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SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the Synaptotagmin-1 (Syt1) binding interface, and I67N, which destabilizes the SNARE-complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusionin vitroand in neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced Readily Releasable Pool (RRP) size, due to increased spontaneous (mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found inSyt1knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y>V48F) than in theSyt1KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and EPSC amplitudes due to an increase in the apparent height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another. Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage—late fusion—shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

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 Ca2+ with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca2+ sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca2+ affinities too low to account for synaptic function. However, synaptotagmin’s Ca2+ affinity increases upon binding the plasma membrane phospholipid PI(4,5)P2 and, vice versa, Ca2+-binding increases synaptotagmin’s PI(4,5)P2 affinity, indicating a stabilization of the Ca2+/PI(4,5)P2 dual-bound syt. We here devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca2+/PI(4,5)P2 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 Ca2+/PI(4,5)P2 affinities and protein copy numbers, reproduced the steep Ca2+ dependency of neurotransmitter release. Our results indicate that each synaptotagmin dual binding Ca2+/PI(4,5)P2 lowers the energy barrier for vesicle fusion by 4.85 kBT and that allosteric stabilization of this state enables the synchronized engagement of 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)P2 (e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca2+/PI(4,5)P2 dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (2+-dependent priming as a prelude to fast and slow exocytosis triggering.

Fusion of vesicles with the plasma membrane and liberation of their contents is a multistep process involving several proteins. Correctly assigning the role of specific proteins and reactions in this cascade requires a measurement method with high temporal resolution. Patch-clamp recordings of cell membrane capacitance in combination with calcium measurements, calcium uncaging, and carbon-fiber amperometry allow for the accurate determination of vesicle pool sizes, their fusion kinetics, and their secreted oxidizable content. Here, we will describe this method in a model system for neurosecretion, the adrenal chromaffin cells, which secrete adrenaline.

The functional consequences of the co-expression of synaptotagmin-1 and synaptotagmin-7 are unclear. We show that when present separately, synaptotagmin-1 and synaptotagmin-7 act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming. The priming effect of Synaptotagmin-7 extends to the Readily Releasable Pool, whose fusion is executed by synaptotagmin-1, indicating synergistic action of the two Ca2+-sensors, although they are only partially colocalized. Synaptotagmin-7 promotes ubMunc13-2-dependent priming and the absence of synaptotagmin-7 renders phorbolesters less effective in stimulating priming, although synaptotagmin-7 independent priming is also observed. Morphologically, synaptotagmin-7 places vesicles in close membrane apposition (< 6 nm); in its absence vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming and sets the stage for fast and slow exocytosis triggering.

Neuronal SNAREs and their key regulators together drive synaptic vesicle exocytosis and synaptic transmission as a single integrated membrane fusion machine. Human pathogenic mutations have now been reported for all eight core components, but patients are diagnosed with very different neurodevelopmental syndromes. We propose to unify these syndromes, based on etiology and mechanism, as “SNAREopathies.” Here, we review the strikingly diverse clinical phenomenology and disease severity and the also remarkably diverse genetic mechanisms. We argue that disease severity generally scales with functional redundancy and, conversely, that the large effect of mutations in some SNARE genes is the price paid for extensive integration and exceptional specialization. Finally, we discuss how subtle differences in components being rate limiting in different types of neurons helps to explain the main symptoms.

Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1β ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1β ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1β:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1β ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.

There was an error in J. Cell Sci. (2019) 132 , [jcs234674][1] ([doi:10.1242/jcs.234674][2]). The merge panels in [Fig. 1][3]G were inadvertently duplicated. The corrected and original panels are shown below; both the online full text and PDF versions of the paper have been corrected. The authors

Synaptotagmin 1 is a presynaptic calcium sensor, regulating SNARE-mediated vesicle exocytosis of transmitter. Increasing evidence indicate roles of SNARE proteins in postsynaptic glutamate receptor trafficking. However, a possible postsynaptic expression of synaptotagmin 1 has not been demonstrated previously. Here, we used postembedding immunogold electron microscopy to determine the subsynaptic localization of synaptotagmin 1 in rat hippocampal CA1 Schaffer collateral synapses. We report for the first time that synaptotagmin 1 is present in rat hippocampal postsynaptic spines, both on cytoplasmic vesicles and at the postsynaptic density. We further investigated whether postsynaptic synaptotagmin 1 is regulated during synaptic plasticity. In a rat model of chronic temporal lobe epilepsy, we found that presynaptic and postsynaptic concentrations of the protein are reduced compared to control animals. This downregulation may possibly be an adaptive measure to decrease both presynaptic and postsynaptic calcium sensitivity in excitotoxic conditions.

Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. Anin vitroassay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In the presence of PI(4,5)P2, vesicle attachment was unaffected by mutation. When expressed inSnap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in anin vitrofusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction.SIGNIFICANCE STATEMENTThe function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Usingin vitroassays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clampingin vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.

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 Ca

In the original publication of the article the author name M. Schupps was incorrect.

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

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.

SNARE-complexes drive the fusion of membrane-bound vesicles with target membranes or with each other (homotypic fusion). The SNARE-proteins are subdivided into Qa, Qb, Qc and R-SNAREs depending on their position in the four-helical SNARE-bundle. Here, we review the SNAP-25 protein sub-family, which includes both the Qb and Qc SNARE-domains within a single protein. In vertebrates, this sub-family consists of SNAP-25, SNAP-23, SNAP-29 and SNAP-47, named for their apparent molecular weights. SNAP-25 and SNAP-23 are specialized for driving regulated exocytosis. SNAP-25 performs this function in the nervous system, and in neuroendocrine cells, where fast Ca2+-dependent triggering is required in order to synchronize release with an electrical signal, whereas SNAP-23 drives regulated exocytosis in most other cases that have been studied, e.g. platelet exocytosis or glucose transporter trafficking. SNAP-25 is regulated by alternative splicing, phosphorylation and by G-protein binding, and it regulates Ca2+-channels, neuronal survival and postsynaptic spine development. SNAP-23 is primarily regulated by phosphorylation within the linker connecting Qb to Qc. Cross-rescue experiments show that SNAP-25 and SNAP-23 can (at least partly) substitute for each other, whereas SNAP-29 and SNAP-47 cannot. SNAP-29 is present on intracellular membranes and performs functions in autophagosome-to-lysosome fusion, among others. An overlapping function for SNAP-47 was described; in addition, SNAP-47 mediates postsynaptic AMPA-receptor insertion. Overall, the presence of two SNARE-domains confers members of this family the ability to associate to different Qa and R-SNAREs and drive diverse membrane fusion reactions; one member of the family, SNAP-25, has been devoted entirely to Ca2+-triggered fusion and has taken on a number of additional, regulatory roles.

Information transfer across CNS synapses depends on the very low basal vesicle fusion rate and the ability to rapidly upregulate that rate upon Ca2+ influx. We show that local electrostatic repulsion participates in creating an energy barrier, which limits spontaneous synaptic transmission. The barrier amplitude is increased by negative charges and decreased by positive charges on the SNARE-complex surface. Strikingly, the effect of charges on the barrier is additive and this extends to evoked transmission, but with a shallower charge dependence. Action potential-driven synaptic release is equivalent to the abrupt addition of ∼35 positive charges to the fusion machine. Within an electrostatic model for triggering, the Ca2+ sensor synaptotagmin-1 contributes ∼18 charges by binding Ca2+, while also modulating the fusion barrier at rest. Thus, the energy barrier for synaptic vesicle fusion has a large electrostatic component, allowing synaptotagmin-1 to act as an electrostatic switch and modulator to trigger vesicle fusion.

The SNARE protein SNAP-25 is well documented as regulator of presynaptic vesicle exocytosis. Increasing evidence suggests roles for SNARE proteins in postsynaptic trafficking of glutamate receptors as a basic mechanism in synaptic plasticity. Despite these indications, detailed quantitative subsynaptic localization studies of SNAP-25 have never been performed. Here, we provide novel electron microscopic data of SNAP-25 localization in postsynaptic spines. In addition to its expected presynaptic localization, we show that the protein is also present in the postsynaptic density (PSD), the postsynaptic lateral membrane and on small vesicles in the postsynaptic cytoplasm. We further investigated possible changes in synaptic SNAP-25 protein expression after hippocampal long-term potentiation (LTP). Quantitative analysis of immunogold-labeled electron microscopy sections did not show statistically significant changes of SNAP-25 gold particle densities 1 h after LTP induction, indicating that local trafficking of SNAP-25 does not play a role in the early phases of LTP. However, the strong expression of SNAP-25 in postsynaptic plasma membranes suggests a function of the protein in postsynaptic vesicle exocytosis and a possible role in hippocampal synaptic plasticity.

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B -inhibition of release during sustained calcium elevations -depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.

Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (SolubleNSFAttachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors., eLife digest Cells in our body communicate by releasing compounds called transmitters that carry signals from one cell to the next. Packages called vesicles store transmitters within the signaling cell. When the cell needs to send a signal, the vesicles fuse with the cell's membrane and release their cargo. For many signaling processes, such as those used by neurons, this fusion is regulated, fast, and coupled to the signal that the cell receives to activate release. Specialized molecular machines made up of proteins and fatty acid molecules called signaling lipids enable this to happen. One signaling lipid called PI(4,5)P2 (short for phosphatidylinositol 4,5-bisphosphate) is essential for vesicle fusion as well as for other processes in cells. It interacts with several proteins that help it control fusion and the release of transmitter. While it is possible to study the role of these proteins using genetic tools to inactivate them, the signaling lipids are more difficult to manipulate. Existing methods result in slow changes in PI(4,5)P2 levels, making it hard to directly attribute later changes to PI(4,5)P2. Walter, Müller, Tawfik et al. developed a new method to measure how PI(4,5)P2 affects transmitter release in living mammalian cells, which causes a rapid increase in PI(4,5)P2 levels. The method uses a chemical compound called “caged PI(4,5)P2” that can be loaded into cells but remains undetected until ultraviolet light is shone on it. The ultraviolet light uncages the compound, generating active PI(4,5)P2 in less than one second. Walter et al. found that when they uncaged PI(4,5)P2 in this way, the amount of transmitter released by cells increased. Combining this with genetic tools, it was possible to investigate which proteins of the release machinery were required for this effect. The results suggest that two different types of proteins that interact with PI(4,5)P2 are needed: one must bind PI(4,5)P2 to carry out its role and the other helps PI(4,5)P2 accumulate at the site of vesicle fusion. The new method also allowed Walter et al. to show that a fast increase in PI(4,5)P2 triggers a subset of vesicles to fuse very rapidly. This shows that PI(4,5)P2 rapidly regulates the release of transmitter. Caged PI(4,5)P2 will be useful to study other processes in cells that need PI(4,5)P2, helping scientists understand more about how signaling lipids control many different events at cellular membranes.

The action potential (AP) is an electrical impulse elicited by depolarization of the neuronal membrane from the resting membrane potential (around -70 mV). It propagates along the axon, allowing for rapid and distant communication. Recently, it was claimed that two APs traveling in opposite direction will pass unhindered through each other (penetrate) upon collision [Gonzalez-Perez et al.Phys. Rev. X 4, 031047 (2014)PRXHAE2160-330810.1103/PhysRevX.4.031047]. We tested this claim under carefully controlled conditions and found that we cannot reproduce penetration. Instead, APs consistently annihilated upon collision. This is consistent with a vast body of literature.

The functions of four of the five proteins in the mammalian uncoordinated-13 (Munc13) family have been identified as priming factors in SNARE-dependent exocytosis. In this issue, Zhang et al. show that the fifth member, BAIAP3, acts in retrograde trafficking by returning secretory vesicle material to the TGN. In its absence, secretory vesicle formation is impaired, leading to accumulation of immature vesicles, or lysosomal vesicle degradation., The functions of four of the five proteins in the mammalian uncoordinated-13 (Munc13) family have been identified as priming factors in SNARE-dependent exocytosis. In this issue, Zhang et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201702099) show that the fifth member, BAIAP3 (brain-specific angiogenesis inhibitor I–associated protein 3), acts in retrograde trafficking by returning secretory vesicle material to the trans-Golgi network. In its absence, secretory vesicle formation is impaired, leading to accumulation of immature vesicles, or lysosomal vesicle degradation.

SNAP-25 regulates Ca2+ channels in an unknown manner. Endogenous and exogenous SNAP-25 inhibit Ca2+ currents indirectly by recruiting syntaxin-1 from clusters on the plasma membrane, thereby making it available for Ca2+ current inhibition. Thus the cell can regulate Ca2+ influx by expanding or contracting syntaxin-1 clusters., SNAP-25 regulates Ca2+ channels, with potentially important consequences for diseases involving an aberrant SNAP-25 expression level. How this regulation is executed mechanistically remains unknown. We investigated this question in mouse adrenal chromaffin cells and found that SNAP-25 inhibits Ca2+ currents, with the B-isoform being more potent than the A-isoform, but not when syntaxin-1 is cleaved by botulinum neurotoxin C. In contrast, syntaxin-1 inhibits Ca2+ currents independently of SNAP-25. Further experiments using immunostaining showed that endogenous or exogenous SNAP-25 expression recruits syntaxin-1 from clusters on the plasma membrane, thereby increasing the immunoavailability of syntaxin-1 and leading indirectly to Ca2+ current inhibition. Expression of Munc18-1, which recruits syntaxin-1 within the exocytotic pathway, does not modulate Ca2+ channels, whereas overexpression of the syntaxin-binding protein Doc2B or ubMunc13-2 increases syntaxin-1 immunoavailability and concomitantly down-regulates Ca2+ currents. Similar findings were obtained upon chemical cholesterol depletion, leading directly to syntaxin-1 cluster dispersal and Ca2+ current inhibition. We conclude that clustering of syntaxin-1 allows the cell to maintain a high syntaxin-1 expression level without compromising Ca2+ influx, and recruitment of syntaxin-1 from clusters by SNAP-25 expression makes it available for regulating Ca2+ channels. This mechanism potentially allows the cell to regulate Ca2+ influx by expanding or contracting syntaxin-1 clusters.

