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1.
EMBO J ; 34(15): 2059-77, 2015 Aug 04.
Article in English | MEDLINE | ID: mdl-26108535

ABSTRACT

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin(-/-) neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin(-/-) phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca(2+) sensor, we demonstrate that snapin-dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca(2+) sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting.


Subject(s)
Lysosomes/metabolism , Models, Neurological , Neurons/physiology , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Vesicular Transport Proteins/metabolism , Animals , Biological Transport/physiology , Blotting, Western , Calcium/metabolism , Cell Fractionation , Cells, Cultured , Circular Dichroism , Dysbindin , Dystrophin-Associated Proteins , Immunohistochemistry , Mice , Microscopy, Electron , Time-Lapse Imaging , Vesicular Transport Proteins/genetics
2.
Neuron ; 67(2): 268-79, 2010 Jul 29.
Article in English | MEDLINE | ID: mdl-20670834

ABSTRACT

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.


Subject(s)
Neurons/metabolism , Neurotransmitter Agents/metabolism , Synapses/physiology , Synaptic Vesicles/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Animals, Newborn , Calcium/metabolism , Cell Membrane/metabolism , Cerebral Cortex/cytology , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Excitatory Postsynaptic Potentials/drug effects , In Vitro Techniques , Liposomes/metabolism , Macrolides/pharmacology , Mutation/genetics , Neurons/drug effects , Neurons/ultrastructure , Neurotransmitter Agents/pharmacology , Peptides/metabolism , Peptides/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Protein Subunits/genetics , Protein Subunits/metabolism , Proteolipids/metabolism , Rats , Rats, Wistar , SNARE Proteins/metabolism , Sequence Alignment/methods , Two-Hybrid System Techniques , Vacuolar Proton-Translocating ATPases/chemistry , Vesicle-Associated Membrane Protein 2/genetics , Vesicle-Associated Membrane Protein 2/metabolism
3.
J Biol Chem ; 285(31): 23665-75, 2010 Jul 30.
Article in English | MEDLINE | ID: mdl-20519509

ABSTRACT

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.


Subject(s)
Calcium/metabolism , Calmodulin/chemistry , Gene Expression Regulation , Membrane Fusion , SNARE Proteins/chemistry , Animals , Calcium/chemistry , Cattle , Cell Membrane/metabolism , Exocytosis , Fluorescence Resonance Energy Transfer , Humans , Kinetics , Liposomes/chemistry , Synaptotagmin I/chemistry , Tetanus Toxin/chemistry
4.
Proc Natl Acad Sci U S A ; 107(8): 3517-21, 2010 Feb 23.
Article in English | MEDLINE | ID: mdl-20133592

ABSTRACT

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.


Subject(s)
Fluorescence Recovery After Photobleaching/methods , Membrane Fusion , SNARE Proteins/metabolism , Animals , Humans , SNARE Proteins/chemistry , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Unilamellar Liposomes/chemistry
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