Compound vesicle fusion increases quantal size and potentiates synaptic transmission.
ABSTRACT: Exocytosis at synapses involves fusion between vesicles and the plasma membrane. Although compound fusion between vesicles was proposed to occur at ribbon-type synapses, whether it exists, how it is mediated, and what role it plays at conventional synapses remain unclear. Here we report the existence of compound fusion, its underlying mechanism, and its role at a nerve terminal containing conventional active zones in rats and mice. We found that high potassium application and high frequency firing induced giant capacitance up-steps, reflecting exocytosis of vesicles larger than regular ones, followed by giant down-steps, reflecting bulk endocytosis. These intense stimuli also induced giant vesicle-like structures, as observed with electron microscopy, and giant miniature excitatory postsynaptic currents (mEPSCs), reflecting more transmitter release. Calcium and its sensor for vesicle fusion, synaptotagmin, were required for these giant events. After high frequency firing, calcium/synaptotagmin-dependent mEPSC size increase was paralleled by calcium/synaptotagmin-dependent post-tetanic potentiation. These results suggest a new route of exocytosis and endocytosis composed of three steps. First, calcium/synaptotagmin mediates compound fusion between vesicles. Second, exocytosis of compound vesicles increases quantal size, which increases synaptic strength and contributes to the generation of post-tetanic potentiation. Third, exocytosed compound vesicles are retrieved via bulk endocytosis. We suggest that this vesicle cycling route be included in models of synapses in which only vesicle fusion with the plasma membrane is considered.
Project description:Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca<sup>2+</sup>-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca<sup>2+</sup>-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca<sup>2+</sup>-dependent priming as a prelude to fast and slow exocytosis triggering.
Project description:Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger--a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.
Project description:Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.
Project description:Neurons that form ribbon-style synapses are specialized for continuous exocytosis. To this end, their synaptic terminals contain numerous synaptic vesicles, some of which are ribbon associated, that have difference susceptibilities for undergoing Ca<sup>2+</sup>-dependent exocytosis. In this study, we probed the relationship between previously defined vesicle populations and determined their fusion competency with respect to SNARE complex formation. We found that both the rapidly releasing vesicle pool and the releasable vesicle pool of the retinal bipolar cell are situated at the ribbon-style active zones, where they functionally interact. A peptide inhibitor of SNARE complex formation failed to block exocytosis from either pool, suggesting that these two vesicle pools have formed the SNARE complexes necessary for fusion. By contrast, a third, slower component of exocytosis was blocked by the peptide, as was the functional replenishment of vesicle pools, indicating that few vesicles outside of the ribbon-style active zones were initially fusion competent. In cone photoreceptors, similar to bipolar cells, fusion of the initial ribbon-associated synaptic vesicle cohort was not blocked by the SNARE complex-inhibiting peptide, whereas a later phase of exocytosis, attributable to the recruitment and subsequent fusion of vesicles newly arrived at the synaptic ribbons, was blocked. Together, our results support a model in which stimulus-evoked exocytosis in retinal ribbon synapses is SNARE-dependent; where vesicles higher up on the synaptic ribbon replenish the rapidly releasing vesicle pool; and at any given time, there are sufficient SNARE complexes to support the fusion of the entire ribbon-associated cohort of vesicles. An important implication of these results is that ribbon-associated vesicles can form intervesicular SNARE complexes, providing mechanistic insight into compound fusion at ribbon-style synapses.
Project description:Neuronal communication is mediated by Ca(2+)-triggered fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin I functions as a Ca(2+) sensor that regulates exocytosis, whereas soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in the vesicle and target membrane assemble into complexes that directly catalyze bilayer fusion. Here we report that, before the Ca(2+) trigger, synaptotagmin interacts with SNARE proteins in the target membrane to halt SNARE complex assembly at a step after donor vesicles attach, or dock, to target membranes. This results in fusion complexes that, when subsequently triggered by Ca(2+), drive rapid, highly efficient lipid mixing. Ca(2+)-independent interactions with SNAREs also predispose synaptotagmin to selectively penetrate the target membrane in response to Ca(2+); we demonstrate that Ca(2+)-synaptotagmin must insert into the target membrane to accelerate SNARE-catalyzed fusion. These findings demonstrate that Ca(2+) converts synaptotagmin from a clamp to a trigger for exocytosis.
Project description:Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.
