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PubMed1998

SVOP, an evolutionarily conserved synaptic vesicle protein, suggests novel transport functions of synaptic vesicles.

We describe a novel synaptic vesicle protein called SVOP that is distantly related to the synaptic vesicle proteins SV2A, SV2B, and SV2C (20-22% sequence identity). Both SVOP and SV2 contain 12 transmembrane regions. However, SV2 is highly glycosylated, whereas SVOP is not. Databank searches revealed that closely related homologs of SVOP are present in Caenorhabditis elegans and Drosophila (48% sequence identity), suggesting that SVOP is evolutionarily ancient. In contrast, no invertebrate orthologs of SV2 were detected. The sequences of SVOP and SV2 exhibit homology with transport proteins, in particular with mammalian organic cation and anion transporters. SVOP and SV2 are more distantly related to eukaryotic and bacterial phosphate, sugar, and organic acid transporters. SVOP is expressed at detectable levels only in brain and endocrine cells where it is primarily localized to synaptic vesicles and microvesicles. SVOP is present in all brain regions, with particularly high levels in large pyramidal neurons of the cerebral cortex. Immunocytochemical staining of adjacent rat brain sections for SVOP and SV2 demonstrated that SVOP and SV2 are probably coexpressed in most neurons. Although the functions of SV2 and SVOP remain obscure, the evolutionary conservation of SVOP, its hydrophobic nature, and its homology to transporters strongly support a role in the uptake of a novel, as yet unidentified component of synaptic vesicles. Thus synaptic vesicles contain two classes of abundant proteins with 12 transmembrane regions that are related to transporters, nonglycosylated SVOP and highly glycosylated SV2, suggesting that the transport functions of synaptic vesicles may be more complex than currently envisioned.

Adrenal Glands↗

PubMed1999

Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons.

Recycling synaptic vesicles are already present in isolated axons of developing neurons (Matteoli et al., Zakharenko et al., 1999). This vesicle recycling is distinct from the vesicular traffic implicated in axon outgrowth. Formation of synaptic contacts coincides with a clustering of synaptic vesicles at the contact site and with a downregulation of their basal rate of exo-endocytosis (Kraszewski et al, 1995; Coco et al., 1998) We report here that tetanus toxin-mediated cleavage of synaptobrevin/vesicle-associated membrane protein (VAMP2), previously shown not to affect axon outgrowth, also does not inhibit synaptic vesicle exocytosis in isolated axons, despite its potent blocking effect on their exocytosis at synapses. This differential effect of tetanus toxin could be seen even on different branches of a same neuron. In contrast, botulinum toxins A and E [which cleave synaptosome-associated protein of 25 kDa. (SNAP-25)] and F (which cleaves synaptobrevin/VAMP1 and 2) blocked synaptic vesicle exocytosis both in isolated axons and at synapses, strongly suggesting that this process is dependent on "classical" synaptic SNAP receptor (SNARE) complexes both before and after synaptogenesis. A tetanus toxin-resistant form of synaptic vesicle recycling, which proceeds in the absence of external stimuli and is sensitive to botulinum toxin F, E, and A, persists at mature synapses. These data suggest the involvement of a tetanus toxin-resistant, but botulinum F-sensitive, isoform of synaptobrevin/VAMP in synaptic vesicle exocytosis before synapse formation and the partial persistence of this form of exocytosis at mature synaptic contacts.

Animals↗

PubMed1996

Colocalization on the same synaptic vesicles of syntaxin and SNAP-25 with synaptic vesicle proteins: a re-evaluation of functional models required?

