scholarly journals Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

2007 ◽  
Vol 404 (3) ◽  
pp. 525-534 ◽  
Author(s):  
Dario Bonanomi ◽  
Laura Rusconi ◽  
Chiara Agnese Colombo ◽  
Fabio Benfenati ◽  
Flavia Valtorta
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Critical Component ◽  
Attachment Protein ◽  
Protein Protein Interaction ◽  
Protein Mutants ◽  
Vesicle Biogenesis ◽  
Exocytic Pathway ◽  
Protein Attachment

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle­-fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein–protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and α-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.

scholarly journals Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

2010 ◽  
Vol 188 (1) ◽  
pp. 145-156 ◽  
Author(s):  
Rubén Fernández-Busnadiego ◽  
Benoît Zuber ◽  
Ulrike Elisabeth Maurer ◽  
Marek Cyrklaff ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Attachment Protein ◽  
Readily Releasable Pool ◽  
Protein Receptor ◽  
Membrane Contact ◽  
Synaptic Vesicle Release ◽  
Protein Attachment

The presynaptic terminal contains a complex network of filaments whose precise organization and functions are not yet understood. The cryoelectron tomography experiments reported in this study indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations are consistent with an exocytosis model in which vesicles are first anchored by long (>5 nm) tethers that give way to multiple short tethers once vesicles enter the readily releasable pool. The formation of short tethers was inhibited by tetanus toxin, indicating that it depends on soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor complex assembly. Vesicles were extensively interlinked via a set of connectors that underwent profound rearrangements upon synaptic stimulation and okadaic acid treatment, suggesting a role of these connectors in synaptic vesicle mobilization and neurotransmitter release.

scholarly journals How do you recognize and reconstitute a synaptic vesicle after fusion?

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1734 ◽  
Author(s):  
Natali L. Chanaday ◽  
Ege T. Kavalali
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Membrane Composition ◽  
Synaptic Vesicle Recycling ◽  
Vesicle Recycling ◽  
Molecular Identity ◽  
Daunting Task ◽  
Vesicle Biogenesis ◽  
Vesicle Cycle

Synaptic vesicle recycling is essential for sustained and reliable neurotransmission. A key component of synaptic vesicle recycling is the synaptic vesicle biogenesis process that is observed in synapses and that maintains the molecular identity of synaptic vesicles. However, the mechanisms by which synaptic vesicles are retrieved and reconstituted after fusion remain unclear. The complex molecular composition of synaptic vesicles renders their rapid biogenesis a daunting task. Therefore, in this context, kiss-and-run type transient fusion of synaptic vesicles with the plasma membrane without loss of their membrane composition and molecular identity remains a viable hypothesis that can account for the fidelity of the synaptic vesicle cycle. In this article, we discuss the biological implications of this problem as well as its possible molecular solutions.

scholarly journals The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts.

1993 ◽  
Vol 123 (3) ◽  
pp. 575-584 ◽  
Author(s):  
M B Feany ◽  
A G Yee ◽  
M L Delvy ◽  
K M Buckley
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Cell Lines ◽  
Synaptic Vesicles ◽  
Vesicle Biogenesis ◽  
Intracellular Vesicles ◽  
Cellular Compartments ◽  
Vesicle Proteins

We expressed the synaptic vesicle proteins SV2, synaptotagmin, and synaptophysin in CHO fibroblasts to investigate the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. SV2-containing vesicles did not have the same sedimentation characteristics as authentic synaptic vesicles, even though transfected SV2 was sorted from endosomal markers. We also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not created synaptic vesicle-like structures. These results have important implications for models of synaptic vesicle biogenesis.

Neurotransmitter Release

2017 ◽  
Author(s):  
Peggy Mason
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Synaptic Cleft ◽  
Fusion Pore ◽  
Synaptic Membrane ◽  
Specialized Region ◽  
Synaptic Vesicle Fusion

The biochemical and physiological processes of neurotransmitter release from an active zone, a specialized region of synaptic membrane, are examined. Synaptic vesicles containing neurotransmitters are docked at the active zone and then primed for release by SNARE complexes that bring them into extreme proximity to the plasma membrane. Entry of calcium ions through voltage-gated calcium channels triggers synaptic vesicle fusion with the synaptic terminal membrane and the consequent diffusion of neurotransmitter into the synaptic cleft. Release results when the fusion pore bridging the synaptic vesicle and plasma membrane widens and neurotransmitter from the inside of the synaptic vesicle diffuses into the synaptic cleft. Membrane from the active zone membrane is endocytosed, and synaptic vesicle proteins are then reassembled into recycled synaptic vesicles, allowing for more rounds of neurotransmitter release.

