): In the cells of metazoan animals, motor proteins that move along microtubules drive the active transport of chromosomes, organelles and other macromolecular cargoes. Motor-based transport processes are fundamental for cell growth, division, signaling, and asymmetry. My lab is investigating the mechanisms that generate and control microtubule-based movements, taking advantage of the sophisticated genetics of Drosophila, its sequenced genome, and a variety of biochemical/cell biological approaches. To understand how motors move their cargoes, 3 aspects of each motor's activity must be defined: how productive motion is derived from chemical energy, how specific motor-cargo linkages are made, and how these two activities are controlled. Our knowledge of mechanochemical mechanisms has progressed rapidly, but linkage and regulatory mechanisms remain poorly understood. Our proposed studies are aimed at identifying motor protein functions, defining specific motor-cargo linkages and probing regulatory mechanisms. We previously discovered that ubiquitous kinesin-I, the founder of the kinesin superfamily, is an important anterograde motor for fast organelle transport in Drosophila axons; its specific cargoes remain uncertain. In the last funding period we used genetic enhancer screens to identify proteins that influence kinesin-l function in axons. Eight different loci were identified: Kinesin light chain, dynein, dynactin, ABL tyrosine kinase, ENMIASP, and 3 unknowns that we will characterize (EK2/1O, EK4, EK5). We propose to study specific motor-cargo relationships and the functions of the new fast axonal transport proteins using a GFP-based single organeIIe tracking approach in live neurons, as well as standard biochemical/cell biological approaches. ABL and ENA are particularly exciting; known to interact with the actin cytoskeleton, they now also appear to regulate kinesin-l in axons. Mapping of EK4 has placed it in a small region that includes 2-3 good candidate genes, one of which is a membrane protein implicated in mitochondrial motility. Pursuing kinesin-l functions in other tissues, we have discovered essential roles in axial patterning during oogenesis. Kinesin-l is required specifically for the localization of the posterior determinant oskar mRNA. It is also needed for Gurken secretion by the oocyte, which signals follicle cells to establish the dorsal pole. We propose to use biochemical interaction tests, immunolocalization, and genetic screens to determine how kinesin-l interacts with the oskar transport complex, and how it facilitates Gurken secretion.
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