ABSTRACT
SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.
Subject(s)
Brain Diseases/genetics , Brain Diseases/physiopathology , Synaptic Transmission/genetics , Synaptosomal-Associated Protein 25/genetics , Adolescent , Amino Acid Sequence , Animals , Cells, Cultured , Child , Child, Preschool , Female , HEK293 Cells , Haploinsufficiency/genetics , Humans , Male , Mice , Mice, Knockout , Mice, Transgenic , Protein Structure, Secondary , Rats , Rats, Sprague-Dawley , Synaptosomal-Associated Protein 25/chemistryABSTRACT
Synaptic vesicle fusion is mediated by SNARE proteins-VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the presynaptic membrane. Chaperones Munc18-1 and Munc13-1 cooperatively catalyze SNARE assembly via an intermediate 'template' complex containing Syntaxin-1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 'linker' region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in ~ 1 : 1 stoichiometry by the self-assembled Munc13-1 nanoclusters.
Subject(s)
Liposomes/metabolism , Nerve Tissue Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Animals , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Liposomes/chemistry , Mice , Models, Molecular , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Optical Tweezers , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/genetics , Syntaxin 1/chemistry , Syntaxin 1/genetics , Vesicle-Associated Membrane Protein 2/chemistry , Vesicle-Associated Membrane Protein 2/geneticsABSTRACT
Central to the exocytotic release of hormones and neurotransmitters is the interaction of four SNARE motifs in proteins on the secretory granule/synaptic vesicle membrane (synaptobrevin/VAMP, v-SNARE) and on the plasma membrane (syntaxin and SNAP25, t-SNAREs). The interaction is thought to bring the opposing membranes together to enable fusion. An underlying motivation for this Viewpoint is to synthesize from recent diverse studies possible new insights about these events. We focus on a recent paper that demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion. Remarkably, lipid-perturbing agents rescue a palmitoylation-deficient mutant whose phenotype includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Unexpectedly, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE "acceptor" complex for synaptobrevin2. This insight, together with biophysical and optical studies from other laboratories, suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the subsequent trans-SNARE complex with the v-SNARE synaptobrevin form within 100 ms before fusion.
Subject(s)
Exocytosis , Membrane Fusion , Synaptosomal-Associated Protein 25/chemistry , Cell Membrane , Qa-SNARE Proteins , R-SNARE ProteinsABSTRACT
The neuronal protein complexin contains multiple domains that exert clamping and facilitatory functions to tune spontaneous and action potential-triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that, prior to fusion, the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitters in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.
Subject(s)
Cell Membrane/metabolism , Exocytosis , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Vesicle-Associated Membrane Protein 2/chemistry , Vesicle-Associated Membrane Protein 2/metabolism , Calcium/metabolism , Cross-Linking Reagents/chemistry , Humans , Light , Membrane Fusion , Models, Biological , Mutant Proteins/metabolism , Neurons/metabolism , Neurotransmitter Agents/metabolism , Protein Binding , Protein Interaction Mapping , Protein Structure, Secondary , Proteolipids/metabolism , Synapses/metabolism , Synaptic Vesicles/metabolismABSTRACT
Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by â¼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.