Synaptic vesicle docking and calcium dependent exocytosis are thought to require the specific interaction of proteins of the synaptic vesicle membrane (such as VAMP/synaptobrevin and synaptotagmin) and their plasma membrane-located counterparts (such as syntaxin and SNAP-25). When isolating synaptic vesicles by glycerol velocity gradient centrifugation we found cosedimentation of the presumptive presynaptic plasma membrane proteins syntaxin and SNAP-25 with synaptic vesicle membrane proteins. In order to further identify the antibody binding organelles we performed an immunoelectron microscopical analysis of synaptosomal profiles. Syntaxin and SNAP-25 were not only associated with the plasma membrane but to a large extent also with synaptic vesicle profiles. In order to answer the question whether the syntaxin and SNAP-25 containing vesicular compartment would also carry classical synaptic vesicle membrane markers we performed double labeling experiments using poly- and monoclonal antibodies. We found colocalization on the same vesicle not only of SNAP-25 and syntaxin but also of SNAP-25 with the synaptic vesicle membrane proteins SV2 and synaptotagmin and of syntaxin with the vesicular membrane protein synaptophysin. Our results demonstrate that syntaxin and SNAP-25 are colocalized with classical vesicle membrane proteins on the same vesicle and suggest that the functional models for the interaction of presynaptic proteins need to be re-evaluated.

Animals↗

PubMed1986

Synaptic vesicle diameters and synaptic cleft widths at the mouse diaphragm in neonates and adults.

Miniature endplate potential (MEPP) amplitude distributions from adult mouse diaphragm junctions show two classes of MEPPs. The larger MEPPs form a bell-shaped distribution (bell-MEPPs) with a variance of 20-30%. The smaller MEPPs form a right-hand skew-distribution composing 1-10% of the MEPPs with a mode 1/10th (s-MEPP class) that of the bell-MEPPs. Junctions of 1-day-old neonates generate mainly s-MEPPs. The MEPP distribution gradually changes to mainly bell-MEPPs during the first 3 weeks of postnatal junctions to those of adults and reinnervated adults. We found no differences between neonates and adults in either synaptic vesicle diameter (56 nm o.d.) or in synaptic cleft widths. Synaptic vesicle diameters were the same for 'touching vesicles'. Junctions of 1-day-old neonates showed no or shallow postsynaptic folds. Postsynaptic folds were essentially developed by the third day although one-quarter of the MEPPs were of the skew-class. Assuming that vesicles are simple containers, we would expect a class of 30-nm o.d. vesicles which would generate the s-MEPP class. Since we found a smooth distribution of synaptic vesicles and no vesicles of 30 nm diameter we conclude that within the constraints of the vesicle hypothesis of transmitter release that s- and bell-MEPPs are from vesicles sized the same and that vesicle volume does not determine the amount of acetylcholine released.

Acetylcholine↗

PubMed1989

Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles.

Much current work on the mechanism of neurosecretion has focused on proteins specific to neural secretory vesicles (synaptic vesicles). We report a calcium-stimulated lipid kinase that co-purifies with rat brain synaptic vesicles. This enzyme activity is found only in membrane fractions that contain synaptic vesicle markers. Based on identification of the lipid product as ceramide 1-phosphate and on the finding that ceramide kinase activity co-purifies with synaptic vesicles, the enzyme is proposed to be a ceramide kinase. Kinase activity is stimulated by micromolar concentrations of calcium. Calcium increases the apparent Vmax of the reaction with little effect on the Km for ceramide. The vesicular localization of this enzyme, the requirement for ATP, and the stimulation of enzyme activity by micromolar calcium suggest that ceramide phosphorylation may be associated with neurotransmitter release.

Animals↗

PubMed1985

The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides.

Coated vesicles have been purified in the past on the basis of their remarkably homogeneous structure, not their function. We have succeeded in isolating two subpopulations of bovine brain coated vesicles that carry specific "cargoes," in this case two synaptic vesicle membrane polypeptides (Mr = 95,000 and 65,000). Monoclonal antibodies that recognize cytoplasmic domains of these polypeptides can penetrate the clathrin coat and recognize them on the outer surface of the coated vesicle membrane. An immunoadsorption technique could therefore be used to fractionate coated vesicles on the basis of their membrane composition. The subpopulations have the normal complement of conventional coated vesicle proteins. Exclusive, however, to the subpopulations that carry synaptic vesicle polypeptides are two new coated vesicle polypeptides (Mr = 38,000 and 29,000).

Animals↗

PubMed1995

Essential functions of synapsins I and II in synaptic vesicle regulation.

Synaptic vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.

Animals↗

PubMed1979

Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex.

Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, gamma-aminobutyric acid, aspartate, glutamate and catecholamines were 4--8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca--Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.

