The objectives are to continue our studies to: 1) identify, isolate and characterize intracellular pools of membrane bound glycoconjugates undergoing fast axonal transport, 2) to characterize different functional compartments of the neuronal cytoskeleton related particularly to phosphorylation of component and associated polypeptides of cytoskeletal organelles. Immunocytochemical analysis will be used to study the distribution of glycoprotein antigens in cerebellum whose biochemical properties mimic the cytochemical properties of specialized smooth membrane systems in Purkinje cell axons and dendrites. We will use specific radioisotope labeling together with pharmacological manipulations of oligosaccharide processing and axonal transport to identify the origin and fate of newly synthesized glycoconjugates destined for fast axonal transport in retinal photoreceptor and ganglion cells. Enzyme-linked cytochemical, autoradiographic and biochemical analysis will be used to begin to characterize oligosaccharide chains of transported glycoconjugates compared to those that have reached the presynaptic terminal of retinal neurons. We will use immunocytochemical and immuno-blot analysis of tissue sections, cultured neurons and isolated fractions of cytoskeletal organelles to study the intracellular compartmentalization of the phospholipid/Ca2+-dependent protein kinase (PKC) and its substrates in axons. Endogenous and exogenous PKC will be used in in vitro phosphorylation experiments for comparison with in vivo labeling patterns to elucidate specific substrates for the enzyme among the cytoskeletal polypeptides. Retinal tissue slices and cultures of retinal and sympathetic ganglion neurons will be used to ask the question whether or not PKC translocates from the cytosol to the plasma membrane of neurons in response to physiological stimulation or pharmacological activation of PKC. We will use non-hydrolyzable analogs of ATP to probe microtubule deficent axons of Myxicola infundibulum for the presence of """"""""kinesin"""""""" type translocator molecules. Kinesin preparations from rat brain will be used to test for the presence of axonal polypeptides associated with the cytoskeleton, particularly the tau proteins and PKC.
