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PubMed2003

Isolation of small agranular synaptic vesicles of rat brain by gel filtration chromatography.

I attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesicles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200 nm. Peak I at the void volume contained large vesicular membranes and coated vesicles besides synaptic vesicles; Peak II consisted almost entirely of small agranular synaptic vesicles of 40-50 nm diameter; and Peak III comprised soluble proteins. Western blotting revealed that components of 72 kDa in peaks I and II reacted with an anti-H(+)-ATPase A-subunit antibody [Moriyama et al. (1995) FEBS Lett. 367, 233-236]. When examined for Mg(2+)-ATPase activity, peak I showed specific activity of 4.52 ( micromol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A(1) [Bowman et al. (1988) PROC: Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H(+)-ATPase as to total Mg(2+)-ATPase, 18-22%, was unchanged, indicating no accumulation of the H(+)-ATPase in peak II even on the chromatography. In brief, the small agranular synaptic vesicles in peak II showed little or no Mg(2+)-ATPase activity, although they reacted with the H(+)-ATPase antibody. The reason for this is obscure. Mg(2+)-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.

Adenosine Triphosphate↗

PubMed2004

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.

Animals↗

PubMed1981

Ultrastructural and morphometric investigations on the mechanism of neurotransmitter release from synaptic vesicles.

The mechanism of neurotransmitter release from synaptic vesicles as the results of neuron excitation is not quite clear. It is believed, that this process may occur by way of exocytosis into the synaptic cleft directly from the cytosol to the cleft. Ultrastructural-morphometric analysis was attempted of the synaptic vesicles from the motor cortex of mice. As stimulation model an audiogenic epileptic seizure was used. The method of ZIO impregnation was applied, which according to Kawana (7) allowed to visualise the presence of a neurotransmitter in synaptic vesicles. Morphometric analysis consisted in counting the synaptic vesicles ZIO-positive, filled with neurotransmitter, and ZIO-negative, empty ones, in 3 successive zones of the synaptic bouton in the initial and end phases of seizures. As the result of excitation the number of ZIO-positive-synaptic vesicles diminished significantly whereas the number of ZIO-negative ones did not increase proportionally. This indicates that the "dark" vesicles do not change into the "clear" ones, but they disappear completely. The applied method did not allow us to precisely establish vesicle translocation toward the synaptic cleft. Nevertheless the considerable reduction of their number with the percentual distribution of vesicles remaining the same within the particular zones of bouton, may indicate, under the assumption of translocation of vesicles, that they disappear close to the cleft, probably by way of exocytosis.

Animals↗

PubMed2003

An essential role of Rab5 in uniformity of synaptic vesicle size.

Rab5 small GTPase is a famous regulator of endocytic vesicular transport from plasma membrane to early endosomes. In neurons, Rab5 is found not only on endocytic vesicles in cell bodies but also on synaptic vesicles in nerve terminals. However, the function of Rab5 on synaptic vesicles remains unclear. Here, we elucidate the function of Rab5 on synaptic vesicles with in vivo and in vitro experiments using Drosophila photoreceptor cells. Functional inhibition of Rab5 with Rab5N142I, a dominant negative version of Drosophila Rab5, induced enlargement of synaptic vesicles. This enlargement was, however, suppressed by enhancing synaptic vesicle recycling under light illumination. In addition, synaptic vesicles prepared from Rab5N142I-expressing flies exhibited homotypic fusion in vitro. These results indicate that Rab5 functions to keep the size of synaptic vesicles uniform by preventing their homotypic fusion. By contrast, Rab5 was not involved in the endocytic reformation of synaptic vesicles, contrary to expectation from its conventional function. Furthermore, we electrophysiologically and behaviourally showed that the function of Rab5 is essential for efficient signal transmission across synapses.

Animals↗

PubMed2006

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo.

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.

Amino Acid Motifs↗

PubMed1980

An antiserum specific for cholinergic synaptic vesicles from electric organ.

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle-specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.

Acetylcholine↗

PubMed1998

Differential regulation of synaptic vesicle protein genes by target and synaptic activity.

Differentiation of presynaptic nerve terminals involves changes in gene expression; these may be regulated by synaptic transmission and/or by contact with the target muscle. To gain insight into the control of presynaptic differentiation, we examined the regulation by target and synaptic activity of synaptic vesicle protein (SVP) genes in the chick ciliary ganglion (CG). In the CG, two SVP genes, synaptotagmin I (syt I) and synaptophysin II (syp II), are coordinately upregulated at the time of target contact. To test the hypothesis that this upregulation is induced by target contact, we examined mRNA levels of syt I and syp II in CGs from embryos in which one eye had been removed before axon outgrowth. As expected, target removal prevented the normal upregulation of syt I mRNA in the deprived ganglion. In contrast, and unexpectedly, syp II mRNA upregulation was not affected. The target dependence of syt I upregulation was not attributable to nerve-muscle transmission, because blockade of this transmission had no effect on SVP mRNA levels. Surprisingly, blockade of synapses onto CG neurons from the brain also did not affect syt I mRNA levels but increased levels of syp II mRNA. We conclude that contact with target induces upregulation of syt I mRNA, which is the case for spinal motor neurons. However, the normal upregulation of syp II mRNA is not controlled by the same signal(s). Instead, our results suggest that these two SVP genes are differentially regulated, both by target contact and by blockade of synaptic transmission.

