REGULATION OF VESICLE TRAFFIC IN NEURONS & RELATED CELLS

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Neurons and neuroendocrine cells possess an intricate cytoplasmic machinery for the transport and release of membrane-bounded organelles. This organelle trafficking serves a multitude of purposes: to supply proteins to the axon, which cannot synthesize its own resident proteins; to deliver secretory products, such as neurotransmitters and catecholamines, to the cell surface; and for retrograde transport of endocytosed trophic factors. Collectively, these types of organelle movements are essential for the development and maintenance of the neuronal phenotype, and for the specialized secretory functions of neuroendocrine cells. Two types of cytoskeletal polymers play pivotal roles in organelle trafficking. Microbutules serve as the tracks for long range organelle motility, while dense meshworks of microfilaments must be traversed by motile organelles near the cell surface and in cytoplasmic regions immediately surrounding microtubules. The broad purpose of the experiments outlined in this proposal is to characterize mechanisms for regulating organelle transport in neuronal and neuroendocrine cells. It is evident that regulation must occur because organelles capable of being transported along microbutules are not always in transit, and often are capable of moving in either direction relative to microtubule polarity. How, then, does an organelle know when to move along a microtubule and which way to travel? Moreover, do changes in the number and organization of microfilaments affect the ability of organelles to travel through the cell, as evidence suggests, or do microfilaments serve different roles in organelle trafficking? Questions such as these will be addressed in the proposed study. Research will focus on three distinct and complementary levels at which regulation is likely to occur. The first Specific Aim concerns the role of phosphorylation in regulating the functions of kinesin. This microtubule- stimulated ATPase is evidently responsible for anterograde transport of membranous organelles in the axon, and for related organelle motility elsewhere in neurons and in other cell types. Recent evidence from the laboratories of the PI and Co-PI has indicated that the kinesin heavy and light chains become phosphorylated in vivo. The effects of phosphorylation on the ATPase, microtubule binding and organelle binding activities of kinesin will be investigated. The second Specific Aim is focused on the role of MAPs in regulating access of kinesin to the microtubule surface, and on the differential abilities of various brain tubulin isoforms to interact with kinesin. The final Specific Aim will deal with a small G protein recently discovered by the PI's laboratory to interact with the cytoskeleton. This G protein will be cloned and sequenced, and its biological role will be characterized by a combination of cell biological and biochemical approaches.