Acetylcholinesterase↗

PubMed1999

Synaptic vesicle biogenesis.

Synaptic vesicles, which have been a paradigm for the fusion of a vesicle with its target membrane, also serve as a model for understanding the formation of a vesicle from its donor membrane. Synaptic vesicles, which are formed and recycled at the periphery of the neuron, contain a highly restricted set of neuronal proteins. Insight into the trafficking of synaptic vesicle proteins has come from studying not only neurons but also neuroendocrine cells, which form synaptic-like microvesicles (SLMVs). Formation and recycling of synaptic vesicles/SLMVs takes place from the early endosome and the plasma membrane. The cytoplasmic machinery of synaptic vesicle/SLMV formation and recycling has been studied by a variety of experimental approaches, in particular using cell-free systems. This has revealed distinct machineries for membrane budding and fission. Budding is mediated by clathrin and clathrin adaptors, whereas fission is mediated by dynamin and its interacting protein SH3p4, a lysophosphatidic acid acyl transferase.

Animals↗

PubMed1993

Synaptic vesicle life cycle and synaptic turnover.

Cholinergic synaptic vesicles contain a mixture of soluble low molecular mass constituents. Besides acetylcholine these include Ca2+, ATP, GTP, small amounts of ADP and AMP, and also the diadenosine polyphosphates Ap4A and Ap5A. In synaptic vesicles isolated from the electric ray these diadenosine polyphosphates occur in mmol concentrations and might represent a novel cotransmitter. The membrane proteins of cholinergic synaptic vesicles presumably are identical to those in other types of electron-lucent synaptic vesicles. A presumptive exception are the transmitter-specific carriers. The life cycle of the synaptic vesicle in intact neurons and in situ was investigated by analysis of all cytoplasmic membrane compartments that share membrane integral proteins with synaptic vesicles. The results suggest that the synaptic vesicle membrane compartment might originate from the trans-Golgi network and, after cycles of exo- and endocytosis in the nerve terminal, might fuse into an endosomal membrane compartment early on retrograde transport. Tracer experiments using membrane proteins and soluble contents suggest that the synaptic vesicle membrane compartment does not intermix with the presynaptic plasma membrane on repeated cycles of exo- and endocytosis if low frequency stimulation is applied. A cDNA has been isolated from the electric ray electric lobe that codes for o-rab3, a small GTP-binding protein highly homologous to mammalian rab3. While abundant in the nerve terminals of the electric organ and at the neuromuscular junction this protein occurs only in limited subpopulations of nerve terminals in electric ray brain. Immunocytochemical analysis using the colloidal gold technique and a monospecific antibody against o-rab3 suggests that the GTP-binding protein remains attached to recycling synaptic vesicles. No evidence was found for a major contribution of an intraterminal endosomal sorting compartment involved in synaptic vesicle recycling.

Acetylcholine↗

PubMed1980

Calcium uptake by cholinergic synaptic vesicles.

Pure synaptic vesicles have been isolated in sucrose-KCl media. They are able to take up calcium in the presence of ATP and Mg. This is based on the following evidence. First, the synaptic vesicle fraction is the gradient peak for calcium uptake. Second, it was not possible to separate ACh and ATP from the uptake peak after refractionation of synaptic vesicles. Third, the fraction appears very pure on morphological and biochemical grounds. The physiological significance of the calcium uptake by synaptic vesicles is discussed.

Acetylcholine↗

PubMed1995

The eighth Datta Lecture. Molecular mechanisms in synaptic vesicle recycling.

Synaptic vesicles are specialized secretory organelles which are involved in the fast, point-to-point signaling typical of synapses. They store and secrete non-peptide neurotransmitters and are continuously regenerated in nerve terminals by exoendocytotic recycling. This recycling represents a highly specialized form of the recycling pathway which occurs at the surface of all cells. Several unique properties make synaptic vesicles a powerful experimental model for studies of vesicular traffic. These unique properties include their abundance in brain, the high specialization of nerve terminals for synaptic vesicle recycling, the possibility of studying their exocytosis at the level of single events by electrophysiology and the availability of toxins which block their recycling. This lecture will summarize current information of molecular mechanisms in synaptic vesicle recycling with emphasis on recent studies carried out in my laboratory on mechanisms of vesicle reformation after exocytosis.

