Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.

Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

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Abstract. In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37~ resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons sew eral days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.S VS^PTIC vesicles (SVs) ~ are highly specialized secretory organelles by which neurons secrete nonpepride neurotransmitters. They are highly homogeneous in size (average diameter, 50 rim) and are clustered at release sites in nerve endings. These clusters represent the key morphological features of synapses. SVs undergo regulated fusion with the plasmalemma, but differ in several important characteristics from secretory granules of the classical regulated secretory pathway which store and secrete peptide hormones and neurotransmitters and which in neurons are referred to as large dense-core vesicles .Specific features of SVs include a unique membrane protein composition, an extremely short latency between the stimulus and the exocytotic response, the occurrence of their exocytosis at highly specialized plasmalemma sites, and their property to undergo exo-endocytotic recycling within the nerve terminal. At each exo-endocytotic cycle, SVs are refilled locally with neurotransmitters (Smith and Augustine, 1988; De CamiUi and Jahn, 1990; S~idhof and Jahn, 1991).Most of these properties were established for SVs in maMichela Matteoli's permanent address is

Since the demonstration that Ca+ influx into the presynaptic terminal is esential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for i ,niting ex ts. D3, D37, D39, and D45 were generated with ethyl methanesulfonate as described (7). Alleles N10, N13, and N28 were generated by imprecise excision of the 177 P-element insertion in syt.

Cloning and characterization of the Drosophila syntaxin-1A gene, syx-1A, reveal that it is present in several tissues but is predominantly expressed in the nervous system, where it is localized to axons and synapses. We have generated an allelic series of loss-of-function mutations that result in embryonic lethality with associated morphological and secretory defects dependent on the severity of the mutant allele. Electrophysiological recordings from partial loss-of-function mutants indicate absence of endogenous synaptic transmission at the neuromuscular junction and an 80% reduction of evoked transmission. Complete absence of syx-1A causes subtle morphological defects in the peripheral and central nervous systems, affects nonneural secretory events, and entirely abolishes neurotransmitter release. These data demonstrate that syntaxin plays a key role in nonneuronal secretion and is absolutely required for evoked neurotransmission.

Neuronal pentraxins (NPs) define a family of proteins that are homologous to C-reactive and acutephase proteins in the immune system and have been hypothesized to be involved in activity-dependent synaptic plasticity. To investigate the role of NPs in vivo, we generated mice that lack one, two, or all three NPs. NP1/2 knock-out mice exhibited defects in the segregation of eye-specific retinal ganglion cell (RGC) projections to the dorsal lateral geniculate nucleus, a process that involves activity-dependent synapse formation and elimination. Retinas from mice lacking NP1 and NP2 had cholinergically driven waves of activity that occurred at a frequency similar to that of wild-type mice, but several other parameters of retinal activity were altered. RGCs cultured from these mice exhibited a significant delay in functional maturation of glutamatergic synapses. Other developmental processes, such as pathfinding of RGCs at the optic chiasm and hippocampal long-term potentiation and long-term depression, appeared normal in NP-deficient mice. These data indicate that NPs are necessary for early synaptic refinements in the mammalian retina and dorsal lateral geniculate nucleus. We speculate that NPs exert their effects through mechanisms that parallel the known role of short pentraxins outside the CNS.

Communicated by Alfred G. Gilman, December 22, 1989 (received for review December 6, 1989) ABSTRACT rab3, a low molecular weight GTP-binding protein, is primarily expressed in brain, where it is present in soluble and membrane-bound forms. Membrane-bound rab3 in brain is exclusively localized on synaptic vesicles, the secretory organelles of the synapse that store and release neurotransmitters. rab3 is also expressed in endocrine tissues such as the adrenal medulla, where it is found together with other synaptic vesicle proteins on microvesicles distinct from chromaffin granules. The tight binding of rab3 to membranes correlates with hydrophobic modifications that are different in the membrane-bound and soluble forms of rab3. The results demonstrate the exclusive targeting of a small GTP-binding protein to secretory vesicles of a subset of the regulated pathway of secretion.