A normal functioning human nervous system requires the interconnection of billions of neurons. Improper formation or maintenance of these connections leads to neurological abnormalities that result in a number of mental diseases and disorders. How are these circuits assembled and integrated? The semaphorins are one of the largest protein families involved in the formation and maintenance of axonal connections. Semaphorins are phylogenetically conserved secreted and transmembrane proteins found in invertebrates and in vertebrates. Many semaphorins utilize plexins, a family of large transmembrane proteins as receptors. How plexins actually transduce semaphorin signals is poorly understood but is of importance for learning how semaphorins sculpt and maintain the nervous system. I recently identified Drosophila MICAL, a large, multidomain, cytosolic protein. MICAL is expressed in axons, interacts with the neuronal plexin A (PlexA) receptor, and is required for Drosophila semaphorin la (Sema-la)/PlexA mediated axon guidance and connectivity. The MICAL protein contains several domains known to interact with the cytoskeletal machinery necessary for axonal extension. Furthermore, MICAL contains a flavoprotein monooxygenase domain, the integrity of which is required for Sema-la-PlexA axonal circuit formation. The presence of this flavoprotein monooxygenase domain implicates for the first time oxidation-reduction signaling mechanisms in semaphorin-mediated axon guidance. This proposal focuses on characterizing the role these unusual proteins play in axonal connectivity. There are three MICAL proteins encoded in the mammalian genome that are highly similar to Drosophila MICAL. My preliminary analyses show that they are also neuronally expressed and are plexin-interacting proteins. Combining in vivo genetic approaches with in vitro gene transfer approaches, I will determine if these vertebrate MICAL proteins play a role in the development of the vertebrate nervous system. My results also suggest that MICALs are excellent candidates for orchestrating the cytoskeletal alterations associated with semaphorin-mediated axonal connectivity. To further define the role played by MICAL in semaphorin/plexin signaling, I will use biochemical approaches to determine whether MICAL interacts with the neuronal cytoskeleton and several proteins that regulate its assembly. The results from the proposed experiments, as well as the conceptual and technical training I will acquire, will supplement my experimental background and promote my transition to an independent, productive research program as the principal investigator of an academic laboratory.