Subject(s)
Molecular Chaperones/metabolism , Munc18 Proteins/metabolism , Nerve Tissue Proteins/metabolism , SNARE Proteins/metabolism , Exocytosis/physiology , Kinetics , Membrane Fusion/physiology , Molecular Chaperones/chemistry , Munc18 Proteins/chemistry , Nerve Tissue Proteins/chemistry , Neurons/metabolism , Optical Tweezers , Protein Binding , Protein Domains , Qa-SNARE Proteins/metabolism , SNARE Proteins/chemistry , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/metabolism , Vesicle-Associated Membrane Protein 2/metabolismABSTRACT
SNAP-25, one of the three SNARE-proteins responsible for synaptic release, can be phosphorylated by Protein Kinase C on Ser-187, close to the fusion pore. In neuroendocrine cells, this phosphorylation event potentiates vesicle recruitment into releasable pools, whereas the consequences of phosphorylation for synaptic release remain unclear. We mutated Ser-187 and expressed two mutants (S187C and S187E) in the context of the SNAP-25B-isoform in SNAP-25 knockout glutamatergic autaptic neurons. Whole-cell patch clamp recordings were performed to assess the effect of Ser-187 phosphorylation on synaptic transmission. Blocking phosphorylation by expressing the S187C mutant did not affect synapse density, basic evoked or spontaneous neurotransmission, the readily-releasable pool size or its Ca2+-independent or Ca2+-dependent replenishment. Furthermore, it did not affect the response to phorbol esters, which activate PKC. Expressing S187C in the context of the SNAP-25A isoform also did not affect synaptic transmission. Strikingly, the - potentially phosphomimetic - mutant S187E reduced spontaneous release and release probability, with the largest effect seen in the SNAP-25B isoform, showing that a negative charge in this position is detrimental for neurotransmission, in agreement with electrostatic fusion triggering. During the course of our experiments, we found that higher SNAP-25B expression levels led to decreased paired pulse potentiation, probably due to higher release probabilities. Under these conditions, the potentiation of evoked EPSCs by phorbol esters was followed by a persistent down-regulation, probably due to a ceiling effect. In conclusion, our results indicate that phosphorylation of Ser-187 in SNAP-25 is not involved in modulation of synaptic release by Ca2+ or phorbol esters.
Subject(s)
Calcium/metabolism , Excitatory Postsynaptic Potentials , Synaptosomal-Associated Protein 25/metabolism , Animals , Cells, Cultured , Mice , Mice, Inbred C57BL , Mutation , Neuronal Plasticity , Neurons/drug effects , Neurons/metabolism , Neurons/physiology , Phorbol Esters/pharmacology , Phosphorylation , Protein Kinase C/metabolism , Rats , Rats, Wistar , Serine/chemistry , Serine/genetics , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/geneticsABSTRACT
Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.
Subject(s)
Cell Membrane/chemistry , Lipid Bilayers/chemistry , Membrane Fusion , Munc18 Proteins , SNARE Proteins/chemistry , Transport Vesicles/chemistry , Amino Acid Sequence , Animals , Caenorhabditis elegans , Cell Membrane/metabolism , Drosophila melanogaster , Humans , Lipid Bilayers/metabolism , Mice , Models, Molecular , Munc18 Proteins/chemistry , Munc18 Proteins/metabolism , Peptides/chemistry , Peptides/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , SNARE Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolism , Transport Vesicles/metabolism , Vesicle-Associated Membrane Protein 2/chemistry , Vesicle-Associated Membrane Protein 2/metabolism , Xenopus laevisABSTRACT
Botulinum neurotoxin serotype A (BoNT/A), marketed commercially as Botox, is the most toxic substance known to man with an estimated intravenous lethal dose (LD50) of 1-2 ng/kg in humans. Despite its widespread use in cosmetic and medicinal applications, no postexposure therapeutics are available for the reversal of intoxication in the event of medical malpractice or bioterrorism. Accordingly, the Centers for Disease Control and Prevention categorizes BoNT/A as a Category A pathogen, posing the highest risk to national security and public health as a result of the ease with which BoNT/A can be weaponized and disseminated. BoNT/A-mediated lethality results from neurons impeded from releasing acetylcholine, which ultimately causes muscle paralysis and possible death by asphyxiation with the loss of diaphragm function. Currently, the only available respite for BoNT/A poisoning is antibody-based therapy; however, this intervention is only effective within 12-24 h postexposure. Small molecule therapeutics remain the only opportunity to reverse BoNT/A intoxication after neuronal poisoning and are urgently needed. Nevertheless, no small molecule BoNT/A inhibitors have reached the clinic or even advanced to clinical trials. This Account highlights the accomplishments and existing challenges facing BoNT/A drug discovery today. Using the comprehensive body of work from our laboratory, we illustrate our nearly two-decade endeavor to discover a clinically relevant BoNT/A inhibitor. Specifically, a discussion on the identification and characterization of new chemical leads, the development of in vitro and in vivo assays, and pertinent discoveries in BoNT/A structural biology related to small molecule inhibition is presented. Lead discovery efforts in our laboratory have leveraged both in vitro high-throughput screening and rational design, and an array of mechanistic strategies for inhibiting BoNT/A has been discovered, including noncovalent inhibition, metal-binding active site inhibition, covalent inhibition, and α- and ß-exosite inhibition. We contrast the strengths and weaknesses of each of these mechanistic strategies and propose the most favorable approach for success. Finally, we discuss multiple serendipitous discoveries of antibotulism small molecules with alternative mechanisms of action. Remaining challenges facing clinically relevant BoNT/A inhibition are presented and analyzed, including the current inability to reconcile toxin half-life (months to greater than one year) in neurons with in vivo pharmaceutical lifetimes and reoccurring inconsistencies between in vitro, cellular, and in vivo translation. Our Account of BoNT/A chemical research emphasizes the present accomplishments and critically analyzes the remaining obstacles for drug discovery. Importantly, we call for an increased focus on the discovery of safe and effective covalent inhibitors of BoNT/A that compete with the inherent half-life of the toxin.
