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PubMed1998

Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila.

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 +/- 5% (means +/- SEM) of control in the sytnull mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examined syt mutants in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation resulted in the expected decrease in evoked release but suppressed the increase in spontaneous release frequency. Motor nerve terminals in this genotype contained more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones was still reduced (33 +/- 4% of control). Our findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the increase in spontaneous release. These results support the hypothesis that synaptotagmin stabilizes the docked state.

Animals↗

PubMed1987

Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein.

We have isolated from a lambda gt11 rat brain cDNA library cDNA clones encoding greater than 95% of the open reading frame and untranslated regions of the mRNA for p38, the most abundant of the integral membrane proteins of the synaptic vesicle. Phage containing cDNA that encoded vesicle proteins were identified by screening fusion proteins with a polyclonal serum to rat brain synaptic vesicles. To identify phage carrying p38 sequences, fusion proteins were used to affinity purify monospecific antibodies from the original heterogeneous serum; antibodies to a 38,000-D protein were then identified by Western blotting. Inserts carrying DNA-encoding p38 sequences were subcloned into plasmid vectors and used to generate cDNA probes for Northern blot analysis. A major transcript of 2.4 kb was expressed specifically in brain and endocrine tissue but not in liver, consistent with the tissue-specific expression of the protein detected by antibody techniques. Using three overlapping clones that encoded fusion proteins, we identified and sequenced approximately 85% of the cDNA. Two additional Eco RI fragments at the 5' end of the mRNA were obtained from a fourth clone identified by screening a second lambda gt11 library with a 5' cDNA probe. The cDNA encoded an open reading frame of 298 amino acids with a 3' untranslated region of 1.4 kb. The protein shares no sequence homology with other Ca2+-binding proteins. The availability of a cDNA clone for an integral synaptic vesicle protein should facilitate studies of its function in transmitter release, its intracellular targeting, and regulation of synaptic vesicle biogenesis during development and regeneration of nerve terminals.

Amino Acid Sequence↗

PubMed1992

Synaptotagmin: a calcium sensor on the synaptic vesicle surface.

Neurons release neurotransmitters by calcium-dependent exocytosis of synaptic vesicles. However, the molecular steps transducing the calcium signal into membrane fusion are still an enigma. It is reported here that synaptotagmin, a highly conserved synaptic vesicle protein, binds calcium at physiological concentrations in a complex with negatively charged phospholipids. This binding is specific for calcium and involves the cytoplasmic domain of synaptotagmin. Calcium binding is dependent on the intact oligomeric structure of synaptotagmin (it is abolished by proteolytic cleavage at a single site). These results suggest that synaptotagmin acts as a cooperative calcium receptor in exocytosis.

Animals↗

PubMed1988

Effects of an inhibitor of the synaptic vesicle acetylcholine transport system on quantal neurotransmitter release: an electrophysiological study.

The drug 2-(4-phenylpiperidino)cyclohexanol (AH5183), which potently inhibits the active transport of acetylcholine (ACh) into synaptic vesicles, was used as a pharmacological tool to study the functional role of synaptic vesicles in quantal transmitter release. Using microelectrode recording techniques, miniature endplate potentials (mepps) and nerve-evoked endplate potentials (epps) were recorded from frog cutaneous pectoris neuromuscular junctions in low Ca2+/high Mg2+ Ringer solution, and in normal Ringer with added D-tubocurarine (D-TC). Stimulation in the presence of AH5183 caused a 40% reduction in quantal size (mepp amplitude), depressed tetanic potentiation, and decreased the number of quanta released with each nerve impulse in the presence of D-TC. All of these effects appeared gradually and only after extended stimulation of the nerve, during which several hundred thousand quanta were released. Consequently, these findings suggest a serial one-time usage of vesicles, with little or no re-entry of recycled vesicles until after a large fraction of the original vesicles has been exhausted. The results primarily show that filling of synaptic vesicles with ACh is crucial for sustaining synaptic transmission, and gives further evidence that the ACh released by nerve impulses originates from these organelles.

Acetylcholine↗

PubMed2004

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. II. Functional effects of the Rab3A-synapsin I interaction.

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760-43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.

Actins↗

PubMed2000

A conserved clathrin assembly motif essential for synaptic vesicle endocytosis.

Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.

Adaptor Protein Complex alpha Subunits↗

PubMed1991

Phylogenetic studies on the synaptic vesicle glutamate transport system.

The ATP-dependent uptake of glutamate into synaptic vesicles isolated form mammalian brains is well characterized. Glutamate uptake requires an electrochemical proton gradient, is specific for glutamate over other amino acids, and is stimulated by chloride. To determine whether these characteristics are fundamental to the vesicular uptake system, vesicles were isolated from the brain and central nervous ganglia of several vertebrate and invertebrate species, which included goldfish, frogs, turtles, pigeons, rats, Drosophila, and crayfish, and these vesicles were assayed for glutamate uptake activity. ATP-dependent glutamate was found in all of the vertebrate species tested, but was not detected in Drosophila or crayfish vesicles. The nature of the vesicular uptake of glutamate was similar among all the vertebrates: the specificity for glutamate remained high, transport was energized by a vacuolar (V)-type ATPase, 2-4 mM chloride stimulated uptake three- to sixfold, and Km for glutamate was between 0.5 and 2 mM. While these major characteristics of the uptake system remained conserved among the vertebrates tested, minor differences were seen in glutamate specificity, the steady-state level of glutamate obtained in the vesicles, and Vmax of the glutamate uptake systems. These results indicate that the synaptic vesicle glutamate uptake system is present throughout the vertebrate class, and that while minor changes in the transport system have occurred, its major functional characteristics, such as stimulation by chloride and strict substrate specificity, have been conserved for over 350-400 million years.

