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Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca 2+ , suggesting Syt rings may clamp fusion as membrane-separating “washers” until Ca 2+ -mediated disassembly triggers fusion and release [J. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 111, 13966–13971 (2014)].). Here, we combined mathematical modeling with experiment to measure the mechanical properties of Syt rings and to test this mechanism. Consistent with experimental results, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently phosphatidylinositol 4,5-bisphosphate (PIP2)-rich plasma membrane (PM) domains, the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca 2+ -sensitive fusion clamp and a high-fidelity sensor for directed docking.

Cellular plasma membranes, in their role as gatekeepers to the external environment, host numerous protein assemblies and lipid domains that manage the movement of molecules into and out of cells, regulate electric potential, and direct cell signaling. The ability to investigate these roles on the bilayer at a single-molecule level in a controlled, in vitro environment while preserving lipid and protein architectures will provide deeper insights into how the plasma membrane works. A tunable silicon microarray platform that supports stable, planar, and asymmetric suspended lipid membranes (SLIM) using synthetic and native plasma membrane vesicles for single-molecule fluorescence investigations is developed. Essentially, a "plasma membrane-on-a-chip" system that preserves lipid asymmetry and protein orientation is created. By harnessing the combined potential of this platform with total internal reflection fluorescence (TIRF) microscopy, the authors are able to visualize protein complexes with single-molecule precision. This technology has widespread applications in biological processes that happen at the cellular membranes and will further the knowledge of lipid and protein assemblies.

Successive fusion events between transport vesicles and their target membranes mediate trafficking of secreted, membrane- and organelle-localised proteins. During the initial steps of this process, termed the secretory pathway, COPII vesicles bud from the endoplasmic reticulum (ER) and fuse with the cis-Golgi membrane, thus depositing their cargo. This fusion step is driven by a quartet of SNARE proteins that includes the cis-Golgi t-SNARE Membrin, encoded by the GOSR2 gene. Mis-sense mutations in GOSR2 result in Progressive Myoclonus Epilepsy (PME), a severe neurological disorder characterised by ataxia, myoclonus and seizures in the absence of significant cognitive impairment. However, given the ubiquitous and essential function of ER-to-Golgi transport, why GOSR2 mutations cause neurological dysfunction and not lethality or a broader range of developmental defects has remained an enigma. Here we highlight new work that has shed light on this issue and incorporate insights into canonical and non-canonical secretory trafficking pathways in neurons to speculate as to the cellular and molecular mechanisms underlying GOSR2 PME. This article is part of a Special Issue entitled: SNARE proteins: a long journey of science in brain physiology and pathology: from molecular.

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.

International audience; Munc13-1 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggest that multiple copies of Munc13-1 form nanoassemblies in active zones of neurons. However, it is not known if such clustering of Munc13-1 is correlated with multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13-1 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13-1, and is indeed required for vesicle binding to bilayers containing Munc13-1. Purified Munc13-1 protein reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ~20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters containing a minimum of 6 copies of Munc13-1 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C 2 C domain of Munc13-1 is not required for Munc13-1 clustering, but is required for efficient vesicle capture. This capture is largely due to a combination of electrostatic and hydrophobic interactions between the C 2 C domain and the vesicle membrane.

During calcium-regulated exocytosis, the constitutive fusion machinery is 'clamped' in a partially assembled state until synchronously released by calcium. The protein machinery involved in this process is known, but the supra-molecular architecture and underlying mechanisms are unclear. Here, we use cryo-electron tomography analysis in nerve growth factor-differentiated neuro-endocrine (PC12) cells to delineate the organization of the release machinery under the docked vesicles. We find that exactly six exocytosis modules, each likely consisting of a single SNAREpin with its bound Synaptotagmins, Complexin, and Munc18 proteins, are symmetrically arranged at the vesicle-PM interface. Mutational analysis suggests that the symmetrical organization is templated by circular oligomers of Synaptotagmin. The observed arrangement, including its precise radial positioning, is in-line with the recently proposed 'buttressed ring hypothesis'.

