Single-vesicle Fusion Assay Research Articles

Observing isolated synaptic vesicle association and fusion ex vivo.

Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca2+-independent and Ca2+-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems.

Molecular determinants of complexin clamping and activation function.

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.

Synergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis.

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 (<100 msec) and synchronous fusion of the docked vesicles.

Inhibition of Airway Epithelial Snare/Synaptotagmin Mediated Membrane Fusion by Hydrocarbon-Stapled Peptides

Airway mucin secretion involves membrane fusion of secretory granules that is mediated by SNAREs (syntaxin-3, SNAP-23, VAMP8), synaptotagmin-2, and other proteins. Mucus hypersecretion is a major cause of airway obstruction in the pathophysiology of asthma and cystic fibrosis. Deletion of synaptotagmin-2 protects mice against airway obstruction in a model of allergic asthma indicating the rationality of synaptotagmin-2 as a target for drug design. The so-called primary interface between the neuronal SNARE complex and synaptotagmin-1 is important for neurotransmitter release (Zhou et al., 2015), and it is also conserved for airway epithelial SNAREs and synaptotagmin-2. We designed a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface. The most α-helical stapled peptide had an affinity to the synaptotagmin-1 C2B domain that is similar to the affinity between the SNARE complex and the C2B domain. A subset of four stapled peptides, which were α-helical as assessed by circular dichroism, inhibit Ca2+-triggered fusion in a single vesicle fusion assay with reconstituted airway SNAREs and synaptotagmin-2. Inclusion of both Munc13-2 and Munc18-2 in the single vesicle fusion assay, the stapled peptide SP9 strongly suppress the Ca2+-triggered fusion even at the physiological Ca2+ concentrations. Moreover, cell penetrating peptide adjunct stapled peptide can be efficiently delivered into cultured epithelial cells and markedly reduced stimulated mucin secretion. Taken together, hydrocarbon-stapled peptide that interfere with the synaptotagmin-2/SNARE complex primary interface may allow modulation of secretory pathways.

Regulation of SNARE‐Dependent Membrane Fusion by Alpha‐Synuclein

Neurotransmitter release is a foundation of all thoughts in central nervous system. Communication between these neurons is mediated by the release and uptake of neurotransmitters through vesicle fusion at the synapses. The SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor) proteins are believed to mediate neurotransmitter release by promoting fusion of neurotransmitter laden presynaptic vesicle with the presynaptic membrane. Defects in the regulation of neurotransmission are believed to be the primary cause for many neurological diseases. The key to a better understanding of neurological diseases may lie in a more complete understanding of the molecular machine responsible for synaptic vesicle fusion. Alpha-synuclein (αS) has long been a protein of great interest because of its pathological aggregation in neuronal plaque and Lewy bodies which are associated with Parkinson's Disease and Lewy Bodies Dementia, respectively. Despite its medical significance, the normal function of αS has been elusive. αS is a 14 kDa soluble protein, abundantly present (several μM) in the presynapse. Thus, not surprisingly, αS has been found to play roles in various aspects of vesicle recycling. Very interestingly, however, αS has emerged as an important regulator for SNARE-dependent vesicle fusion. We used an in vitro single-vesicle fusion assay which allowed for the precise variation of the αS concentration. This assay provided the opportunity to dissect the impact of αS on individual vesicle fusion steps such as docking and lipid mixing along the membrane fusion pathway. The assay is effective in analyzing single fusion events in a natural, millisecond timescale between vesicle and a supported-bilayer. Using this assay, we have found that αS enhances vesicle docking by a factor of 10 at excessive 20 μM which is as much as 4 times higher than a normal cellular concentration. Detailed analysis reveals that the effect of αS on SNARE-dependent membrane fusion is largely on vesicle docking with no significant changes in lipid mixing observed. It also suggests that αS plays a role in accelerating and stabilizing large fusion pore formation. αS may be pathologically involved in neurological diseases, but it also is a crucial SNARE regulator by enhancing presynaptic vesicle docking and stabilizing the membrane fusion pore. αS may be pathologically involved in neurological diseases, but it also is a crucial SNARE regulator This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alpha-Synuclein Continues to Enhance SNARE-Dependent Vesicle Docking at Exorbitant Concentrations.

Recently, Parkinson’s disease-associated α-synuclein (αS) has emerged as an important regulator for SNARE-dependent vesicle fusion. However, it is controversial if excessive accumulation of αS, even in the absence of aggregation, impairs neurotransmission. Here we use a single vesicle fusion assay with ms time resolution capable of dissecting the impact of αS on each step of membrane fusion. Unlike the previous results from various in vitro, cellular, and in vivo studies, we find that non-aggregated αS promotes vesicle merger even at exorbitant concentrations. The enhancement has been seen as much as 13 fold. Delving into the kinetics of the intermediate states for vesicle fusion reveals that αS stimulates vesicle docking without altering the dynamics of bilayer merger (lipid mixing). However, minute amounts of soluble aggregated species abolish SNARE-dependent bilayer merger completely. Thus, the results show that excessive accumulation of non-aggregated αS may not be toxic for neurotransmitter release.

