The Abl and Arg nonreceptor tyrosine kinases regulate neuronal migration and morphogenesis in the developing mouse brain. Abl and Arg relay information from cell surface receptors to promote cytoskeletal rearrangements. The goal of this proposal is to understand the molecular mechanisms by which Abl and Arg regulate cytoskeletal structure and function in developing neurons.
Our first aim i s to elucidatetheroles for Abl and Arg in neuronal development. Our morphometric analysis of dye-filled neurons has shown that dendrite arbors are reduced in arg-/- hippocampal neurons relative to wild type. Neurons are more tightly packed in the cerebral cortices of brain-specific-abl-/-arg-/- double knockout mice. This phenotype may reflect a severe deficit in dendrite arborization. We also find that a subset of cerebellar granular neurons fail to migrate normally in the brain-specific-abl-/-arg-/- mice. We will study neuronal differentiation in knockout mice and in explanted neurons from these mice to examine how mutations in genes encoding components of Abl- and Arg-signaling pathways affect dendrite morphogenesis and cerebellar granular neuron migration.
Our second aim i s to understand how Arg regulates cytoskeletal structure in developing neurons. Arg overexpression leads to increased membrane ruffling and decreased cell motility in fibroblasts and increased neurite branching in cultured cortical neurons. We have identified two distinct pathways by which Arg can regulate cytoskeletal structure: 1) the Arg C-terminal half can bundle F-actin and crosslink F-actin bundles to microtubules; and 2) Arg can phosphorylate and activate the p190 Rho GTPase-activating protein (p190RhoGAP) to inhibit the cytoskeletal regulator Rho. We will express Arg and/or p190RhoGAP mutants in cultured fibroblasts to dissect how these two pathways contribute to the regulation of cytoskeletal structure and function.
Our third aim i s to determine how Abl and Arg kinase activities are regulated in developing neurons. We have shown that in vitro phosphorylation of Abl and Arg at several sites can regulate their kinase activities, but it is unclear how Abl and Arg become phosphorylated in developing neurons. Progress in this area has been limited because the cell surface receptors that regulate Abl and Arg kinase activity in developing neurons have not been identified. Genetic and biochemical experiments have identified a small number of candidate receptors (the Robo receptors, TrkB, integrin ?5) that are likely to regulate Abl and Arg kinase activity in developing neurons. We will determine whether these receptors regulate Abl and Arg kinase activity and examine whether Abl and Arg mediate migratory or morphogenetic signals from these receptors.
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