Subject(s)
Botulinum Toxins, Type A/antagonists & inhibitors , Botulism/drug therapy , Protease Inhibitors/therapeutic use , Animals , Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Catalytic Domain , Humans , Mice , Protein Binding , Proteolysis , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/metabolismABSTRACT
Botulinum is a deadly bacterial toxin that causes neuroparalytic disease. However, appropriate tools to detect trace toxic proteins are scarce. This study presents a bead-based diffusometric technique for the rapid, simple, and quantitative detection of biological toxins. Functionalized particles called nano-immunosensors were fabricated by forming sandwiched immunocomplexes comprising Au nanoparticles (AuNPs), toxic proteins, and antibodies on fluorescent probe particles. Particle diffusivity tended to decline with increasing concentration of the target proteins. Calibration curves of purified botulinum toxins (0.01-500 ng/mL) were obtained from whole milk and bovine serum, and results suggested that measurement was independent of the background matrix. The activity of botulinum toxin was evaluated by coating synaptosomal-associated protein 25 (SNAP-25) on fluorescent probe particles. AuNP-conjugated antibodies attached to the probe particles when SNAP-25 proteins were cleaved by active botulinum. Thus, toxicity could be detected from slight changes in diffusivity. A short measurement time of 2 min and a limit of detection of 10 pg/mL were achieved. The nano-immunosensors demonstrated rapid biosensing capability and met the demands of onsite screening for food safety, medical instrument hygiene, and cosmetic surgery products.
Subject(s)
Biosensing Techniques/methods , Botulinum Toxins, Type A/blood , Immunoassay/methods , Metal Nanoparticles/chemistry , Animals , Antibodies, Monoclonal, Murine-Derived/immunology , Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/immunology , Food Contamination/analysis , Gold/chemistry , Limit of Detection , Mice , Milk/chemistry , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/immunologyABSTRACT
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex comprises synaptosome-associated protein of 25 kDa (SNAP25), syntaxin-1a (syx-1), and synaptobrevin 2, which is essential for many physiologic processes requiring membrane fusion. Several studies imply that the loop region of SNAP25 plays important roles in SNARE-complex assembly. However, why and how the flexible loop facilitates the complex assembly remains poorly understood because it is purposely deleted in almost all structural studies. By using NMR spectroscopy and circular dichroism spectropolarimetry, we characterized SNAP25 structure and interactions with other SNAREs in aqueous buffer and in the membrane. We found that the N-terminal of the SNAP25 loop region binds with membrane, and this interaction induced a disorder-to-order conformational change of the loop, resulting in enhanced interaction between the C-terminal of the SNAP25 loop and syx-1. We further proved that SNARE-complex assembly efficiency decreased when we disrupted the electrostatic interaction between C-terminal of the SNAP25 loop and syx-1, suggesting that the SNAP25 loop region facilitates SNARE-complex assembly through promoting prefusion SNARE binary complex formation. Our work elucidates the role of the flexible loop and the membrane environment in SNARE-complex assembly at the residue level, which helps to understand membrane fusion, a fundamental transport and communication process in cells.-Jiang, X., Zhang, Z., Cheng, K., Wu, Q., Jiang, L., Pielak, G. J., Liu, M., Li, C. Membrane-mediated disorder-to-order transition of SNAP25 flexible linker facilitates its interaction with syntaxin-1 and SNARE-complex assembly.
