The neuron has no rival as a cell type that specializes in transport of membrane-bounded organelles (MBOs) along microtubles (MTs). This distinctive trait of the neuron is dictated by its unique morphological and functional properties. During neurogenesis, for example, the formation of dendrites and axons requires sustained delivery of nascent plasma membrane proteins and lipids into growing neurites. These membrane precursors are packages in vesicle whose surface-associated motor molecules move the vesicle along MTs to the distal tips of neurites, where fusion of the vesicles with the plasma membrane promotes neurite elongation. In differentiated neurons, the maintenance of axons is equally reliant upon MTs. Because the axon lacks protein synthesis machinery, but can be more than 10,000 times as long as the diameter of the perikaryon from which it emanates, the axon must constantly be supplied with new resident proteins to replace those that age and degrade. New axonal proteins are synthesized in the perikaryon, and many, like those destined for incorporation into the axolemma, are packaged in vesicles that use motor proteins to move great distances along MTs toward the axon terminal. Likewise, the precursors of synaptic vesicles (SVs), the ultimate specialized products of neurons, are delivered from the cell body, where they are manufactured, to the distal end of the axon by MT- based transport. Finally, endosomal and pre-lysosomal vesicles carry degraded axonal components and edocytosed neurotrophic factors along axonal MTs in the retrograde direction, toward the perikaryon. This renewal application comprises 3 Specific Aims based on progress made during the present funding period. 1) Reconstituted motility systems will be sued to identify and characterize regulatory factors for MBO transport in brian and adrenal chromaffin cells, with emphasis on transport of SVs and chromaffin granules. The hypothesis that MBO transport along MTs is regulated in an organelle-specific manner will be tested. 2) The structure of a newly discovered complex of MTs and protein phosphatase 2A (PP2A) will be determined. A hypothesis to be tested is that MT-bound PP2A regulates MBO transport along MTs by controlling the phosphorylation state of components of the transport machinery or by regulating MT stability. 3) The role of MTs in maintaining the structure of caveolae will be tested. New data indicate that caveolin, a resident protein of caveolae, cycles constitutively between the plasma membrane and the Golgi by a mechanism that requires MTs for transport toward, but not away from the Golgi. A test will be made of the hypothesis that other resident caveolar proteins also normally more between the plasma membrane and the golgi by a MT- dependent cycle. The hypothesis that dynein is the motor for transport of caveolin to the golgi will be also tested, and the mechanism for caveolin transport from the Golgi back to the plasma membrane will be determined.
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