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At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel’s intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation. At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.

Chemical synaptic transmission relies on neurotransmitter release from presynaptic release sites and on transmitter-sensing by the postsynaptic cell. Presynaptic plasticity increasing neurotransmitter release achieves two fundamental nervous system functions: It tunes some synapses to be more responsive to millisecond repetitive activation and it maintains signals when postsynaptic transmitter sensitivity is reduced. How enhanced neurotransmitter release is achieved in these phenomena, termed short-term facilitation and homeostatic potentiation, remains unknown. We combine mathematical modeling and experimental analysis ofDrosophilaneuromuscular junction model synapses to elucidate the molecular mechanisms underlying these forms of plasticity. Our results indicate that both phenomena depend on a rapid increase in the participation of neurotransmitter release sites which is controlled by the regulatory domains of the evolutionarily conserved (M)Unc13A protein that bind Ca2+/Calmodulin and diacylglycerol. Mutation of the Calmodulin binding (CaM) domain increased baseline transmission and impaired both short-term facilitation and acute homeostatic potentiation. Mathematical modeling indicated that these defects result from too many release sites participating at rest combined with the inability to plastically further increase their number. Super-resolution microscopy revealed that this coincided with a redistribution of Unc13A’s functionally essential MUN domain closer to the synaptic plasma membrane, which may constitute the molecular switch to increase release site participation. Similar consequences (enhanced baseline transmission, block of both short-term facilitation and homeostatic potentiation) were caused by the acute pharmacological activation of the C1 domain of wildtype Unc13A using phorbol esters. This treatment had no effect on Unc13A CaM domain mutants, indicating that both the CaM and C1 domains activate a binary release site switch. Thus, our findings indicate that Unc13A regulatory domains are tuned to integrate a multitude of signals on various timescales to switch release site participation for synaptic plasticity.

Significance Every cell produces thousands of lipid species, but studying the function of individual lipids in living cells is almost impossible with existing methodologies. Addressing this experimental bottleneck, we developed a strategy to quantify dissociation constants for lipid–protein interactions and transmembrane flip-flop rates of native lipids in live-cell experiments. Using a combination of plasma membrane-specific photochemical probes and mathematical modeling, we demonstrate that, for diacylglycerols as a model lipid class, the inherent lipid structural diversity caused by variations in acyl chain composition determines lipid protein affinities and transbilayer kinetics. In fact, subtle chemical differences change these values by orders of magnitude. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales., Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol–protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.

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.

Peripheral nerves contain sensory and motor neuron axons coated by glial cells whose interplay ensures function, but molecular details are lacking. SNARE-proteins mediate the exchange and secretion of cargo by fusing vesicles with target organelles, but how glial SNAREs contribute to peripheral nerve function is largely unknown. We, here, identify non-neuronal Synaptobrevin (Syb) as the essential vesicular SNARE in Drosophila peripheral glia to insulate and metabolically supply neurons. We show that tetanus neurotoxin light chain (TeNT-LC), which potently inhibits SNARE-mediated exocytosis from neurons, also impairs peripheral nerve function when selectively expressed in glia, causing nerve disintegration, defective axonal transport, tetanic muscle hyperactivity, impaired locomotion, and lethality. While TeNT-LC disrupts neural function by cleaving neuronal Synaptobrevin (nSyb), it targets non-neuronal Synaptobrevin (Syb) in glia, which it cleaves at low rates: Glial knockdown of Syb (but not nSyb) phenocopied glial TeNT-LC expression whose effects were reverted by a TeNT-LC-insensitive Syb mutant. We link Syb-necessity to two distinct glial subtypes: Impairing Syb function in subperineurial glia disrupted nerve morphology, axonal transport, and locomotion, likely, because nerve-isolating septate junctions (SJs) could not form as essential SJ components (like the cell adhesion protein Neurexin-IV) were mistargeted. Interference with Syb in axon-encircling wrapping glia left nerve morphology and locomotion intact but impaired axonal transport, likely because neural metabolic supply was disrupted due to the mistargeting of metabolite shuffling monocarboxylate transporters. Our study identifies crucial roles of Syb in various glial subtypes to ensure glial-glial and glial-neural interplay needed for proper nerve function, animal motility, and survival.