Amino Acid Transport System X-AG↗

PubMed1983

Pharmacological characterization of the acetylcholine transport system in purified Torpedo electric organ synaptic vesicles.

A wide variety of pharmacologically active compounds was surveyed for effects on active transport of [3H]acetylcholine by synaptic vesicles isolated from the electric organ of Torpedo californica. In over 80 compounds tested, inhibitors of a wide range of potencies were found. The most potent inhibitor was 2-(4-phenylpiperidino)cyclohexanol (AH5183), which half-inhibited transport at 40 nM. This compound had been predicted by Marshall [Br. J. Pharmacol. 38:503-516 (1970)] to block acetylcholine storage by vesicles in vivo. The synaptic vesicle active transport system is shown to be pharmacologically distinct from other cholinergic systems. The site of action of AH5183 and other potent inhibitors is not certain, but the possibility of trivial action on the vesicle ATPase or a vesicle proton gradient was eliminated. The results constitute new evidence supporting vesicle exocytosis as the source of evoked acetylcholine release by nerve terminals. AH5183 appears to be the prototype for a new family of anticholinergics. The possibility that some drugs that exhibit secondary anticholinergic effects act in part by antagonizing acetylcholine storage is discussed.

Acetylcholine↗

PubMed1989

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.

Animals↗

PubMed1990

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.

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.

Amino Acid Sequence↗

PubMed1981

Synaptic vesicle exocytosis in goldfish photoreceptor cells.

The photoreceptor cell endings of goldfish retinas were examined using ultrathin and freeze-fracture techniques. The synaptic vesicles could be seen lying closely packed in rows on each side of the synaptic ribbon, and the vesicles nearest to the end of the ribbon could be seen to be in direct contact with the presynaptic membrane. The openings of the synaptic vesicles to the synaptic cleft could also be observed at the presynaptic membrane nearest to the end of the ribbon, that is, 100 nm distant from the center of the apex. In the freeze-fractured replicas of the presynaptic membrane, the band-shaped membrane particle aggregation was seen at the apex and a row of the crater-like or circular protuberances were observed at each side of the apex. These structures were considered to be changes of the presynaptic membrane organizations according to the synaptic vesicle exocytosis. It may be possible that the synaptic vesicle exocytosis in the photoreceptor cells occurs at the sites of the presynaptic membrane nearest to the apical end of the synaptic ribbon, and that the exocytotic sites form a line on each side of the apex.

Animals↗

PubMed1998

Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire.

In a temperature-sensitive Drosophila mutant, shibire, synaptic vesicles are completely depleted in nerve terminals after stimulation at 34 degrees C, but upon returning to 22 degrees C, endocytosis resumes. In this study, synaptic vesicles in the boutons of nerve terminals at the mutant neuromuscular junction were loaded with a fluorescent dye, FM1-43, during vesicle reformation at 22 degrees C after complete depletion at 34 degrees C. We found two distinct pools of synaptic vesicles, namely an exo/endo cycling pool, located in the periphery of the bouton, and a reserve pool, located in its center. Cytochalasin D treatment eliminated the reserve pool and reduced synaptic transmission evoked by high frequency stimulation. Thus, the reserve pool may play a crucial role for sustaining high frequency synaptic transmission.

Actins↗

PubMed1997

Axonal transport of synaptic vesicle proteins in the rat optic nerve.

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the "stop-flow/nerve crush" method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80-90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10-20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin.

Animals↗

PubMed1990

A study in rat brain cortex synaptic vesicles of endogenous ligands for N-methyl-D-aspartate receptors.

The presence of endogenous ligands for the N-methyl-D-aspartate receptor was looked for in highly purified rat brain cortex synaptic vesicles, the contents of which were extracted and fractionated by gel filtration on Sephadex G-10, or by three different high-voltage electrophoresis procedures. The presence of endogenous ligands was detected by their ability to compete with 50 nM L-[3H]glutamate for binding to whole rat brain N-methyl-D-aspartate receptors. The receptor preparations used were those present in purified postsynaptic densities, in which the quisqualate receptors were blocked by 10 microM quisqualate. Synaptic vesicles had a high content of N-methyl-D-aspartate receptor ligands, which on fractionation always coincided with glutamate or aspartate. A variable and very small amount of a highly acidic endogenous ligand was also found. The latter substance did not coincide in the electrophoresis with homocysteic, cysteic, quinolinic, cysteine sulphinic or homocysteine sulphinic acids, or with N-acetyl-aspartyl-glutamic acid, S-sulphocysteine or sulphoserine. We also found that a single centrifugation, in 0.25 M sucrose, 25 mM Tris-citrate, pH 7.1, of purified synaptic vesicles, at 135,000 gmax for 45 min, led to a 51% loss of endogenous glutamate, but did not change their aspartate content. Thus, in uncentrifuged vesicles the glutamate/aspartate ratio was 9.4, while in centrifuged ones the ratio was 3.9 ATP markedly enhanced L-[3H]glutamate uptake into synaptic vesicles, but did not change the binding of L-[3H]aspartate. Differences in labelled aspartate and glutamate efflux from the vesicles were also found.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1991

Presynaptic alpha-latrotoxin receptor components interact with protein p65 of synaptic vesicle membranes.