Synaptotagmin-1 and -7 constitute the main calcium sensors mediating SNARE-dependent exocytosis in mouse chromaffin cells, but the role of a closely related calcium-binding protein, Doc2b, remains enigmatic. We investigated its role in chromaffin cells using Doc2b knock-out mice and high temporal resolution measurements of exocytosis. We found that the calcium dependence of vesicle priming and release triggering remained unchanged, ruling out an obligatory role for Doc2b in those processes. However, in the absence of Doc2b, release was shifted from the readily releasable pool to the subsequent sustained component. Conversely, upon overexpression of Doc2b, the sustained component was largely inhibited whereas the readily releasable pool was augmented. Electron microscopy revealed an increase in the total number of vesicles upon Doc2b overexpression, ruling out vesicle depletion as the cause for the reduced sustained component. Further experiments showed that, in the absence of Doc2b, the refilling of the readily releasable vesicle pools is faster, but incomplete. Faster refilling leads to an increase in the sustained component as newly primed vesicles fuse while the [Ca2+]ifollowing stimulation is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily releasable pool after relaxation of the calcium signal. In sum, Doc2b favors fast, synchronized release, and limits out-of-phase secretion.

The molecular mechanisms for calcium-triggered membrane fusion have long been sought for, and detailed models now exist that account for at least some of the functions of the many proteins involved in the process. Key players in the fusion reaction are a group of proteins that, upon binding to calcium, trigger the merger of cargo-filled vesicles with the plasma membrane. Low-affinity, fast-kinetics calcium sensors of the synaptotagmin family - especially synaptotagmin-1 and synaptotagmin-2 - are the main calcium sensors for fast exocytosis triggering in many cell types. Their functions extend beyond fusion triggering itself, having been implicated in the calcium-dependent vesicle recruitment during activity, docking of vesicles to the plasma membrane and priming, and even in post-fusion steps, such as fusion pore expansion and endocytosis. Furthermore, synaptotagmin diversity imparts distinct properties to the release process itself. Other calcium-sensing proteins such as Munc13s and protein kinase C play important, but more indirect roles in calcium-triggered exocytosis. Because of their higher affinity, but intrinsic slower kinetics, they operate on longer temporal and spatial scales to organize assembly of the release machinery. Finally, the high-affinity synaptotagmin-7 and Doc2 (Double C2-domain) proteins are able to trigger membrane fusion in vitro, but cellular measurements in different systems show that they may participate in either fusion or vesicle priming. Here, we summarize the properties and possible interplay of (some of) the major C2-domain containing calcium sensors in calcium-triggered exocytosis. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

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. SIGNIFICANCE STATEMENT 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.

Although it has been known for almost two decades that the ternary complex of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) constitutes the functional unit driving membrane fusion, our knowledge about the dynamical arrangement and organization of SNARE proteins and their complexes before and during vesicle exocytosis is still limited. Here, we review recent progress in this expanding field with emphasis on the question of fusion complex stoichiometry, i.e., how many SNARE proteins and complexes are needed for the fusion of a vesicle with the plasma membrane.