Project description:Synaptic vesicle protein 2 (SV2), one of the first synaptic vesicle proteins identified, is characterized by multiple transmembrane regions that exhibit homology to sugar transporters, and by a highly glycosylated intravesicular sequence. Deletion of SV2 causes postnatal lethality in mice, primarily because of fulminant epilepsy. At the cellular level, deletion of SV2 impairs neurotransmitter release, but its function is unknown, and even the exact point at which release is affected in SV2-deleted synapses remains unclear. Using electrophysiological approaches, we now examine at what step in exocytosis the deletion of SV2 impairs release. Our data demonstrate that deletion of SV2 produces a decrease in evoked synaptic responses without causing changes in mini frequency, mini amplitude, the readily releasable pool of vesicles, or the apparent Ca(2+) sensitivity of vesicle fusion. These findings indicate that a previously unidentified step may couple priming of synaptic vesicles to Ca(2+) triggering of fusion, and that SV2 acts in this step to render primed synaptic vesicles fully Ca(2+) responsive. To investigate the structural requirements for this function of SV2, we used rescue experiments. We demonstrate that conserved charged residues within the transmembrane regions and the intravesicular glycosylation of SV2 are required for its normal folding and trafficking. In contrast, the conserved putative synaptotagmin-binding sequence of SV2 is fully dispensable. Viewed together, these observations suggest that SV2 functions in a maturation step of primed vesicles that converts the vesicles into a Ca(2+)- and synaptotagmin-responsive state.
Project description:Background:Calcium signaling and membrane fusion play key roles in exocytosis of drug-containing vesicles through the blood-brain barrier (BBB). Identifying the role of synaptotagmin-like protein4-a (Slp4-a) in the presence of Ca2+ ions, at the pre-fusion stage of a vesicle with the basolateral membrane of endothelial cell, can reveal brain drug transportation across BBB. Methods:We utilized molecular dynamics (MD) simulations with a coarse-grained PACE force field to investigate the behaviors of Slp4-a with vesicular and endothelial membranes at the pre-fusion stage of exocytosis since all-atom MD simulation or experiments are more time-consuming and expensive to capture these behaviors. Results:The Slp4-a pulls lipid membranes (vesicular and endothelial) into close proximity and disorganizes lipid arrangement at contact points, which are predictors for initiation of fusion. Our MD results also indicate that Slp4-a needs Ca2+ to bind with weakly-charged POPE lipids (phosphatidylethanolamine). Conclusions:Slp4-a is an important trigger for membrane fusion in BBB exocytosis. It binds to lipid membranes at multiple binding sites and triggers membrane disruption for fusion in calcium-dependent case. General significance:Understanding the prefusion process of the vesicle will help to design better drug delivery mechanisms to the brain through formidable BBB.
Project description:Hippocampal neurons in tissue culture develop functional synapses that exhibit considerable variation in synaptic vesicle content (20-350 vesicles). We examined absolute and fractional parameters of synaptic vesicle exocytosis of individual synapses. Their correlation to vesicle content was determined by activity-dependent discharge of FM-styryl dyes. At high frequency stimulation (30 Hz), synapses with large recycling pools released higher amounts of dye, but showed a lower fractional release compared to synapses that contained fewer vesicles. This effect gradually vanished at lower frequencies when stimulation was triggered at 20 Hz and 10 Hz, respectively. Live-cell antibody staining with anti-synaptotagmin-1-cypHer 5, and overexpression of synaptopHluorin as well as photoconversion of FM 1-43 followed by electron microscopy, consolidated the findings obtained with FM-styryl dyes. We found that the readily releasable pool grew with a power function with a coefficient of 2/3, possibly indicating a synaptic volume/surface dependency. This observation could be explained by assigning the rate-limiting factor for vesicle exocytosis at high frequency stimulation to the available active zone surface that is proportionally smaller in synapses with larger volumes.
Project description:Neurotransmitter release requires the direct coupling of the calcium sensor with the machinery for membrane fusion. SNARE proteins comprise the minimal fusion machinery, and synaptotagmin I, a synaptic vesicle protein, is the primary candidate for the main neuronal calcium sensor. To test the effect of synaptotagmin I on membrane fusion, we incorporated it into a SNARE-mediated liposome fusion assay. Synaptotagmin I dramatically stimulated membrane fusion by facilitating SNAREpin zippering. This stimulatory effect was topologically restricted to v-SNARE vesicles (containing VAMP 2) and only occurred in trans to t-SNARE vesicles (containing syntaxin 1A and SNAP-25). Interestingly, calcium did not affect the overall fusion reaction. These results indicate that synaptotagmin I can directly accelerate SNARE-mediated membrane fusion and raise the possibility that additional components might be required to ensure tight calcium coupling.