The long term goal of this project is to elucidate the in vivo function of microtubule proteins in mitosis and cellular morphogenesis. The specific approach makes use of the model system, Drosophila melanogaster, and utilizes biochemical, molecular biological, and genetic analyses to elucidate the in vivo functions of proteins characterized in vitro. This application focuses on one member of each of two classes of proteins, microtubule-associated proteins and microtubule-motility proteins. The first molecule that we will study is the 205K microtubule-associated protein, which is a constituent of the mitotic spindle and interphase cytoskeleton. The in vivo function of the 205K MAP in mitosis and morphogenesis will be determined by phenotypic analysis of 205K MAP mutants. In addition, if we find that the 205K MAP has a role in microtubule function in mitosis or morphogenesis, we will probe its role in detail through the use of in vitro mutagenesis. The second protein that we will analyze is kinesin. Kinesin is a molecule that generates microtubule movement in vitro, and which current evidence suggests is a mitotic spindle component and an element of the axonal transport system. The in vivo function of kinesin will primarily be studied by analysis of kinesin mutants, and by inhibition of kinesin expression in cultured cells using inducible expression of anti-sense kinesin RNA. To develop sufficient understanding of kinesin so that mutations can be generated in vitro and studied in vivo, and to identify domains required for in vitro functions, we will also analyze the structure and function of kinesin by sequencing and deletion analysis. To elucidate the function of other mitotic spindle components, we will use genetic analysis to study new mitotic spindle proteins. These new proteins will be identified by three methods: a) production and analysis of monoclonal antibodies; b) nucleotide homology to existing genes; and c) homology to consensus elements of existing genes such as those encoding microtubule- binding regions and nucleotide binding regions. Genetic and phenotypic analysis of mutations disrupting these proteins can then be used to study their in vivo roles. Elucidation of the in vivo functions of the 205K MAP, kinesin, and other mitotic spindle proteins will provide considerable information about the mechanisms of mitosis and cellular morphogenesis, and may shed light on human diseases such as cancer and birth defects.
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