International audience; Synaptic vesicle fusion is mediated by SNARE proteins-VAMP2 on the vesicle and Syntaxin-1/ SNAP25 on the pre-synaptic 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 nano-clusters. Neuronal communication involves the controlled release of neurotransmitters stored in synaptic vesicles (SV) into the neuronal synapse [1-4]. This process is tightly regulated to ensure that the message is timely and precise [3, 4]. SV fusion is catalyzed by the synaptic SNARE (soluble N-ethyl maleimide sensitive factor attachment protein) proteins-VAMP2 on the vesicle membrane (v-SNARE) and Syntaxin-1 and SNAP25 on the plasma membrane (t-SNAREs) [5, 6]. When the vesicle approaches the plasma membrane (PM), the helical SNARE motifs of the cognate v-and t-SNAREs constitutively assemble into a ternary complex that initially bridges and ultimately fuses the two membranes [5-7]. The SNARE complex nucleates into a four-helix bundle at its membrane distal end and progressively assembles ("zippers") towards the membranes, exerting a potent force that ultimately drives the membranes together to fuse into one [6, 8, 9]. Though efficient, under in vitro conditions, the fusion process is artificially slow with its rate being limited by the intrinsic rate of nucleation of SNARE complex [10-12]. In vivo, nucleation is greatly accelerated by two cooperating , specialized molecular chaperones, Munc13 and Munc18 [3, 4, 13, 14].

Significance Neuronal cells maintain a small number of synaptic vesicles (SVs) that are “primed,” i.e., ready to release upon receiving the triggering signal to allow for tightly regulated and rapid release of neurotransmitters. The proteins involved in this process are known, but how they assemble and operate together is poorly understood. Here we report the visualization of protein organization under primed SVs in cultured neurons under native conditions. Using cryoelectron tomography analysis, we find that there is a symmetric arrangement of exactly six protein densities at the docking interface, suggesting the fusion machinery is well ordered and prearranged for fast and precise release of neurotransmitters., Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is a fundamental process that is central to all information processing in the brain. This relies on tight coupling of the SV fusion to action potential-evoked presynaptic Ca2+ influx. This Ca2+-evoked release occurs from a readily releasable pool (RRP) of SVs docked to the plasma membrane (PM). The protein components involved in initial SV docking/tethering and the subsequent priming reactions which make the SV release ready are known. Yet, the supramolecular architecture and sequence of molecular events underlying SV release are unclear. Here, we use cryoelectron tomography analysis in cultured hippocampal neurons to delineate the arrangement of the exocytosis machinery under docked SVs. Under native conditions, we find that vesicles are initially “tethered” to the PM by a variable number of protein densities (∼10 to 20 nm long) with no discernible organization. In contrast, we observe exactly six protein masses, each likely consisting of a single SNAREpin with its bound Synaptotagmins and Complexin, arranged symmetrically connecting the “primed” vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca2+ influx.

Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator. Fil: Gruget, Clémence. Ecole Normale Supérieure; Francia Fil: Bello, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Coleman, Jeff. University of Yale. School of Medicine; Estados Unidos Fil: Krishnakumar, Shyam S.. University of Yale. School of Medicine; Estados Unidos Fil: Perez, Eric. Ecole Normale Supérieure; Francia Fil: Rothman, James E.. University of Yale. School of Medicine; Estados Unidos Fil: Pincet, Frederic. Ecole Normale Supérieure; Francia. University of Yale. School of Medicine; Estados Unidos Fil: Donaldson, Stephen H.. Ecole Normale Supérieure; Francia

Synaptic vesicle fusion is mediated by membrane-bridging complexes formed by SNARE proteins - VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the pre-synaptic membrane. Accumulating evidence suggest that chaperones Munc18-1 and Munc13-1 co-operatively catalyze SNARE assembly via an intermediate ‘template’ complex containing Syntaxin-1 and VAMP2. How SNAP25 is chaperoned into this nascent complex 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 presumably bound in ~1:1 stoichiometry by the self-assembled Munc13-1 nanoclusters. Our data suggests that Munc13-1’s capacity to bind all three synaptic SNARE proteins likely underlie its chaperone function.

Munc13 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggested that multiple copies of Munc13 form nanoassemblies in active zones of neurons. However, it is not known if such clustering is an inherent self-assembly property of Munc13 or whether Munc13 clusters indirectly by multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13, and is indeed required for vesicle binding to bilayers containing Munc13. Pure Munc13 reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to ∼20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters a minimum of 6 copies of Munc13 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13 is not required for Munc13 clustering, but is required for efficient vesicle capture.