Biological Transport↗

PubMed1989

The role of coated vesicles in recycling of synaptic vesicle membrane.

The uptake of extracellular tracers into synaptic nerve terminals has been a phenomenon of persistent interest. Uptake is into synaptic vesicles, hence vesicles spend part of their life in continuity with the plasma membrane, as expected if exocytosis underlies the quantal discharge of neurotransmitters. However, exactly how or when synaptic vesicles acquire extracellular tracers has not been unambiguously determined. Two schools of thought have developed, one holding that vesicles acquire tracers directly via a reversible exo/endocytotic sequence in which they consistently maintain their biochemical identity during their transient continuity with the plasma membrane, the other holding that synaptic vesicles acquire tracers indirectly, via the formation of clathrin-coated vesicles which are spatially and temporally separate from exocytosis and reverse a temporary loss of the vesicles' individual identity upon merger with the plasma membrane. Efforts to distinguish between these two alternatives have generated an interesting diversity of electron microscopic experiments, many of which are reviewed here. However, definitive determination of which view is correct may ultimately require direct visualization of synaptic vesicle turnover in living nerve terminals. To this end, we here review the results of visualizing endocytosis in tissue cultured cells, where light microscopy can provide sufficient resolution to reveal membrane dynamics in living cells. This has allowed visual discrimination of two different types of endocytosis, one clathrin-mediated (coated vesicle formation) and the other actin-mediated (macropinocytosis). Current work is also reviewed which aims at determining experimental methods for inhibiting each type of endocytosis selectively. Hypertonicity and severe cytoplasmic acidification turn out to inhibit coated vesicle formation, while cytochalasin D and mild cytoplasmic acidification selectively inhibit macropinocytosis. Applied to nerves, these various treatments affect synaptic vesicle turnover in a manner that supports the notion that synaptic vesicle membrane recycles via the "indirect" route of coated vesicle formation.

Animals↗

PubMed1995

The non-specific ion channel in Torpedo ocellata fused synaptic vesicles.

1. Synaptic vesicles were isolated and fused into large structures with a diameter of more than 20 microns to characterize their ionic channels. The 'cell'-attached and inside-out configurations of the patch clamp technique were used. 2. Two types of ion channels were most frequently observed: a low conductance chloride channel and a high conductance non-specific channel. 3. The non-specific channel has a main conducting state and a substate. The main conducting state has a slope conductance of 246 +/- 15 pS (+/- S.E.M., n = 15), in the presence of different combinations of KCl and potassium glutamate. 4. From the reversal potentials of the current-voltage (I-V) relation, it was concluded that this channel conducts both Cl- and K+. 5. The non-specific channel is highly voltage dependent: under steady-state voltages it has a high open probability near 0 mV and does not inactivate; when the membrane is hyperpolarized (pipette side more positive), the open probability decreases dramatically. 6. Voltage pulses showed that upon hyperpolarization (from holding potentials between -20 and + 20 mV), the channels deactivated; when the membrane was stepped back to the holding potential, the channels reactivated rapidly. 7. In a number of experiments, when the pipette side was made more negative than the bath, the open probability also decreased. 8. Frequently, a substate with a conductance of about 44 +/- 4% (+/- S.E.M., n = 3) of the main state was detected. 9. We speculate that this non-specific ion channel may have different roles at the various stages of the life cycle of the synaptic vesicle. When the synaptic vesicle is an intracellular structure, it might help its transmitter-concentrating capacity by dissipating the polarization. After fusion with the surface membrane, it might constitute an additional conductance pathway, taking part in frequency modulation of synaptic transmission.

Animals↗

PubMed2006

STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.

Animals↗

PubMed2001

Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming.

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.

Alternative Splicing↗

PubMed1997

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles.

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimide-sensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.

Adenosine Triphosphatases↗

PubMed2002

Signals involved in targeting membrane proteins to synaptic vesicles.

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood. 2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified. 3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.

Animals↗