Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes.

Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. Here, using adrenal chromaffin cells, we identify the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, we show that the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, lead us to propose that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes.

Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities.

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.

Glial Synaptobrevin mediates peripheral nerve insulation, neural metabolic supply, and is required for motor function.

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.

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses.

The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.

The SNARE protein vti1a functions in dense-core vesicle biogenesis.

The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short-term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca(2+)-channel trafficking, but is dispensable for transmitter release.

Doc2b synchronizes secretion from chromaffin cells by stimulating fast and inhibiting sustained release.

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 [Ca(2+)]i following 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.

A sequential vesicle pool model with a single release sensor and a Ca(2+)-dependent priming catalyst effectively explains Ca(2+)-dependent properties of neurosecretion.

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca(2+) dependence, but also upstream steps depend on Ca(2+). After deletion of the Ca(2+) sensor for fast release - synaptotagmin-1 - slower Ca(2+)-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca(2+) sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca(2+)-dependent action: a Ca(2+)-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca(2+)-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca(2+)-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca(2+)-dependent fusion from the NRP. We conclude that the elusive 'alternative Ca(2+) sensor' for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca(2+)-dependent properties of secretion without assuming parallel pools or sensors.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

Role of the synaptobrevin C terminus in fusion pore formation.

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.

Rate-limiting recovery processes in neurotransmission under sustained stimulation.

At active zones of chemical synapses, an arriving electric signal induces the fusion of vesicles with the presynaptic membrane, thereby releasing neurotransmitters into the synaptic cleft. After a fusion event, both the release site and the vesicle undergo a recovery process before becoming available for reuse again. Of central interest is the question which of the two restoration steps acts as the limiting factor during neurotransmission under high-frequency sustained stimulation. In order to investigate this problem, we introduce a non-linear reaction network which involves explicit recovery steps for both the vesicles and the release sites, and includes the induced time-dependent output current. The associated reaction dynamics are formulated by means of ordinary differential equations (ODEs), as well as via the associated stochastic jump process. While the stochastic jump model describes the dynamics at a single active zone, the average over many active zones is close to the ODE solution and shares its periodic structure. The reason for this can be traced back to the insight that recovery dynamics of vesicles and release sites are statistically almost independent. A sensitivity analysis on the recovery rates based on the ODE formulation reveals that neither the vesicle nor the release site recovery step can be identified as the essential rate-limiting step but that the rate-limiting feature changes over the course of stimulation. Under sustained stimulation, the dynamics given by the ODEs exhibit transient changes leading from an initial depression of the postsynaptic response to an asymptotic periodic orbit, while the individual trajectories of the stochastic jump model lack the oscillatory behavior and asymptotic periodicity of the ODE-solution.

Spontaneous neurotransmission at evocable synapses predicts their responsiveness to action potentials.

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.

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca2+ channels drives sustained active zone 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.

Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation.

Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca2+/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.

Allosteric stabilization of calcium and phosphoinositide dual binding engages several synaptotagmins in fast exocytosis.

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 state. Here, we 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 engaging in Ca2+/PI(4,5)P2 dual-binding lowers the energy barrier for vesicle fusion by ~5 kBT and that allosteric stabilization of this state enables the synchronized engagement of several (typically three) synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin's allosteric properties may show dominant-negative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)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.

For our brains and nervous systems to work properly, the nerve cells within them must be able to 'talk' to each other. They do this by releasing chemical signals called neurotransmitters which other cells can detect and respond to. Neurotransmitters are packaged in tiny membrane-bound spheres called vesicles. When a cell of the nervous system needs to send a signal to its neighbours, the vesicles fuse with the outer membrane of the cell, discharging their chemical contents for other cells to detect. The initial trigger for neurotransmitter release is a short, fast increase in the amount of calcium ions inside the signalling cell. One of the main proteins that helps regulate this process is synaptotagmin which binds to calcium and gives vesicles the signal to start unloading their chemicals. Despite acting as a calcium sensor, synaptotagmin actually has a very low affinity for calcium ions by itself, meaning that it would not be efficient for the protein to respond alone. Synpatotagmin is more likely to bind to calcium if it is attached to a molecule called PIP₂, which is found in the membranes of cells The effect also occurs in reverse, as the binding of calcium to synaptotagmin increases the protein's affinity for PIP₂. However, how these three molecules ­ synaptotagmin, PIP₂, and calcium ­ work together to achieve the physiological release of neurotransmitters is poorly understood. To help answer this question, Kobbersmed, Berns et al. set up a computer simulation of 'virtual vesicles' using available experimental data on synaptotagmin's affinity with calcium and PIP₂. In this simulation, synaptotagmin could only trigger the release of neurotransmitters when bound to both calcium and PIP₂. The model also showed that each 'complex' of synaptotagmin/calcium/PIP₂ made the vesicles more likely to fuse with the outer membrane of the cell ­ to the extent that only a handful of synaptotagmin molecules were needed to start neurotransmitter release from a single vesicle. These results shed new light on a biological process central to the way nerve cells communicate with each other. In the future, Kobbersmed, Berns et al. hope that this insight will help us to understand the cause of diseases where communication in the nervous system is impaired.

A coiled coil trigger site is essential for rapid binding of synaptobrevin to the SNARE acceptor complex.

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.

Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca2+ channel distances.

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.

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.

Homeostatic scaling of active zone scaffolds maintains global synaptic strength.

Synaptic terminals grow and retract throughout life, yet synaptic strength is maintained within stable physiological ranges. To study this process, we investigated Drosophila endophilin (endo) mutants. Although active zone (AZ) number is doubled in endo mutants, a compensatory reduction in their size homeostatically adjusts global neurotransmitter output to maintain synaptic strength. We find an inverse adaptation in rab3 mutants. Additional analyses using confocal, STED, and electron microscopy reveal a stoichiometric tuning of AZ scaffolds and nanoarchitecture. Axonal transport of synaptic cargo via the lysosomal kinesin adapter Arl8 regulates AZ abundance to modulate global synaptic output and sustain the homeostatic potentiation of neurotransmission. Finally, we find that this AZ scaling can interface with two independent homeostats, depression and potentiation, to remodel AZ structure and function, demonstrating a robust balancing of separate homeostatic adaptations. Thus, AZs are pliable substrates with elastic and modular nanostructures that can be dynamically sculpted to stabilize and tune both local and global synaptic strength.

Rapid active zone remodeling consolidates presynaptic potentiation.

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.

Regulation of synaptic release-site Ca2+ channel coupling as a mechanism to control release probability and short-term plasticity.

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.

Vesicle release site organization at synaptic active zones.

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.