Subject(s)
Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Cell Membrane/metabolism , Circular Dichroism , Cysteine/chemistry , Humans , Liposomes , Multiprotein Complexes/chemistry , Phosphorylation , Protein Binding , Protein Conformation , Protein Domains , Protein Interaction Mapping , Protein Processing, Post-Translational , Recombinant Proteins/metabolism , Static Electricity , Synaptosomal-Associated Protein 25/chemistryABSTRACT
Exocytosis of synaptic vesicles and dense-core vesicles requires both the Munc13 and CAPS (Ca2+-dependent activator proteins for secretion) proteins. CAPS contains a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-binding region (called the DAMH domain), which has been found to be essential for SNARE-mediated exocytosis. Here we report a crystal structure of the CAPS-1 DAMH domain at 2.9-Å resolution and reveal a dual role of CAPS-1 in SNARE complex formation. CAPS-1 plays an inhibitory role dependent on binding of the DAMH domain to the MUN domain of Munc13-1, which hinders the ability of Munc13 to catalyze opening of syntaxin-1, inhibiting SNARE complex formation, and a chaperone role dependent on interaction of the DAMH domain with the syntaxin-1/SNAP-25 complex, which stabilizes the open conformation of Syx1, facilitating SNARE complex formation. Our results suggest that CAPS-1 facilitates SNARE complex formation via the DAMH domain in a manner dependent on sequential and cooperative interaction with Munc13-1 and SNARE proteins.
Subject(s)
Nerve Tissue Proteins/chemistry , Synaptosomal-Associated Protein 25/chemistry , Syntaxin 1/chemistry , Animals , Crystallography, X-Ray , HEK293 Cells , Humans , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , PC12 Cells , Protein Domains , Protein Structure, Quaternary , Rats , Sf9 Cells , Spodoptera , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/genetics , Syntaxin 1/metabolismABSTRACT
Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Munc18 Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/chemistry , Nerve Tissue Proteins/chemistry , Synaptosomal-Associated Protein 25/chemistry , Synaptotagmins/chemistry , Animals , CHO Cells , Calcium/chemistry , Cricetinae , Cricetulus , Cryoelectron Microscopy , Cytoplasm/chemistry , Fluorescence Resonance Energy Transfer , Kinetics , Mutation , R-SNARE Proteins/chemistry , Rats , Syntaxin 1/chemistryABSTRACT
8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), which is the second messenger in nitric oxide/reactive oxygen species redox signaling, covalently binds to protein thiol groups (called S-guanylation) and exerts various biological functions. Synaptosomal associated protein 25 (SNAP-25), a member of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, plays an important role in the process of membrane fusion. We previously showed that SNAP-25 is S-guanylated at cysteine 90. In addition, we revealed that S-guanylation of SNAP-25 increases SNARE complex formation, but decreases the affinity of SNARE complex for complexin. Since SNAP-25 plays a critical role in regulating exocytosis, it is important to elucidate the physiological or pathophysiological meanings of S-guanylation of this protein. Here we describe a protocol for detecting 8-nitro-cGMP and S-guanylated proteins in cells by immunocytochemistry, and methods to detect SNARE complex in 8-nitro-cGMP-treated cells.
Subject(s)
Cyclic GMP/analogs & derivatives , Protein Structure, Quaternary , Synaptosomal-Associated Protein 25/chemistry , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Line, Tumor , Cyclic GMP/chemistry , Cysteine/chemistry , Humans , Immunohistochemistry , Membrane Fusion , Native Polyacrylamide Gel Electrophoresis/instrumentation , Native Polyacrylamide Gel Electrophoresis/methods , Synaptosomal-Associated Protein 25/metabolismABSTRACT
In this chapter, we introduce a nanodisc-based experimental platform to study Ca2+-triggered membrane interaction of synaptotagmin-1. We describe and discuss in detail how to assemble this soluble mimetic of the docked vesicle-plasma membrane junction, with fluorescently labeled synaptotagmin-1 bound to trans SNAREpins assembled between nanodiscs and present the stopped-flow rapid mixing method used to monitor the conformational dynamics of Ca2+-activation process on a millisecond timescale.
