During the current grant period we established that kinesin and cytoplasmic dynein are the motors most likely responsible for anterograde and retrograde, fast axonal transport (respectively) as well as microtubule- dependent organelle movements within cultured cells. In the next grant period we propose to define the composition of the organelle motility complex and to clarify how motility is regulated. Our in vitro studies of reconstituted organelle transport have defined an organelle motility complex which contains the motor, soluble accessory factor(s) and an organelle integral membrane protein. One accessory factor for cytoplasmic dynein is a 150 Kda protein which often copurifies with the motor. A kinesin accessory factor, which binds to kinesin, will also activate cytoplasmic dynein-dependent movements. In both the squid axonal and chick brain systems, extensive extraction of organelles with KI or sodium carbonate does not prevent organelle motility or motor binding to the organelles. This grant is the logical extension of those studies. We propose to define the components of attachment sites for motors on organelle surfaces using our polyclonal and monoclonal antibody probes against the two motors. These binding sites will be purified for reconstitution studies and production of antibody probes, which would be useful for cloning and identification in other organelles and tissues. With these probes for the motors, the accessory factors and the binding sites, we will analyze the systems in which motility is regulated and determine the mechanisms for directionality and activity regulation. We will continue our biophysical, biochemical and structural studies of the motors to understand the mechanisms by which they convert ATP energy into motive force. From all our studies, we hope to obtain a functional understanding of the molecular mechanisms of organelle motility and their regulation.
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