Animals↗

PubMed1998

A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex.

Reconstitution of synaptic vesicle formation in vitro has revealed a pathway of synaptic vesicle biogenesis from endosomes that requires the heterotetrameric adaptor complex AP3. Because synaptic vesicles have a distinct protein composition, the AP3 complex should selectively recognize some or all of the synaptic vesicle proteins. Here we show that one element of this recognition process is the v-SNARE, VAMP-2, because tetanus toxin, which cleaves VAMP-2, inhibited the formation of synaptic vesicles and their coating with AP3 in vitro. Mutant tetanus toxin and botulinum toxins, which cleave t-SNAREs, did not inhibit synaptic vesicle production. AP3-containing complexes isolated from coated vesicles could be immunoprecipitated by a VAMP-2 antibody. These data imply that AP3 recognizes a component of the fusion machinery, which may prevent the production of inert synaptic vesicles.

Adaptor Protein Complex 3↗

PubMed1995

Variation in the number, location and size of synaptic vesicles provides an anatomical basis for the nonuniform probability of release at hippocampal CA1 synapses.

Synaptic vesicles, synaptic clefts and postsynaptic areas were measured in three dimensional reconstructions at representative axonal boutons in hippocampal area CA1. Both docked and non-docked vesicles were counted and measured. Small boutons on thin spines had about 2-6 docked vesicles from a pool of more than 200 vesicles. Medium-sized boutons on medium-sized mushroom-shaped dendritic spines contained about 13-16 docked vesicles from a pool of more than 450 vesicles. A large bouton synapsing with a large mushroom-shaped dendritic spine had two clusters of vesicles totaling more than 1000 vesicles. The postsynaptic density was segmented into two discrete zones under the two clusters of vesicles and 36 docked vesicles were distributed over its surfaces. Two multiple-synapse boutons contained more than 500 vesicles with 2-12 docked vesicles observed at each of the two postsynaptic densities on each bouton. This nonuniform number of docked vesicles provides an anatomical basis for the non-uniform probability of release that occurs across hippocampal synapses of different sizes. In addition, the volume of each synaptic vesicle was determined to be 0.4-5.2% of the total volume of the reconstructed synaptic clefts into which they presumably release their contents. However, since each vesicle contains more than 10 times the concentration of glutamate needed to saturate the postsynaptic receptors, these data also support the hypothesis that release a single synaptic vesicle will activate all of the postsynaptic receptors.

Animals↗

PubMed2006

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques.

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.

Animals↗

PubMed1994

Phosphorylation of dynamin I and synaptic-vesicle recycling.

In nerve terminals, neurotransmitters are packaged in synaptic vesicles, and released by exocytosis. Empty synaptic vesicles are rapidly recycled for reuse by endocytosis. Much progress has been made in identifying the proteins involved in synaptic-vesicle trafficking, but the mechanism and regulation of endocytosis have largely remained an enigma. One approach to defining regulatory proteins that might be involved is to study stimulus-dependent phosphorylation events in nerve terminals. This has led to the identification of dephosphin, which is quantitatively dephosphorylated by nerve-terminal depolarization. Sequencing reveals that dephosphin is identical with dynamin I, a GTP-binding protein that functions in endocytosis. Phosphorylation and dephosphorylation of nerve-terminal dynamin I/dephosphin regulates its intrinsic GTPase activity in parallel with the regulation of synaptic-vesicle recycling. Therefore, phosphorylation and dephosphorylation of dynamin I might provide a Ca(2+)-dependent switch for endocytosis in the synaptic-vesicle pathway.

Animals↗

PubMed2006

Synaptic vesicles: an organelle comes of age.

Synaptic vesicles mediate the release of neurotransmitters at nerve terminals. In this issue of Cell, Takamori et al. (2006) analyze the lipid and protein components of synaptic vesicles, providing the most comprehensive description of synaptic vesicles to date.

Animals↗

PubMed1994

Putative synaptic vesicle nucleotide transporter identified as glyceraldehyde-3-phosphate dehydrogenase.

Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N-ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins on AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.

Amino Acid Sequence↗

PubMed2003

Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo.

Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. Here, we test directly the role of Syt I during endocytosis in vivo. We use quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (synapto-pHluorin) to measure the kinetics of endocytosis in sytI-null Drosophila. We then combine live imaging of the synapto-pHluorins with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, we demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein.

Animals↗

PubMed2004

Synaptic vesicle cycling at type-identified diaphragm neuromuscular junctions.

Differences in neuromuscular transmission and neuromuscular junction morphology exist across muscle fiber types. We hypothesized that these fiber-type differences are reflected in the size of the cycling synaptic vesicle pool. Synaptic vesicle cycling at type-identified rat diaphragm neuromuscular junctions was examined by fluorescently labeling presynaptic vesicles with FM4-64. We found that FM4-64 fluorescence uptake was higher at presynaptic terminals of type I/IIa fibers than type IIx/IIb fibers. However, no fiber-type differences in the rate of FM4-64 destaining were found with repetitive nerve stimulation. Synaptic vesicle density at active zones was examined by transmission electron microscopy. In accordance with FM4-64 uptake, synaptic vesicle density was greater at type I/IIa than IIx/IIb fibers. These results demonstrate differences in synaptic vesicle cycling across diaphragm muscle fiber types, which may underlie previously observed differences in neuromuscular transmission across diaphragm muscle fiber types. In the diaphragm, motor units comprising type I and type IIa fibers are most frequently recruited with a duty cycle of approximately 40%. Motor units comprising IIx/IIb fibers are infrequently recruited and only for short durations. The capacity for synaptic vesicle release and cycling at different muscle fiber types matches the functional requirements of these motor units. If the demand for recruitment of motor units comprising IIx/IIb fibers increases, for example, with mechanical loading, there is an increased risk for neuromuscular transmission failure that my relate to the capacity for synaptic vesicle release and cycling. Muscle fiber type-specific adaptations should be considered when examining neuromuscular disorders.

Animals↗

PubMed1988

Synaptic vesicles from mammalian brain: large-scale purification and physical and immunochemical characterization.

Purification of synaptic vesicles directly from homogenates of mammalian brain is compared with a classical method based on osmotic lysis of brain synaptosomes. The direct method affords increased yield and purity of synaptic vesicles prepared under isoosmotic conditions. Antigen SV2 and the antigens (primarily synaptophysin) recognized by rabbit antiserum R10, raised to purified rat brain synaptic vesicles, are localized specifically on approximately 40-nm-diameter microsomal vesicles from rat brain. Rat brain synaptic vesicles have equilibrium densities of approximately 1.11 g/ml on Nycodenz density gradients, 1.12 g/ml on glycerol/Nycodenz, and 1.07 g/ml on Ficoll gradients. Both SV2 and the R10 antigens are enriched approximately 50-fold in purified rat brain synaptic vesicles. Synaptic vesicles purified from rat or cow brain show active uptake of [3H]norepinephrine that is reserpine sensitive and dependent on ATP and Mg2+. Synaptic vesicles exhibiting [3H]norepinephrine uptake comigrate with approximately 40-nm-diameter synaptic vesicles carrying SV2 or R10 antigens during permeation chromatography. After the Sephacryl S-1000 chromatography step, [3H]-norepinephrine uptake activity is purified approximately 90-fold. Highly purified brain synaptic vesicles should facilitate studies at the molecular level of the roles of these organelles in neurotransmission at mammalian synapses.

Animals↗

PubMed2004

Isoosmotic isolation of rat brain synaptic vesicles, some of which contain tyrosine hydroxylase.

Rat brain synaptic vesicles were isoosmotically isolated and examined for Mg(2+)-ATPase [EC 3.6.1.3.] and tyrosine hydroxylase [EC 1.14.16.2.] associated with the synaptic vesicles. Synaptosomes in 0.32 M sucrose were disrupted by freezing and thawing treatment, and the cytosol fraction was fractionated on a Sephacryl S-500 column with a mean exclusion size of 200 nm. Peak I at the void volume was a mixture of large vesicular membranes, small amounts of synaptic vesicles and coated vesicles, etc. Peak II consisted of non- and granulated synaptic vesicles of 35-40 nm diameter, and peak III of soluble proteins. The synaptic vesicles in peak II reacted with antibodies against the H(+)-ATPase A-subunit, vesicular acetylcholine transporter, and vesicular monoamine transporter. However, they showed little Mg(2+)-ATPase activity. Tyrosine hydroxylase was observed in either peak II or III on blotting with an anti-tyrosine hydroxylase antibody. These results imply that tyrosine hydroxylase exists in soluble and bound forms to synaptic vesicles in nerve terminals.

Acetylcholine↗

PubMed1998

Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes.

Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degreesC had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.

Adenosine Triphosphate↗