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, “spontaneous” transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses.

Peripheral nerves contain motoneuron axons coated by glial cells, which essentially contribute to function but cellular reactions remain poorly understood. We here identify non-neuronal Synaptobrevin (Syb) as the essential vesicular SNARE in glia to insulate and metabolically supplyDrosophilamotoneurons. Interfering with Syb-functionality by glial knockdown, or glial expression of tetanus neurotoxin light chain (TeNT-LC) caused motonerve disintegration, blocked axonal transport, induced tetanic muscle hyperactivity and caused lethal paralysis. Surprisingly, not the established TeNT-LC-target, neuronal Synaptobrevin (nSyb), is the relevant SNARE, but non-neuronal Synaptobrevin (Syb): Knockdown of Syb- (but not nSyb-) phenocopied glial TeNT-LC expression whose effects were reverted by a TeNT-LC-insensitive Syb mutant. We link Syb-necessity to two distinct glia: to establish nerve insulating septate junctions in subperineurial glia and to integrate monocarboxylate transporters along the nerve in wrapping glia for motoneuronal metabolic supply. Our study identifies crucial roles of Syb in glial subtypes for nerve function and pathology, animal motility and survival.

SummaryNeuronal synapses transduce information via the consecutive action of three transducers: voltage-gated Ca2+-channels, fusion-competent synaptic vesicles, and postsynaptic receptors. Their physical distance is thought to influence the speed and efficiency of neurotransmission. However, technical limitations have hampered resolving their nanoscale arrangement. Here, we developed a new method for live-labeling proteins for electron microscopy (EM), revealing that release-competent vesicles preferentially align with Ca2+-channels and postsynaptic AMPA receptors within 20-30 nm and thereby forming a transsynaptic tripartite nanocomplex. Using functional EM, we show that single action potentials cause vesicles within the nanocomplex to fuse with a 50% probability. The loss of the presynaptic scaffold disrupts the formation of the tripartite transducers. Strikingly, the forced transsynaptic alignment of the Ca2+-channel subunit α2δ1 and AMPA receptors suffice to restore neurotransmission in a scaffold lacking synapse. Our results demonstrate a synaptic transducer nanocomplex that actively contributes to the organization of central synapses.

Synaptic transmission relies on the rapid fusion of neurotransmitter-containing synaptic vesicles (SVs), which happens in response to action potential (AP)-induced Ca2+ influx at active zones (AZs). A highly conserved molecular machinery cooperates at SV-release sites to mediate SV plasma membrane attachment and maturation, Ca2+ sensing, and membrane fusion. Despite this high degree of conservation, synapses - even within the same organism, organ or neuron - are highly diverse regarding the probability of APs to trigger SV fusion. Additionally, repetitive activation can lead to either strengthening or weakening of transmission. In this review, we discuss mechanisms controlling release probability and this short-term plasticity. We argue that an important layer of control is exerted by evolutionarily conserved AZ scaffolding proteins, which determine the coupling distance between SV fusion sites and voltage-gated Ca2+ channels (VGCC) and, thereby, shape synapse-specific input/output behaviors. We propose that AZ-scaffold modifications may occur to adapt the coupling distance during synapse maturation and plastic regulation of synapse strength.