Significance Release of neurotransmitters relies on submillisecond coupling of synaptic vesicle fusion to the triggering signal: AP-evoked presynaptic Ca2+ influx. The key player that controls exocytosis of the synaptic vesicle is the Ca2+ sensor synaptotagmin 1 (Syt1). While the Ca2+ activation of Syt1 has been extensively characterized, how Syt1 reversibly clamps vesicular fusion remains enigmatic. Here, using a targeted mutation combined with fluorescence imaging and electrophysiology, we show that the structural feature of Syt1 to self-oligomerize provides the molecular basis for clamping of spontaneous and asynchronous release but is not required for triggering of synchronous release. Our findings propose a mechanistic model that explains how Syt1 oligomers regulate different modes of transmitter release in neuronal synapses., Synaptotagmin 1 (Syt1) synchronizes neurotransmitter release to action potentials (APs) acting as the fast Ca2+ release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. While the Syt1 Ca2+ activation mechanism has been well-characterized, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerization provides the molecular basis for the Syt1 clamping function. This follows from the investigation of a designed mutation (F349A), which selectively destabilizes Syt1 oligomerization. Using a combination of fluorescence imaging and electrophysiology in neocortical synapses, we show that Syt1F349A is more efficient than wild-type Syt1 (Syt1WT) in triggering synchronous transmitter release but fails to clamp spontaneous and synaptotagmin 7 (Syt7)-mediated asynchronous release components both in rescue (Syt1−/− knockout background) and dominant-interference (Syt1+/+ background) conditions. Thus, we conclude that Ca2+-sensitive Syt1 oligomers, acting as an exocytosis clamp, are critical for maintaining the balance among the different modes of neurotransmitter release.

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved, namely the vesicular fusion machinery (SNARE proteins) and the regulatory proteins (Synaptotagmin-1 and Complexin) are well-known. Here, we use a reconstituted single-vesicle fusion assay to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act independently but synergistically to establish Ca2+-regulated fusion. Under physiologically-relevant conditions, we find that Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary binding interface, to introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to independently arrest the remaining free ‘peripheral’ SNAREpins to produce stably clamped vesicles. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free ‘peripheral’ SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (

Quantal neurotransmitter release at nerve terminals is tightly regulated by the presynaptic Ca2+ concentration. Here, we summarise current advances in understanding how the interplay between presynaptic Ca2+ dynamics and different Ca2+ release sensors shapes action potential-evoked release on a timescale from hundreds of microseconds to hundreds of milliseconds. In particular, we review recent studies that reveal the synergistic roles of the low Ca2+ affinity/fast release sensors synaptotagmins 1, 2 and 9 and the high affinity/slow release sensor synaptotagmin 7 in the regulation of synchronous and asynchronous release and of short-term synaptic plasticity. We also examine new biochemical and structural data and outline a working model that could potentially explain the cooperative roles of different synaptotagmins in molecular terms.

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by step-wise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 kBT, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations.

We report 2 families with undiagnosed recessive presynaptic congenital myasthenic syndrome (CMS). Whole exome or genome sequencing identified segregating homozygous variants in VAMP1: c.51_64delAGGTGGGGGTCCCC in a Kuwaiti family and c.146G>C in an Israeli family. VAMP1 is crucial for vesicle fusion at presynaptic neuromuscular junction (NMJ). Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. We assessed the effect of the nonsense mutation on mRNA levels and evaluated the NMJ transmission in VAMP1lew/lew mice, observing neurophysiological features of presynaptic impairment, similar to the patients. Taken together, our findings highlight VAMP1 homozygous mutations as a cause of presynaptic CMS. Ann Neurol 2017;81:597–603

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. Here we report the cryo-electron microscopy structure of the Syt1–SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg2+)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the ‘primary’ interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1–SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1., The neuronal Ca2+-sensor Synapotagmin-1 (Syt1) interacts with SNARE proteins and lipid membranes and synchronizes neurotransmitter release in response to Ca2+-influx. Here the authors provide insights into the underlying molecular mechanism by determining the cryo-EM structure of the Syt1–SNARE complex on a lipid membrane at 10 Å resolution.