Subject(s)
Lipid Bilayers/metabolism , Nanostructures/chemistry , Synaptotagmin I/metabolism , Calcium/metabolism , Cysteine/genetics , Fluorescent Dyes/chemistry , Lipid Bilayers/chemistry , Membrane Fusion , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrum Analysis/instrumentation , Spectrum Analysis/methods , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/isolation & purification , Synaptosomal-Associated Protein 25/metabolism , Synaptotagmin I/chemistry , Synaptotagmin I/genetics , Synaptotagmin I/isolation & purification , Syntaxin 1/chemistry , Syntaxin 1/isolation & purification , Syntaxin 1/metabolismABSTRACT
During exocytosis, vesicles fuse with the plasma membrane and release their contents. The fusion pore is the initial, nanometer-sized connection between the plasma membrane and the cargo-laden vesicle. A growing body of evidence points toward the fusion pore being a regulator of exocytosis, but the shortcomings of current experimental techniques to investigate single-fusion pores make it difficult to study factors governing pore behavior. Here we describe an assay that fuses v-SNARE-reconstituted nanodiscs with cells ectopically expressing "flipped" t-SNAREs to monitor dynamics of single fusion pores in a biochemically defined system using electrical recordings. We also describe a fluorescence microscopy-based approach to monitor nanodisc-cell fusion that is much simpler to employ, but cannot resolve single pores.
Subject(s)
Biological Assay/methods , Nanostructures/chemistry , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/metabolism , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Membrane/chemistry , Cell Membrane/metabolism , Exocytosis , Genetic Engineering , HeLa Cells , Humans , Membrane Fusion , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Patch-Clamp Techniques/instrumentation , Patch-Clamp Techniques/methods , Secretory Vesicles/chemistry , Secretory Vesicles/metabolism , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/genetics , Syntaxin 1/chemistry , Syntaxin 1/geneticsABSTRACT
Cultured bovine chromaffin cells have been tested as a successful neuroendocrine model to study the secretory process. Changes in the dynamics of the secretory vesicles and the exocytotic machinery microdomains could be studied in control and stimulated conditions using appropriate molecular tools such as fluorescent SNARE protein expression or fluorochrome vesicular labeling in these neuroendocrine cells. Since most of these changes occur in or near the plasma membrane, the use of the total internal reflection fluorescent microscopy (TIRFM) and the implement of particle motion analysis could be essential tools to study the structural and dynamic changes of secretory machinery related with its function in this exocytotic cell model.
Subject(s)
Chromaffin Cells/metabolism , Secretory Vesicles/metabolism , Synaptosomal-Associated Protein 25/metabolism , Adrenal Medulla/cytology , Animals , Cattle , Exocytosis , Fluorescent Dyes/chemistry , Membrane Fusion , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Primary Cell Culture/instrumentation , Primary Cell Culture/methods , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Secretory Vesicles/chemistry , Synaptosomal-Associated Protein 25/chemistry , Synaptosomal-Associated Protein 25/isolation & purificationABSTRACT
Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the presynaptic membrane. Vesicles are attached to the membrane via the SNARE complex, comprising the vesicle associated protein synaptobrevin (Syb), the membrane associated protein syntaxin (Syx), and the cytosolic protein SNAP25, that together form a four-helical bundle. The full assembly of Syb onto the core SNARE bundle promotes vesicle fusion. We investigated SNARE assembly using a coarse-grained model of the SNARE complex that retains chemical specificity. Steered force-control simulations of SNARE unzippering were used to set up initial disassembled states of the SNARE complex. From these states, the assembly process was simulated. We find that if Syb is in helical form and proximal to the other helices, then the SNARE complex assembles rapidly, on a microsecond time-scale, which is well within in vivo synaptic vesicle fusion time scales. Assembly times grow exponentially with a separation distance between Syb and Syx C-termini. Our results indicate that for biologically relevant rapid assembly of the SNARE complex, Syb should be in helical form, and the SNARE constituent helices brought into proximity, possibly by an agent, such as a chaperone.