Chemical synaptic transmission relies on the Ca2+-induced fusion of transmitter-laden vesicles whose coupling distance to Ca2+ channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Here, using electron- and super-resolution microscopy at the Drosophila neuromuscular junction we quantitatively map vesicle:Ca2+ channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. We show that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca2+-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. We propose activity-dependent inhibition of vesicle un-priming or release site activation as novel facilitation mechanisms., eLife digest Cells in the nervous system of all animals communicate by releasing and sensing chemicals at contact points named synapses. The ‘talking’ (or pre-synaptic) cell stores the chemicals close to the synapse, in small spheres called vesicles. When the cell is activated, calcium ions flow in and interact with the release-ready vesicles, which then spill the chemicals into the synapse. In turn, the ‘listening’ (or post-synaptic) cell can detect the chemicals and react accordingly. When the pre-synaptic cell is activated many times in a short period, it can release a greater quantity of chemicals, allowing a bigger reaction in the post-synaptic cell. This phenomenon is known as facilitation, but it is still unclear how exactly it can take place. This is especially the case when many of the vesicles are not ready to respond, for example when they are too far from where calcium flows into the cell. Computer simulations have been created to model facilitation but they have assumed that all vesicles are placed at the same distance to the calcium entry point: Kobbersmed et al. now provide evidence that this assumption is incorrect. Two high-resolution imaging techniques were used to measure the actual distances between the vesicles and the calcium source in the pre-synaptic cells of fruit flies: this showed that these distances are quite variable – some vesicles sit much closer to the source than others. This information was then used to create a new computer model to simulate facilitation. The results from this computing work led Kobbersmed et al. to suggest that facilitation may take place because a calcium-based mechanism in the cell increases the number of vesicles ready to release their chemicals. This new model may help researchers to better understand how the cells in the nervous system work. Ultimately, this can guide experiments to investigate what happens when information processing at synapses breaks down, for example in diseases such as epilepsy.

Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) we show that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation., Synaptic plasticity ensures functionality during perturbations and enables memory formation. Here, the authors describe homeostatic functional and nano-modular active zone modifications for immediate and long-lasting enhancement of neurotransmitter release, and identify Unc13 as a presynaptic molecular target for homeostatic potentiation and learning.

Significance Highly regulated and precise positioning of Ca 2+ channels at the active zone (AZ) controls Ca 2+ nanodomains at release sites. Their exact localization affects vesicular release probability (P VR ) and is important for proper synaptic transmission during repetitive stimulation. We provide a detailed analysis of synaptic transmission combined with superresolution imaging of the AZ organization in mouse hippocampal synapses lacking Rab-interacting molecule-binding protein 2 (RIM-BP2). By dual- and triple-channel time-gated stimulated emission depletion (gSTED) microscopy, we directly show that RIM-BP2 fine-tunes voltage-gated Ca 2+ channel 2.1 (Ca V 2.1) localization at the AZ. We reveal that RIM-BP2 likely regulates the Ca 2+ nanodomain by positioning Ca V 2.1 channels close to synaptic vesicle release sites. Loss of RIM-BP2 reduces P VR and alters short-term plasticity.

Cunningham and Littleton preview work from Goel et al. that describes a mechanism by which neurons regulate synaptic output after alterations in synapse size or active zone number., How neurons stabilize their overall synaptic strength following conditions that alter synaptic morphology or function is a key question in neuronal homeostasis. In this issue, Goel et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807165) find that neurons stabilize synaptic output despite disruptions in synapse size, active zone number, or postsynaptic function by controlling the delivery of active zone material and active zone size.

Summary: High-throughput electron microscopy has started to reveal synaptic connectivity maps of single circuits and whole brain regions, for example, in the Drosophila olfactory system. However, efficacy, timing, and frequency tuning of synaptic vesicle release are also highly diversified across brain synapses. These features critically depend on the nanometer-scale coupling distance between voltage-gated Ca2+ channels (VGCCs) and the synaptic vesicle release machinery. Combining light super resolution microscopy with in vivo electrophysiology, we show here that two orthogonal scaffold proteins (ELKS family Bruchpilot, BRP, and Syd-1) cluster-specific (M)Unc13 release factor isoforms either close (BRP/Unc13A) or further away (Syd-1/Unc13B) from VGCCs across synapses of the Drosophila olfactory system, resulting in different synapse-characteristic forms of short-term plasticity. Moreover, BRP/Unc13A versus Syd-1/Unc13B ratios were different between synapse types. Thus, variation in tightly versus loosely coupled scaffold protein/(M)Unc13 modules can tune synapse-type-specific release features, and “nanoscopic molecular fingerprints” might identify synapses with specific temporal features. : Fulterer et al. demonstrates that the scaffold proteins Bruchpilot and Syd-1 cluster (M)Unc13 release factor isoforms either close (BRP/Unc13A) or further away (Syd-1/Unc13B) from voltage-gated Ca2+ channels in the Drosophila olfactory system. These scaffold/release factor “modules” varied significantly between different synapse types, thereby tuning release features toward depression or facilitation. Keywords: neurotransmitter release, positional priming, munc13, Bruchpilot, Syd-1, synapse diversity, olfactory system, Drosophila, nanoscopy, synapse physiology, active zone

