Intellectual Merit Criteria: The Drosophla model system has been instrumental in identifying many of the molecules and interactions involved in steering growth cones to their targets during nervous system development. Much work in this area has focused on the dynamic role of the cytoskeleton in directional growth cone movement; how axon guidance receptors signal to the cytoskeleton and how cytoplasmic proteins affect cytoskeletal dynamics. One of the key cytoplasmic regulators of cytoskelton dynamics is the Abelson tyrosine kinase (Abl). Genetic work from the Liebl and Seeger laboratories has shown that the guanine-nucleotide-exchange-factor protein Trio as well as the transmembrane neuronal cell adhesion molecule Neurotactin (Nrt) and Neurotactin's ligand Amalgam (Ama) are integrated into Abl-mediated signaling networks during growth cone outgrowth. Much of this proposal is designed to identify the molecular bases of these genetic interactions. Throughout this proposal Dr. Liebl exploits the strengths of the Drosophila model system. For example, we have identified a protein:protein interaction between Trio and Abl involving these proteins' SH3 domains. He proposes a structure/function analysis of Trio, assaying the ability of Trio deletion constructs, such as an SH3-deleted version of Trio, to rescue aspects of the trio mutant phenotype. We have identified Trio as a phosphotyrosine containing protein. He proposes to both mapping the Trio's major tyrosine phosphorylation site(s), we propose and to determine the in vivo relevance of Trio's tyrosine phosphorylation by testing for the rescue of trio mutant phenotypes with a version of Trio in which tyrosine phosphorylation sites have been mutated to phenylalanine. We have also initiated a genetic screen in which proteins that interact with Trio may be identified via mutation. Under this proposal we will map and clone the genes for these proteins. The cytoplasmic domain of Nrt is essential for its role as an adhesion molecule, and is Nrt's link into Abl signaling networks. A structure/function analysis of Nrt's cytodomain will be carried out integrating a Drosophila cell culture adhesion assay system, in vivo rescue of nrt mutant phenotypes, and comparative genomic approaches utilizing the Drosophila pseudoobsura and Anopheles gambiae (mosquito) genomes. The results of these and other proposed experiments will allow him to begin to understand the molecular underpinnings of the trio, Abl and nrt, Abl genetic interactions and the roles of these specific molecules in the growth cone. Through this work, a fuller, more accurate understanding of the molecular machinery controlling growth cone guidance will emerge.
Broader Impacts Criteria: This is a collaborative proposal between Dr. Liebl at Denison University, an undergraduate, liberal arts college, and Dr. Seeger at Ohio State University, a major research institution. This research collaboration has been active over the past five years. During this time undergraduate students at both Denison and Ohio State have been exposed to substantive research projects. The most recent publications resulting from this collaboration, in Neuron and Development, included ten undergraduate co-authors, seven of whom were women. Members of the Liebl and Seeger labs meet regularly to discuss data and exchange materials. Seeger's graduate students have "shadowed" Liebl to gain insight into a career at a liberal arts institution. Denison undergraduates are welcomed into Seeger's Ohio State lab in order to do experiments not technically possible in Liebl's Denison facilities. Thus this collaboration has proved to be a rich training ground for both graduate and undergraduate researchers, with nine undergraduates from Liebl's lab, and four undergraduates from Seeger's lab going on to post-graduate (M.D. and/or Ph.D., D.V.M.) study over the past six years. Both Liebl and Seeger regularly teach undergraduate courses with laboratory sections. Having an active research program and ongoing exposure to the intellectual culture of a major research institution has kept Liebl's teaching up-to-date. Being exposed to the liberal arts culture where teaching excellence is of high priority has invigorated and informed Seeger's teaching.