Dynamin Mediates Membrane Vesiculation

1998 ◽  
Vol 4 (S2) ◽  
pp. 1022-1023
Author(s):  
Sharon M. Sweitzer ◽  
Jenny E. Hinshaw
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Mechanism Of Action ◽  
Synaptic Vesicles ◽  
Dominant Negative ◽  
Coated Pits ◽  
Vesicle Recycling ◽  
Membrane Vesiculation ◽  
Helical Tubes

Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis and synaptic vesicle recycling; however its mechanism of action is unknown. The requirement for dynamin was first elucidated by the discovery that the shibire gene product in Drosophila melanogaster was homologous to mammalian dynamin-1 (1,2). The shibire flies exhibit a depletion of synaptic vesicles and an accumulation of collared clathrin-coated pits at the plasma membrane of their nerve termini (3). It was later demonstrated that endocytosis was inhibited by the overexpression of dominant negative mutants of dynamin (4,5), and that purified dynamin can self-associate to form spirals which resemble the collars of shibire and structures seen in synaptosomes treated with GTPγS (6,7). These observations led to the speculation that dynamin pinches the clathrin-coated bud from the plasma membrane. In support of this hypothesis, we show that purified recombinant dynamin can bind to a lipid bilayer in a regular and repeating pattern to form helical tubes which vesiculate upon the addition of GTP.

scholarly journals The Vesicle- and Target-SNARE Proteins That Mediate Glut4 Vesicle Fusion Are Localized in Detergent-insoluble Lipid Rafts Present on Distinct Intracellular Membranes

2002 ◽  
Vol 277 (51) ◽  
pp. 49750-49754 ◽  
Author(s):  
Luke H. Chamberlain ◽  
Gwyn W. Gould
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Lipid Rafts ◽  
Significant Proportion ◽  
Vesicle Fusion ◽  
Spatial Integration ◽  
Attachment Protein ◽  
Intracellular Membranes ◽  
Syntaxin 4 ◽  
Protein Attachment

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 (∼35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-β-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.

scholarly journals The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

2000 ◽  
Vol 11 (8) ◽  
pp. 2591-2604 ◽  
Author(s):  
Victor V. Faundez ◽  
Regis B. Kelly
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Enzymatic Reaction ◽  
Casein Kinase ◽  
Small Gtpase ◽  
Casein Kinase I ◽  
Vesicle Membranes ◽  
Vesicle Biogenesis ◽  
Hinge Domain

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes ( Faundez et al., 1998 ). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the β3 subunit of the complex by a kinase similar to casein kinase 1α. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the β3A and β3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

scholarly journals A new life for an old pump: V-ATPase and neurotransmitter release

2014 ◽  
Vol 205 (1) ◽  
pp. 7-9 ◽  
Author(s):  
Stefano Vavassori ◽  
Andreas Mayer
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Snare Complex ◽  
Spontaneous Release ◽  
Synaptic Vesicle Fusion ◽  
Snare Complex Formation

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca2+ ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca2+-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

The molecular mechanisms of the mammalian exocyst complex in exocytosis

2006 ◽  
Vol 34 (5) ◽  
pp. 687-690 ◽  
Author(s):  
S. Wang ◽  
S.C. Hsu
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Secretory Vesicles ◽  
Attachment Protein ◽  
Trafficking Pathway ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Associated Proteins ◽  
Protein Attachment

Exocytosis is a highly ordered vesicle trafficking pathway that targets proteins to the plasma membrane for membrane addition or secretion. Research over the years has discovered many proteins that participate at various stages in the mammalian exocytotic pathway. At the early stage of exocytosis, co-atomer proteins and their respective adaptors and GTPases have been shown to play a role in the sorting and incorporation of proteins into secretory vesicles. At the final stage of exocytosis, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and SNARE-associated proteins are believed to mediate the fusion of secretory vesicles at the plasma membrane. There are multiple events that may occur between the budding of secretory vesicles from the Golgi and the fusion of these vesicles at the plasma membrane. The most obvious and best-known event is the transport of secretory vesicles from Golgi to the vicinity of the plasma membrane via microtubules and their associated motors. At the vicinity of the plasma membrane, however, it is not clear how vesicles finally dock and fuse with the plasma membrane. Identification of proteins involved in these events should provide important insights into the mechanisms of this little known stage of the exocytotic pathway. Currently, a protein complex, known as the sec6/8 or the exocyst complex, has been implicated to play a role at this late stage of exocytosis.

scholarly journals The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

1996 ◽  
Vol 133 (6) ◽  
pp. 1237-1250 ◽  
Author(s):  
K Takei ◽  
O Mundigl ◽  
L Daniell ◽  
P De Camilli
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Nerve Terminals ◽  
Vesicle Budding ◽  
Vesicle Cycle ◽  
Single Vesicle

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

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