The goals of this project are to decipher the mechanisms that regulate the actin and microtubule cytoskeletons, the structures underlying neural cell behaviors including morphology, polarity, adhesion, process elongation, motility, navigation, connectivity, and plasticity. To change their size, shape, and connectivity, neurons require actin and tubulin proteins to assemble together into long polymers (F-actin and microtubules, respectively) – and numerous extracellular stimuli have now been identified that alter the assembly and organization of these cytoskeletal structures. Yet, we still know little of how these extracellular cues exert their precise effects on the cytoskeleton. To better understand these mechanisms, my lab has been focusing on one of the largest families of extracellular cues, the Semaphorins (Semas) – which alter neuronal behaviors by eliciting destabilizing effects on both F-actin and microtubules. Our strategy has been to use model organisms and screening approaches to search for proteins that work in the signal transduction cascade utilized by Semas and their Plexin receptors. Among the proteins that we have identified, is a new family of intracellular proteins called the MICALs that are required for Sema/Plexin signal transduction. Now, work in my lab during the previous funding cycle of this R01 has revealed that the MICALs employ a previously unknown Redox signaling system to control the actin cytoskeleton. Namely, we have found that Mical is a novel F-actin disassembly factor – and our results reveal that Sema/Plexin-mediated reorganizations of the actin cytoskeleton can be precisely achieved in space and time through activation of Mical. We have also found that the MICALs belong to a class of oxidoreductase (Redox) enzymes and that Mical employs its Redox enzymatic activity to alter the properties of F-actin. Our work has gone on to identify that Mical uses F-actin as a direct substrate and post- translationally oxidizes conserved amino acids on actin, simultaneously dismantling F-actin and decreasing polymerization. Moreover, we find that this Sema/Plex/Mical-mediated Redox regulation of actin is reversible (by a protein called SelR/MsrB) – and that this specific reversible Redox actin regulatory system directs multiple different biological processes in neuronal and non-neuronal tissues. Therefore, I hypothesize that Sema/Plexin guidance cues utilize a reversible Redox signaling mechanism composed of Mical and SelR to directly and spatiotemporally coordinate cytoskeletal remodeling to drive cellular form and function. I propose to test this hypothesis by following-up on several lines of preliminary observations that illuminate critical molecular mechanisms of Sema/Plexin/Mical-mediated cytoskeletal reorganization including 1) specific types of F-actin/networks of F-actin that are most responsive to Sema/Plex/Mical effects, 2) molecular interactions that allow Sema/Plexins to coordinate the disassembly of the actin and...