Our broad goal is to understand how cells deliver critical components to specific destinations. Such transport processes, driven by molecular motors that walk on cytoskeletal filaments, are essential in almost all cells of humans and other complex organisms. The proposed work will be done in the oocytes and neurons of Drosophila, a powerful experimental model system for studying transport and human disease mechanisms. Oocytes provide a unique opportunity to study a simplified transport mechanism. Fast streaming, a continuous movement of ooplasm, needs only one motor, kinesin-1, the motor is in a constitutively """"""""on"""""""" state, and its linkage to cargo organelles may be relatively direct. Genetic, dominant negative, and biochemical approaches will be used to identify parts of kinesin-1 and associated proteins that link its movement to the movement of ooplasm. The results will illuminate fundamental aspects of motor- cargo linkage and help illuminate developmental pattern formation. Within neurons, results indicate that multiple types of motors cooperate in axonal transport of mitochondria and the large dense-core vesicles (DCVs) that bear peptide neurotransmitters. Time-lapse microscopy, digital organelle tracking in whole nervous systems, and powerful statistical approaches will be used in combination with genetic mutations to determine the specific sets of motors that transport mitochondria and DCVs. Novel fast-acting temperature- sensitive mutations and a fast thermal microscope stage will be engineered for rigorous tests of direct motor functions and motor-motor interactions. Also in neurons, novel regulators of mitochondrial transport will be identified and their mechanistic contributions investigated. This project uses as a springboard, our discovery that a JNK scaffolding, kinesin-binding protein (APLIP1) strongly influences retrograde mitochondrial transport. A novel genetic screen will be used to identify additional components of that control mechanism and their roles will be studied using genetics, biochemistry, and molecular approaches. Because organelle movement is such an important factor in the function and survival of neurons, defining the underlying linkage and control mechanisms is critical for understanding causes of neurodegeneration. Manipulation of transport mechanisms has great promise for future therapies that will slow the onset of the debilitating symptoms of ALS, Alzheimer's and other neurodegenerative diseases. ? ? ?
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