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 Ca2+-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. DOI: http://dx.doi.org/10.7554/eLife.10635.001, eLife digest Mammals have adrenal glands, which secrete the stress hormone adrenaline as well as other hormones into the bloodstream. These molecules are produced in chromaffin cells, where they are packaged into compartments called large dense-core vesicles (LDCVs). To release the hormones into the bloodstream, the vesicles bind to and fuse with the membrane that surrounds the cell. This process – which is called exocytosis – is triggered by increases in the level of calcium ions inside the cells. Exocytosis also enables nerve cells to release chemical signals at junctions (known as synapses) with other nerve cells. These signals are packaged within another type of vesicle called 'synaptic' vesicles, which also release their contents by fusing with the cell membrane. However, it is not clear whether the two types of vesicle carry out exocytosis in the same way. Exocytosis requires that the vesicles physically attach to the membrane and undergo a process termed 'priming', which enables them to fuse quickly with the membrane in response to an increase in calcium ion levels. In synaptic vesicles, both of these processes – physical membrane attachment and priming – appear to occur in a single step that requires a family of proteins called the Munc13 proteins. Here, Man et al. investigate whether the Munc13 proteins are also essential for LDCV exocytosis in the chromaffin cells of mice. The experiments reveal that in contrast to synaptic vesicles, the initial binding of LDCVs to membranes does not require Munc13 proteins. However, the loss of one member of the family called Munc13-2 dramatically reduces the fusion of LDCVs with the membrane of chromaffin cells. Further experiments reveal that different Munc13 proteins differ in their ability to drive the exocytosis of LDCVs. Man et al. use a mathematical model of LDCV exocytosis, which reveals that Munc13 plays an important role in the first part of the priming step. Together, these findings show that synaptic vesicles and LDCVs use different mechanisms to bind to membranes, but are primed for fusion in a similar way. DOI: http://dx.doi.org/10.7554/eLife.10635.002

Regulated neurotransmitter secretion depends on Ca 2+ sensors, C2 domain proteins that associate with phospholipids and soluble N -ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes to trigger release upon Ca 2+ binding. Ca 2+ sensors are thought to prevent spontaneous fusion at rest (clamping) and to promote fusion upon Ca 2+ activation. At least eight, often coexpressed, Ca 2+ sensors have been identified in mammals. Accumulating evidence suggests that multiple Ca 2+ sensors interact, rather than work autonomously, to produce the complex secretory response observed in neurons and secretory cells. In this review, we present several working models to describe how different sensors might be arranged to mediate synchronous, asynchronous and spontaneous neurotransmitter release. We discuss the scenario that different Ca 2+ sensors typically act on one shared vesicle pool and compete for binding the multiple SNARE complexes that are likely to assemble at single vesicles, to exert both clamping and fusion-promoting functions.

Exocytosis requires formation of SNARE [soluble N-ethylmaleimide - sensitive factor attachment protein (SNAP) receptor] complexes between vesicle and target membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured chromaffin cells. Simultaneous expression of wild-type SNAP-25 and a mutant unable to support exocytosis progressively altered fusion kinetics and fusion-pore opening, indicating that both proteins assemble into heteromeric fusion complexes. Expressing different wild-type:mutant ratios revealed a third-power relation for fast (synchronous) fusion and a near-linear relation for overall release. Thus, fast fusion typically observed in synapses and neurosecretory cells requires at least three functional SNARE complexes, whereas slower release might occur with fewer complexes. Heterogeneity in SNARE-complex number may explain heterogeneity in vesicular release probability.

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight alpha-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1.SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil "trigger site." This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1.SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.

The SNARE-complex consisting of synaptobrevin-2/VAMP-2, SNAP-25 and syntaxin-1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap-25 null mice rescued with mutants challenging the C-terminal, N-terminal and middle domains of the SNARE-bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub-domains of the SNARE-bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C-terminal end of the SNARE-bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C-terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N-terminal end of the SNARE-bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N-terminal deletion delays vesicle priming after a high-frequency train. We propose that the stability of N-terminal two-thirds of the SNARE-bundle has a function for vesicle priming and limiting spontaneous release.

Fast exocytosis of synaptic vesicles differs from other membrane fusion reactions by being under tight temporal control by the intracellular calcium concentration. This is achieved by subjecting the SNARE-dependent fusion pathway to additional layers of control, both upstream and downstream of the assembly of the fusogenic SNARE-complex. Here, I review conflicting views on the function of the core fusion machinery consisting of the SNAREs, Munc18, complexin, and synaptotagmin. Munc18 controls docking of vesicles to the plasma membrane and initial SNARE-complex assembly, whereas complexin and synaptotagmin cooperate in holding the SNARE complex in an intermediate release-ready or cocked state. Different effects of complexin and synaptotagmin shape the energy landscape for fusion and make final fusion calcium triggered. The final steps are fusion pore formation and expansion, which allow release of the water-soluble vesicle content. The fusion pore remains the most elusive part of the exocytosis pathway, owing to its short lifetime.

SummaryBefore 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 Ca2+ 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 Ca2+ triggered release of transmitters.