International audience; The buttressed-ring hypothesis, supported by recent cryo-electron tomography analysis of docked synaptic-like vesicles in neuroendocrine cells, postulates that prefusion SNAREpins are stabilized and organized by Synaptotagmin (Syt) ring-like oligomers. Here, we use a reconstituted single-vesicle fusion analysis to test the prediction that destabilizing the Syt1 oligomers destabilizes the clamp and results in spontaneous fusion in the absence of Ca 2+. Vesicles in which Syt oligomerization is compromised by a ring-destabilizing mutation dock and diffuse freely on the bilayer until they fuse spontaneously, similar to vesicles containing only v-SNAREs. In contrast, vesicles containing wild-type Syt are immobile as soon as they attach to the bilayer and remain frozen in place, up to at least 1 h until fusion is triggered by Ca 2+ .

Here we introduce ApoE-based Nanolipoprotein particles (NLP) – soluble, discoidal bilayer mimetic of ~23 nm in diameter, as fusion partners to study the dynamics of fusion pores induced by SNARE proteins. Using in vitro lipid mixing and content release assays, we report that NLPs reconstituted with synaptic v-SNARE VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25) partner, with the resulting fusion pore opening directly to the external buffer. Efflux of encapsulated fluorescent dextrans of different size shows that unlike the smaller nanodiscs, these larger NLPs accommodate the expansion of the fusion pore to at least ~ 9 nm and dithionite quenching of fluorescent lipid introduced in vNLP confirms that the NLP fusion pores are short-lived and eventually reseal. The NLPs also have capacity to accommodate larger number of proteins and using vNLPs were defined number of VAMP2 protein, including physiologically relevant copy numbers, we find that 3–4 copies of VAMP2 (minimum 2 per face) are required to keep a nascent fusion pore open and the SNARE proteins act cooperatively to dilate the nascent fusion pore.

In this chapter, we introduce a nanodisc-based experimental platform to study Ca

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.

International audience; Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca2+) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca2+‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+ and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca2+‐activation process in synaptic transmission.

Synaptotagmin-1 (Syt1) is the primary calcium sensor (Ca

Summary Mutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders., Graphical Abstract, Highlights • N-terminal proline-rich domain of PRRT2 selectively blocks SNARE complex assembly • PRRT2 moderates the synaptic vesicle docking/priming process • Paroxysmal dyskinesia-associated mutations in PRRT2 disrupt SNARE-modulatory function, PRRT2 is linked to several paroxysmal neurological disorders. Coleman et al. identify a crucial role for PRRT2 as a regulator of the synaptic vesicle priming process as it directly binds and influences SNARE complex assembly. A disease-related mutation in PRRT2 disrupts this function, revealing a possible molecular mechanism underlying pathogenesis.

Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 'MUN' domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work-bench on which SNAREpins are templated. This 'buttressed-ring hypothesis' affords straightforward answers to many principal and long-standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential.

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion). DOI: http://dx.doi.org/10.7554/eLife.22964.001

Although action potentials propagate along axons in an all-or-none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog–digital modulation is depolarization-mediated inactivation of presynaptic Kv1-family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of episodic ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels.

Previously, we showed that synaptotagmin1 (Syt1) forms Ca2+-sensitive ring-like oligomers on membranes containing acidic lipids and proposed a potential role in regulating neurotransmitter release (Zanetti et al., 2016). Here, we report that Syt1 assembles into similar ring-like oligomers in solution when triggered by naturally occurring polyphosphates (PIP2 and ATP) and magnesium ions (Mg2+). These soluble Syt1 rings were observed by electron microscopy and independently demonstrated and quantified using fluorescence correlation spectroscopy. Oligomerization is triggered when polyphosphates bind to the polylysine patch in C2B domain and is stabilized by Mg2+, which neutralizes the Ca2+-binding aspartic acids that likely contribute to the C2B interface in the oligomer. Overall, our data show that ring-like polymerization is an intrinsic property of Syt1 with reasonable affinity that can be triggered by the vesicle docking C2B-PIP2 interaction and raise the possibility that Syt1 rings could pre-form on the synaptic vesicle to facilitate docking.

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded.