Subject(s)
Qa-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Molecular Dynamics Simulation , Monte Carlo Method , Protein Binding , Protein Conformation, alpha-Helical , Protein Folding , Qa-SNARE Proteins/chemistry , R-SNARE Proteins/chemistry , Synaptosomal-Associated Protein 25/chemistryABSTRACT
ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin's function and even 'superclamp' tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build-up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Exocytosis/genetics , Nerve Tissue Proteins/chemistry , Synaptic Vesicles/chemistry , Synaptosomal-Associated Protein 25/chemistry , Adaptor Proteins, Vesicular Transport/genetics , Animals , Calcium/chemistry , Cell Membrane/chemistry , Cell Membrane/genetics , Membrane Fusion/genetics , Mice , Nerve Tissue Proteins/genetics , Protein Binding , Protein Domains/genetics , SNARE Proteins/chemistry , SNARE Proteins/genetics , Synaptic Vesicles/genetics , Synaptosomal-Associated Protein 25/geneticsABSTRACT
As a SNARE binding protein, tomosyn has been reported to negatively regulate synaptic exocytosis via arresting syntaxin-1 and SNAP-25 into a nonfusogenic product that precludes synaptobrevin-2 entry, raising the question how the assembly of the SNARE complex is achieved. Here, we have investigated new functions of tomosyn in SNARE complex formation and SNARE-mediated vesicle fusion. Assisted by NSF/α-SNAP, syntaxin-1 escapes tomosyn arrest and assembles into the Munc18-1/syntaxin-1 complex. Munc13-1 then catalyzes the transit of syntaxin-1 from the Munc18-1/syntaxin-1 complex to the SNARE complex in a manner specific to synaptobrevin-2 but resistant to tomosyn. Our data suggest that tomosyn ensures SNARE assembly in a way amenable to tight regulation by Munc18-1 and Munc13-1.
Subject(s)
Multiprotein Complexes/chemistry , Munc18 Proteins/chemistry , Nerve Tissue Proteins/chemistry , R-SNARE Proteins/chemistry , Synaptosomal-Associated Protein 25/chemistry , Humans , Multiprotein Complexes/metabolism , Munc18 Proteins/metabolism , Nerve Tissue Proteins/metabolism , R-SNARE Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/chemistry , Syntaxin 1/metabolism , Vesicle-Associated Membrane Protein 2/chemistry , Vesicle-Associated Membrane Protein 2/metabolismABSTRACT
Botulinum neurotoxins (BoNTs; serotypes A-G) are metalloproteases, which cleave and inactivate cellular proteins essential for neurotransmitter release. In bacterial cultures, BoNTs are secreted as a complex of the neurotoxin and a group of neurotoxin associated proteins (NAPs). Under physiological condition (pH 7.4), this complex is believed to be dissociated to separate the neurotoxin from NAPs. BoNT consists of a 50 kDa light (L) chain (LC or catalytic domain) and a 100 kDa heavy (H) chain (or HC) linked through a disulfide bond and other non-covalent interactions. The cell intoxication involves three major steps; binding, membrane translocation and inhibition of neurotransmitter release. The last step of intoxication, endopeptidase activity, is very unique and specific that can be used for detection of the complex and isolated forms of the toxin. A fluorescent tag-labeled synthetic peptide (SNAPtide) derived from a segment of SNAP-25, an intracellular substrate of BoNT/A, is used to detect and assay the endopeptidase activity of BoNT/A. The detection of the signal is based on the change in the fluorescence energy transfer after selective cleavage of the peptide by the BoNT/A. In this report, we demonstrate that SNAPtide as a commonly used substrate widely differ in reaction with BoNT/A complex, BoNT/A, and BoNT/A light chain. These findings have implications for assays used in detection, and in screening potential inhibitors.