Purified preparations of bovine brain alpha-latrotoxin receptor contain proteins of 39 kDa and 65 kDa. Sequence analysis shows that the 65 kDa protein corresponds to p65, a synaptic vesicle membrane protein previously identified in rat synaptic vesicles, and that the 39 kDa protein is a proteolytic fragment of the 65 kDa protein. The 39 kDa and 65 kDa proteins appear in receptor samples because of their specific interaction with components of the alpha-latrotoxin receptor. This interaction may represent an essential step in perfusion and/or the fusion of synaptic vesicle membranes with the plasma membrane of the presynaptic terminal, both of which are final steps in the exocytosis of neurotransmitters.

Amino Acid Sequence↗

PubMed1983

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

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.

Agar↗

PubMed2004

Synapsin and synaptic vesicle protein expression during embryonic and post-natal lens fiber cell differentiation.

PURPOSE: Reorganization of cytoskeleton and membrane biogenesis are dynamically coordinated during lens fiber cell differentiation and development to produce an organ with precise dimensions and optical properties. Cargo vesicle trafficking is fundamental to cell elongation and has also been implicated in degenerative disease mechanisms. Alzheimer precursor protein (AbetaPP) acts with kinesin, synapsin, and synaptic vesicle proteins to mediate cargo vesicle transport and membrane fusion in neurons. In our previous studies we demonstrated that AbetaPP is also a key element in lens fiber cell formation, and in early-onset cataract that occurs along with early-onset Alzheimer disease in Down syndrome. In the present study we examine lens expression and regulation of a complement of genes associated with cargo and synaptic vesicle transport in neurons. METHODS: RT-PCR, immunoblot, and immunohistochemical methods were used to characterize expression of AbetaPP and kinesin associated motor proteins, synapsins, and synaptic vesicle proteins in mouse and rat embryonic, post-natal, and adult lenses. Phospho-specific anti-synapsin antibodies were used to determine the distributions of site-1 phosphorylated and dephosphorylated synapsin protein. RESULTS: We demonstrate that a substantial complement of cargo and synaptic vesicle proteins involved in AbetaPP mediated vesicle transport are expressed in lenses along the anterior-posterior axis of fiber cells in embryonic and adult lenses, consistent with vesicles, actin filaments, and neuron-like arrangement of microtubules in lenses shown by others. We identify temporal regulation of synapsins I, II, and III during embryonic and post-natal lens development consistent with their roles in neurons. Regulation of vesicle cytoskeleton attachment, actin polymerization, and the capacity to stimulate cell differentiation by synapsins are governed in large part by phosphorylation at a conserved Ser9 residue (site-1). We demonstrate discrete distributions of Ser9 phospho- and dephospho-synapsins along the axial length of rapidly elongating embryonic lens fiber cells, and decreased levels of site-1 phosphorylated synapsins in adult lenses. CONCLUSIONS: The present findings demonstrate several fundamental parallels between lens and neuron vesicle trafficking cell biology and development, and suggest that more extensive AbetaPP related vesicle trafficking disease mechanisms may be shared by lens and brain.

Amyloid beta-Protein Precursor↗

PubMed1983

Changes in acetylcholine concentration, miniature end-plate potentials and synaptic vesicles in frog neuromuscular preparations during lanthanum treatment.

ACh content and synaptic ultrastructure were compared in neuromuscular preparations (sartorius muscle of Rana esculenta) incubated in control saline and in saline containing 1 mM LaCl3. ACh concentrations remained constant for 6 hr in control preparations. La3+ caused a 38% depletion of ACh within the first 30 min with subsequent recovery to 120% of control values within 3-4 hr. Recovery was prevented by hemicholinium-3. At 23 degrees C La3+ caused complete loss of synaptic vesicles: no depletion was seen at 4 degrees C. Initially MEPP frequency increased 300- to 700-fold (23 degrees C), then declined. Mean vesicle diameter did not change, but SD increased. As the frequency of MEPPs declined, the percentage of s-MEPPs greatly increased. La3+ had a postsynaptic effect which increased the amplitudes of both s-MEPPs and bell-MEPPs within a few seconds. The s-MEPP mean did not change during the course of La3+ treatment although the bell-MEPP mean usually decreased. How the decrease in synaptic vesicles, decrease in MEPP frequencies, and changes in ACh levels relate to changes in the percentage of different classes of quanta is discussed.

Acetylcholine↗