The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx. DOI: http://dx.doi.org/10.7554/eLife.17262.001, eLife digest Reliable communication between neurons is essential for the brain to work properly. This is accomplished by tightly controlling how chemical messengers, called neurotransmitters, move between neurons. Neurotransmitters are typically packaged into bubble-like structures called synaptic vesicles and are released only when the neuron receives an input electrical signal. A set of proteins orchestrates the release of the neurotransmitters from the neuron, which happens after the synaptic vesicles fuse with the cell membrane. Synaptotagmin, a protein found on the surface of the synaptic vesicle, plays many roles in neurotransmitter release. It helps to attach the synaptic vesicle to the cell membrane and also prevents the vesicles from fusing to the membrane in the absence of an appropriate input signal. Most importantly, it detects when the electrical signal arrives at the neuron by binding to calcium ions that flood the cell following the input signal. This triggers the rapid fusion of the vesicles to the cell membrane. It is not clear how Synaptotagmin is able to carry out its different roles and in particular, control how neurotransmitters are released as calcium ions enter the cell. Zanetti et al. have now used a technique called negative stain electron microscopy to investigate how Synaptotagmin molecules taken from mammals arrange themselves on the surface of a membrane. In this technique, individual Synaptotagmin proteins on the surface of a synthetic membrane are chemically marked and their structure is imaged using an electron beam. Using this approach under conditions resembling those in cells, Zanetti et al. found that 15–20 copies of Synaptotagmin came together and formed ring-like structures on the membrane surface. These ring structures were rapidly broken apart when calcium ions were added to them. Further investigations suggest that the ring structures form when synaptic vesicles first attach to a membrane. Overall, it appears that the Synaptotagmin rings act as washers or spacers to prevent the vesicle from fusing to the cell membrane until the rings are disrupted by the arrival of calcium ions. Future studies are now needed to investigate whether the ring structures form inside cells and whether they act together with other proteins involved in neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.17262.002

A key step during neurotransmitter or hormone release (exocytosis) is the formation of a nanometer-sized fusion pore that connects the plasma membrane to the synaptic or secretory vesicle. The pore can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics affect vesicle recycling, amount and kinetics of released cargo, and activation of downstream events. The fusion pore is clearly a key intermediate; however, factors regulating its dynamics are poorly understood, in large part due to a lack of biochemically defined single-pore assays.We have developed a novel assay that probes single fusion pores formed between ∼16 nm flat lipid bilayer nanodiscs (NDs) reconstituted with neuronal/exocytotic vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) proteins and cells engineered to express cognate “flipped” target membrane t-SNAREs on their surfaces, with the SNARE domain facing the extracellular medium. Conductance through single, voltage-clamped fusion pores directly reports sub-millisecond pore dynamics. Pore currents fluctuated, briefly returned to baseline multiple times, and disappeared ∼6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that putative interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, the same modifications that reduced pore nucleation rates dramatically prolonged pore lifetimes. Our results indicate that rod-shaped, post-fusion cis-SNARE complexes that poorly fit the highly curved fusion site rapidly vacate the pore region, leaving pore properties to be determined by lipids. In contrast, mutants deficient in TMD-zippering prevented pore resealing for >60 s, possibly due to their post-fusion Y-shape. Thus, post-fusion geometry of the proteins may determine pore stability, analogous to the well-known effects of lipid geometry on highly curved fusion intermediates.

Synaptic exocytosis relies on assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins into a four-helix bundle to drive membrane fusion. SNARE assembly occurs by zippering in a stepwise manner, beginning with slow N-terminal association and ending at the C-terminal with a rapid power stroke that lowers the barrier to membrane fusion. Imbalances in this process cause severe neuropathy. In particular, dominant missense mutations I67N and I67T in the neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25b) cause cortical hyperexcitability, myasthenia, ataxia, and intellectual disability. Recent electrophysiological measurements indicate that I67T mutation impairs spontaneous and evoked release in murine cortical slices. Similarly, transfection of bovine chromaffin cells with SNAP-25b I67N impairs depolarization-evoked release. However, the molecular mechanism underlying these phenotypes is unclear. Here, we used single-molecule optical tweezers to monitor in real-time the step-wise assembly of SNARE complex harboring SNAP-25b I67T/N. These measurements yielded the assembly energetics and kinetics of single SNARE complex molecules. Comparison between wild type and I67T or I67N mutant revealed that both disease-causing mutants significantly reduced the folding energy of C-terminal SNARE assembly by ∼10 kT, but did not affect N-terminal assembly. Such disruption of the C-terminal power stroke is expected to impair membrane fusion and is consistent with the corresponding phenotypes. In sum, our finding constitutes compelling evidence that SNAP-25b mutations cause neuropathy by greatly reducing the SNARE C-terminal folding energy and thus inhibiting neurotransmitter release.