MOLECULAR BASIS OF AXONAL TRANSPORT

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The general aim of this research project is to develop a more complete understanding of the molecular interactions which determine how organelles are transported along microtubules in axons. Our proposed investigations are particularly concerned with how kinesin and dynein two soluble, force-generating ATPases that promote movement in opposite directions along microtubules, interact with specific populations of organelles, programmed to move either toward or away from the cell body. In recent experiments, the movement of purified organelles depended on the presence of an axoplasmic cytosol fraction containing many proteins in addition to kinesin and dynein. The specific aim of this proposal is to identify, more precisely, the particular cytosolic factors required for organelle movement. We will isolate, by AMP-PNP induced microtubule affinity, followed by salt or nucleotide induced release, a subset of cytosolic proteins form axoplasm, sufficient to promote organelle movement. Our proposed experiments will determine whether purified dynein and kinesin from axoplasm can alone drive organelle movement, or whether soluble "accessory factors" are also required, as suggested by previous studies. We will also explore the possibility, suggested by recent experiments, that both dynein and kinesin are required for retrograde organelle movement. We will do so in part by recombining the purified components and testing their ability to promote directed movement of organelles; in addition, electron microscopic studies of organelles will localize kinesin and dynein, to determine whether they are bound together on the organelle surface. Finally, nm-scale motion analysis, which previously indicated that kinesin-coated beads track along single protofilaments, while dynein-coated beads "wander" over the microtubule surface, will be applied to organelle movement to test if these same features of kinesin and dynein driven movement are apparent. Tracking of organelle movement at the nm level will provide an assay for molecular interactions between particular proteins in the reconstituted system.