DESCRIPTION (provided by applicant): The goals of this project are to characterize a new biological mechanism that has direct application to the regulation of the actin cytoskeleton - the structure underlying neural cell behaviors including morphology, polarity, adhesion, process elongation, motility, navigation, connectivity, and plasticity. In order to change their size, shape, and connectivity, neurons require actin proteins to assemble together into long filaments. Adjusting the length and organization of these actin filaments (F-actin) specifies the direction of movement and enables cells to precisely connect and communicate with one another. A number of extracellular cues have now been identified that control actin dynamics, but we know little of how these signals present outside of cells exert their precise effects within cells. Semaphorins (Semas) are one of the largest families of these guidance cues and they regulate cellular behaviors by eliciting destabilizing effects on F-actin that include a loss of F- actin and the decreased ability to polymerize new F-actin. Importantly, recent breakthroughs have identified cell-surface receptors and intracellular proteins that are essential for Sema-mediated effects on actin but we still know little of the molecular mechanisms that directly regulate F-actin in response to Semas. To identify these molecules and mechanisms we have identified proteins that associate with the Sema receptor Plexin, including a novel family of cytosolic proteins called the MICALs. There is one MICAL gene in invertebrates and three MICAL genes in mammals and they control axon guidance, synaptogenesis, dendritic pruning, and other morphological changes mediated Semas/Plexins. Indeed, our recently published results reveal that MICAL provides a long-sought-after direct link between Semas/Plexins and the modification of the actin cytoskeleton. We find that MICAL directly disassembles F-actin and is both necessary and sufficient for regulating actin dynamics downstream of Semas/Plexins. These new results provide an underlying logic through which Sema- mediated reorganizations of the actin cytoskeleton can be precisely achieved in space and time: through direct Sema-Plexin activation of the novel actin disassembly factor MICAL. Interestingly, MICALs also contain an oxidoreductase (Redox) enzymatic moiety and our results strongly suggest that MICAL utilizes its Redox activity to alter F-actin, implicating for the first time a role for specific Redox signaling events in actin cytoskeletal regulation. Therefore, I hypothesize that MICAL enzymes are a novel family of phylogenetically conserved actin disassembly factors that utilize a previously uncharacterized reversible Redox signaling mechanism to directly regulate actin dynamics. To test this hypothesis, I propose to combine genetics, cell culture, and cell biological approaches using both invertebrate and vertebrate model systems with biochemical, structural, and high-resolution imaging assays utilizing purified MICAL and actin proteins. Understanding how this unusual family of enzymes, the MICALs (which are unlike any proteins that have ever been characterized) causes F-actin to disassemble will reveal new strategies to regulate neural cell biology and behavior. PUBLIC HEALTH RELEVANCE: Our nervous system controls such remarkable abilities as learning, speaking, and walking only because our neurons communicate in highly organized networks. The goal of this proposal is to characterize the biochemical mechanisms that enable neurons to find and connect with one another during development and maintain these proper connections through-out adulthood. Understanding how these neuronal networks are assembled, integrated, and maintained will reveal fundamental mechanisms underlying thought, emotion, and behavior, identify therapeutic strategies for neurological diseases and addictive behaviors, and contribute to healthy recovery following neural trauma.

Our nervous system controls such remarkable abilities as learning, speaking, and walking only because our neurons communicate in highly organized networks. The goal of this proposal is to characterize the biochemical mechanisms that enable neurons to find and connect with one another during development and maintain these proper connections through-out adulthood. Understanding how these neuronal networks are assembled, integrated, and maintained will reveal fundamental mechanisms underlying thought, emotion, and behavior, identify therapeutic strategies for neurological diseases and addictive behaviors, and contribute to healthy recovery following neural trauma.