The long range goal of this research is to provide a better understanding of the mechanism of movement of intracellular organelles along microtubules. Such movement plays a special role in the process of fast axonal transport in nerve cells. This process provides one means for the movement of newly synthesized materials from their site of synthesis in the body of a nerve cell to the synapse at the end of the axon. Similar motility processes, however, also are likely to play an important roles in all eukaryotic cells. For example, the directed movement of membranous organelles has been implicated in the extension of the endoplasmic reticulum and mitochondria away from the nuclear region and in the directed movement of some classes of secretory vesicles towards the plasma membrane. The protein kinesin has recently been isolated and shown to be a motor for driving movement along microtubules in the anterograde direction (corresponding to movement in a nerve cell away from the nuclear region and toward the periphery). The energy for this movement is derived from hydrolysis of adenosine triphosphate (ATP), and purified kinesin has ATPase activity which is stimulated by microtubules.
The aim of this project is to determine the detailed enzymatic mechanism of ATP hydrolysis by the kinesin/microtubule motility system with emphasis on how hydrolysis is coupled to movement. Recent progress indicates that kinesin undergoes a large conformational change as a function of ionic strength and a major component for the upcoming grant period is the further characterization of this conformational change and its possible significance for the regulation of kinesin. An important approach will be the characterization of individual domains of kinesin and their interaction. These domains will be obtained by limited proteolysis and by expression of fragments of kinesin cDNA. Resolution of these issues will allow a more detailed kinetic analysis to be undertaken on the forms of kinesin which are properly activated. Extensive use will be made of steady state and single turnover kinetics and immunological approaches. The combined information which will be available from these studies will allow the formulation of a detailed model for mechanism of motility induced by kinesin and its role in cellular processes.
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