton. My studies suggest that multivalent interactions between the signaling proteins can, in general, contribute to spatiotemporal regulation of cellular processes by clustering of molecules at the plasma membrane. Precise spatiotemporal regulation of protein activity is a key feature of cellular signaling pathways. While contemporary tools in biology have limited control in space and time, recent developments in optogenetics have demonstrated that light can be used as a general tool to investigate protein function in vivo. Many optogenetic tools, however, indirectly regulate protein activity by modulating expression level or cellular localization. Other optogenetic tools with direct control of protein activity have limited applications. Here I demonstrate a method based on fragment complementation that can be generally applicable to a protein of interest based on the light-dependent interaction of Phytochrome B (PhyB) and phytochrome interacting factor 3 (Pif3). My method allows subcellular control of protein function via light-induced reconstitution of a split protein. In particular, I show that light-dependent activation of SopE, a bacterial GEF protein, induces membrane ruffling in a living cell. Furthermore, as an application to inter-molecular regulation between two different molecules, I induced light-mediated interactions between Rho-GTPase Cdc42 and its effector Wiskott-Aldrich Syndrome Protein (WASp) to promote filopodia formation in HeLa cells. Using these optogenetic tools, I demonstrate a general method that can be used to study a broad range of proteins and protein-protein interactions.