CHARACTERIZATION OF SYNAPTIC VESICLES AND RELATED NEURONAL FEATURES IN NERVE GROWTH-FACTOR AND RAS ONCOGENE DIFFERENTIATED PC12 CELLS

PC12 cells can differentiate into neuron-like cells after treatment wi th either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene, The concomitant treatment of NGF p lus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most s trikingly, the number of synaptic vesicle (SV) clusters, These SV clus ters in PC12 cell neurites closely resemble those in the presynaptic t erminals of neurons, Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configu ration, except that the ones in the doubly treated group occur in high er frequency and have more vesicles, The synaptic nature of these vesi cle clusters was demonstrated by their regulated depletion after potas sium stimulation, Furthermore, these vesicle clusters stained positive ly for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC), Such SV clusters in a cell line are very u seful for characterizing the regulated release of SVs and the distribu tion of SV-related antigens in intact cells, Analysis by SDS-gel elect rophoresis and immunoblotting indicated that synapsin I levels are hig her in all three differentiated groups compared to untreated cells; wh ereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment, Possible convergence and/or diverge nce on the mechanisms of NGF and ras differentiation in PC12 cells are discussed. (C) 1995 Wiiey-Liss, Inc.