The discovery of a new class of mechanochemical ATPases, the kinesins, with properties suitable for a motor for fast axonal transport opened an exciting new area of investigation for neurobiologists interested in the molecular mechanisms of fast axonal transport. An understanding of the molecular basis for movement of membrane bounded organelles is critical to our understanding of many aspects of neuronal function and neuropathology. Functions as diverse as generation and maintenance of synaptic structures, regulation of neural activity, and neuronal development and regeneration depend critically on fast axonal transport processes. Moreover, many neuropathological conditions including diabetic and toxic neuropathies, amyotrophic lateral sclerosis, and degenerative of the central nervous system may involve defects of axonal transport. A multidisciplinary approach will be used to evaluate the molecular and function of neuronal kinesins. Electron microscopic, biophysical and biochemical methods will evaluate the functional architecture of kinesin and characterize conformational changes associated with hydrolysis of ATP and translocation of membranous organelles. Molecular genetics approaches are designed to evaluate the primary sequence, gene structure, and expression of kinesin in the nervous system. A library of monoclonal antibodies will be used for quantitative immunochemistry and immunocytochemistry of kinesin. Particular attention will be paid to determining the cellular and subcellular distribution of kinesin isotypes in neuronal tissues. Comparisons of transport will be made between three models for analysis of motility in neurons: isolated axoplasm from the squid giant axon, neuronal cell culture, and an in vitro reconstituted model for organelle translocation in order to identify the biochemical and structural features of the neuron that are involved in the movement of material in fast axonal transport. The discovery of multiple isoforms suggest several specific mechanisms for regulation of both anterograde and retrograde fast axonal transport including posttranslational modifications of kinesin and different genetic isotypes. Factors that modulate the binding of kinesin to membrane bounded organelles and microtubules will be identified and characterized. Experiments proposed in this application represent an integrated approach leading to the first detailed picture of the mechanisms of fast axonal transport at the molecular level.
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