Munc18-1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18-1 forms a tight complex with the ...SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18-1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18-1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18-1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25-deficient mice are used. We conclude that Munc18-1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.
Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the ...strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca
2+ levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca
2+ triggered release of transmitters.
Secretory vesicles dock at their target in preparation for fusion. Using single‐vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles ...dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18‐1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking‐deficient munc18‐1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t‐SNARE and Munc18‐1 binding partner syntaxin, but not the v‐SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18‐1/syntaxin‐dependent, strongly tethered, fusion‐competent state.
Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the ...docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. Here, using adrenal chromaffin cells, we identify the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, we show that the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, lead us to propose that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes.
Synaptic communication relies on the fusion of synaptic vesicles with the plasma membrane, which leads to neurotransmitter release. This exocytosis is triggered by brief and local elevations of ...intracellular Ca
2+
with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca
2+
sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca
2+
affinities too low to account for synaptic function. However, synaptotagmin’s Ca
2+
affinity increases upon binding the plasma membrane phospholipid PI(4,5)P
2
and, vice versa, Ca
2+
binding increases synaptotagmin’s PI(4,5)P
2
affinity, indicating a stabilization of the Ca
2+
/PI(4,5)P
2
dual-bound state. Here, we devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca
2+
/PI(4,5)P
2
dual binding lowers the energy barrier for vesicle fusion and that (2.) the effect of multiple synaptotagmins on the energy barrier is additive. The model, which relies on biochemically measured Ca
2+
/PI(4,5)P
2
affinities and protein copy numbers, reproduced the steep Ca
2+
dependency of neurotransmitter release. Our results indicate that each synaptotagmin engaging in Ca
2+
/PI(4,5)P
2
dual-binding lowers the energy barrier for vesicle fusion by ~5 k
B
T and that allosteric stabilization of this state enables the synchronized engagement of several (typically three) synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin’s allosteric properties may show dominant-negative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)P
2
(e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca
2+
/PI(4,5)P
2
dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.
For our brains and nervous systems to work properly, the nerve cells within them must be able to ‘talk’ to each other. They do this by releasing chemical signals called neurotransmitters which other cells can detect and respond to.
Neurotransmitters are packaged in tiny membrane-bound spheres called vesicles. When a cell of the nervous system needs to send a signal to its neighbours, the vesicles fuse with the outer membrane of the cell, discharging their chemical contents for other cells to detect. The initial trigger for neurotransmitter release is a short, fast increase in the amount of calcium ions inside the signalling cell. One of the main proteins that helps regulate this process is synaptotagmin which binds to calcium and gives vesicles the signal to start unloading their chemicals.
Despite acting as a calcium sensor, synaptotagmin actually has a very low affinity for calcium ions by itself, meaning that it would not be efficient for the protein to respond alone. Synpatotagmin is more likely to bind to calcium if it is attached to a molecule called PIP
2
, which is found in the membranes of cells The effect also occurs in reverse, as the binding of calcium to synaptotagmin increases the protein’s affinity for PIP
2
. However, how these three molecules – synaptotagmin, PIP
2
, and calcium – work together to achieve the physiological release of neurotransmitters is poorly understood
.
To help answer this question, Kobbersmed, Berns et al. set up a computer simulation of ‘virtual vesicles’ using available experimental data on synaptotagmin’s affinity with calcium and PIP
2
. In this simulation, synaptotagmin could only trigger the release of neurotransmitters when bound to both calcium and PIP
2
. The model also showed that each ‘complex’ of synaptotagmin/calcium/PIP
2
made the vesicles more likely to fuse with the outer membrane of the cell – to the extent that only a handful of synaptotagmin molecules were needed to start neurotransmitter release from a single vesicle.
These results shed new light on a biological process central to the way nerve cells communicate with each other. In the future, Kobbersmed, Berns et al. hope that this insight will help us to understand the cause of diseases where communication in the nervous system is impaired.
Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th ...helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324-339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil-coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324-339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca(2+) dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.
The essential postdocking role of Munc18-1 in vesicular exocytosis has remained elusive, but recent data led to the hypothesis that the extension of helix 12 in Munc18 within domain 3a leads to synaptobrevin-2/VAMP2 interaction and SNARE complex formation. Using both lack-of-function and gain-of-function mutants, we here report that the conformation of helix 12 predicts vesicle priming and secretory amplitude in living chromaffin cells. The effects of mutants on secretion could not be explained by differences in syntaxin-1 chaperoning/localization or vesicle docking, and the fusion kinetics and calcium dependence were unchanged, indicating that the effect of helix 12 extension is specific for the vesicle-priming step. We conclude that a conformational change within helix 12 is responsible for the essential postdocking role of Munc18-1 in neurosecretion.
During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate PI(4,5)P2 level in ...bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase I, or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release.
It is currently unknown whether the molecular steps of large dense-core vesicle (LDCV) docking and priming are identical to the corresponding reactions in synaptic vesicle (SV) exocytosis. Munc13s ...are essential for SV docking and priming, and we systematically analyzed their role in LDCV exocytosis using chromaffin cells lacking individual isoforms. We show that particularly Munc13-2 plays a fundamental role in LDCV exocytosis, but in contrast to synapses lacking Munc13s, the corresponding chromaffin cells do not exhibit a vesicle docking defect. We further demonstrate that ubMunc13-2 and Munc13-1 confer Ca(2+)-dependent LDCV priming with similar affinities, but distinct kinetics. Using a mathematical model, we identify an early LDCV priming step that is strongly dependent upon Munc13s. Our data demonstrate that the molecular steps of SV and LDCV priming are very similar while SV and LDCV docking mechanisms are distinct.
Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of ...this fusion machinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target-SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimulatory for vesicle fusion and/or additional essential interactions were identified between Munc18-1 and the fusion machinery. Here, we provide evidence for both notions by dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild-type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step.
Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)-dependent exocytosis pathway at an intermediate "cocked" state, from which fusion can be triggered by Ca²⁺. ...It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin-SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca²⁺-triggered C-terminal assembly and membrane fusion.
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