Information transfer between nerve cells (neurons) forms the basis of behavior, emotion, and survival. Signal transduction from one neuron to another occurs at synapses, and relies on both electrical and chemical signal propagation. At chemical synapses, incoming electrical action potentials trigger the release of chemical neurotransmitters that are sensed by the connected cell and here reconverted to an electrical signal. The presynaptic conversion of an electrical to a chemical signal is an energy demanding, highly regulated process that relies on a complex, evolutionarily conserved molecular machinery. Here, we review the biophysical characteristics of this process, the current knowledge of the molecules operating in this reaction and genetic specializations that may have evolved to shape inter-neuronal signaling.

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.

Summary Neural information processing depends on precisely timed, Ca 2+ -activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

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

The energy required to fuse synaptic vesicles with the plasma membrane (‘activation energy’) is considered a major determinant in synaptic efficacy. From reaction rate theory, we predict that a class of modulations exists, which utilize linear modulation of the energy barrier for fusion to achieve supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced by hypertonic solutions. We show that complexinI/II deficiency or phorbol ester stimulation indeed affects responses to hypertonic solution in a supralinear manner. An additive vs multiplicative relationship between activation energy and fusion rate provides a novel explanation for previously observed non-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release. DOI: http://dx.doi.org/10.7554/eLife.05531.001, eLife digest Information is carried around our nervous system by cells called neurons, which are connected to each other by junctions known as synapses. Within the neurons, there are many small compartments known as synaptic vesicles that are essential to the transfer of information from one neuron to the next. When one neuron is activated, the synaptic vesicles fuse with the membrane surrounding the cell to release molecules called neurotransmitters, which cross the synapse and activate the next neuron. Vesicle fusion is carefully regulated to control the speed and amount of neurotransmitter release, which determines the level of activation of the next neuron. Vesicle fusion requires energy, much of which is provided by a set of proteins found in the synapse. The minimum amount of energy required—called the activation energy—is influenced by many factors, including the shape of the cell's membrane at the synapse. It is thought that altering the activation energy required for fusion may control the activity of synapses, but it is not possible to directly measure this in living cells. To bypass this problem, Schotten, Meijer, Walter et al. established a new method to study vesicle fusion. This method combines a mathematical model with experimental data of the activity of synapses. First, the neurons were placed in a solution containing the sugar sucrose, which triggered vesicle fusion by lowering the activation energy. The increase in vesicle fusion was smaller in neurons that lacked two proteins called complexin I and complexin II—which control vesicle fusion—than in the normal neurons. A molecule called phorbol ester is also able to activate the release of neurotransmitters. When cells were treated with both sucrose and phorbol ester, the speed of vesicle fusion was greater. The experiments show that the effects of sucrose, phorbol ester, and the complexins multiply together to dramatically alter vesicle fusion. Schotten, Meijer, Walter et al. suggest a new model for how the activation energy of vesicle fusion controls the transfer of information across synapses. This might shed new light on how the efficiency of vesicle fusion is altered when neurons are highly active, which is thought to have strong implications for how information is processed in the brain. DOI: http://dx.doi.org/10.7554/eLife.05531.002

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis., Synaptic assembly depends on trans-synaptic Neurexin/Neuroligin signalling. Here, Muhammad et al. show that Spinophilin, a pre-synaptic scaffolding protein, interacts with Neurexin, in competition with Syd-1, to regulate the formation and function of synaptic active zones at Drosophila neuromuscular junctions.