Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes

doi:10.1083/jcb.96.5.1355

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Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes

P De Camilli, SM Harris Jr, WB Huttner and P Greengard

Synapsin I (protein I) is a major neuron-specific endogenous substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases that is widely distributed in synapses of the central and peripheral nervous system (De Camilli, P., R. Cameron, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354). We have now carried out a detailed analysis of the ultrastructural localization of synapsin I in the synapse. For this purpose we have developed a novel immunocytochemical technique that involves the labeling of isolated synaptosomes immobilized in a thin agarose gel. Special fixation conditions were designed to maximize accessibility of synapsin I to marker molecules. Immunoferritin and immunoperoxidase studies of this preparation indicated that synapsin I is localized in the presynaptic compartment and that it is present in close to 100% of all nerve endings. Immunoferritin labeling also indicated that, inside the nerve ending, synapsin I is specifically associated with the cytoplasmic surface of synaptic vesicles. In agreement with these immunoferritin results, the labeling produced by immunoperoxidase was compatible with a specific association of synapsin I with synaptic vesicle membranes. However, at variance with the very specific distribution of immunoferritin, immunoperoxidase reaction product was also found on other membranes of the terminals, presumably as a result of its diffusion over a short distance from the synaptic vesicles. Anti-synapsin I immunoperoxidase staining of tissue sections for electron microscopy produced an uneven labeling of terminals of the neuropile, in agreement with results of a previous study (Bloom, F. E., T. Ueda, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA. 76:5982-5986). A comparison with results obtained in isolated synapses indicates that the limited labeling of nerve endings in tissue sections results from limited and uneven penetration by marker molecules. The specific association of synapsin 1 with synaptic vesicle membranes in the great majority of nerve terminals suggests a prominent role for this phosphoprotein in the regulation of synaptic vesicle function.
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