Botulinum Toxins a and E Inflict Dynamic Destabilization on T-SNARE to Impair Snare Assembly and Membrane Fusion

Botulinum toxins (BoNT) A and E block neurotransmitter release by specifically cleaving the C- terminal ends of SNAP-25, a plasma membrane SNARE protein. We find that SNAP-25A and E, the cleavage products of BoNT A and E respectively, terminate membrane fusion via completely different mechanisms. Combined studies of single molecule FRET and single vesicle fusion assays reveal that SNAP-25E is incapable of supporting SNARE pairing and thus, vesicle docking. In contrast, SNAP-25A facilitates robust SNARE pairing and vesicle docking with somewhat reduced SNARE zippering, which leads to severe impairment of fusion pore opening. The EPR results show that the discrepancy between SNAP-25A and E might stem from the extent of the dynamic destabilization of the t-SNARE core at the N-terminal half which plays a pivotal role in nucleating SNARE complex formation. Thus, the results provide insights into the structure/dynamics-based mechanism by which BoNT A and E impair membrane fusion.

Botulinum Toxins A and E Inflict Dynamic Destabilization on t-SNARE to Impair SNARE Assembly and Membrane Fusion

Botulinum toxins (BoNTs) A and E block neurotransmitter release by specifically cleaving the C- terminal ends of SNAP-25, a plasma membrane SNARE protein. Here, we find that SNAP-25A and E, the cleavage products of BoNT A and E, respectively, terminate membrane fusion via completely different mechanisms. Combined studies of single-molecule FRET and single-vesicle fusion assays reveal that SNAP-25E is incapable of supporting SNARE pairing and thus, vesicle docking. In contrast, SNAP-25A facilitates robust SNARE pairing and vesicle docking with somewhat reduced SNARE zippering, which leads to severe impairment of fusion pore opening. The electron paramagnetic resonance results show that the discrepancy between SNAP-25A and E might stem from the extent of the dynamic destabilization of the t-SNARE core at the N-terminal half, which plays a pivotal role in nucleating SNARE complex formation. Thus, the results provide insights into the structure/dynamics-based mechanism by which BoNT A and E impair membrane fusion.

Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay

Homotypic fusion of endoplasmic reticulum membranes is driven by atlastin GTPases; however, the underlying mechanism remains largely unknown. Here, using a FRET-based single-vesicle fusion assay with liposomes bearing the yeast atlastin Sey1p, we investigated the molecular mechanisms of atlastin-mediated membrane tethering and fusion. Although Sey1p-bearing proteoliposomes frequently underwent membrane tethering in a GTP hydrolysis-dependent manner as reported in studies using bulk assays, only a small fraction of the tethered liposomes proceeded to fusion. Strikingly, the rest of the tethered liposomes failed to fuse or dissociate. This stable tethering, however, did not require continued GTP hydrolysis because GTP omission and magnesium chelation did not disrupt tethering. Interestingly, an increased Sey1p density on the membrane markedly accelerated tethering but barely affected the fusion rate of the tethered liposomes, indicating that Sey1p requires additional factors to support efficient fusion in vivo. Finally, the assay also revealed that Sey1p-mediated liposome fusion occurs through hemifusion, suggesting the mechanistic conservation between biological membrane fusion events despite the existence of diverse fusogens.

SNARE-Mediated Single-Vesicle Fusion Events with Supported and Freestanding Lipid Membranes

In vitro single-vesicle fusion assays are important tools to analyze the details of SNARE-mediated fusion processes. In this study, we employed planar pore-spanning membranes (PSMs) prepared on porous silicon substrates with large pore diameters of 5 μm, allowing us to compare the process of vesicle docking and fusion on the supported parts of the PSMs (s-PSMs) with that on the freestanding membrane parts (f-PSM) under the exact same experimental conditions. The PSMs harbor the t-SNARE ΔN49-complex to investigate the dynamics and fusogenicity of single large unilamellar vesicles doped with the v-SNARE synaptobrevin 2 by means of spinning-disc confocal microscopy with a time resolution of 10 ms. Our results demonstrate that vesicles docked to the s-PSM were fully immobile, whereas those docked to the f-PSM were mobile with a mean diffusion coefficient of 0.42 μm2/s. Despite the different dynamics of the vesicles on the two membrane types, similar fusion kinetics were observed, giving rise to a common fusion mechanism. Further investigations of individual lipid mixing events on the s-PSMs revealed semi-stable post-fusion structures.

Visualization of SNARE-Mediated Hemifusion between Giant Unilamellar Vesicles Arrested by Myricetin.

Neurotransmitters are released within a millisecond after Ca2+ arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca2+-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca2+ triggering. It has been shown that the flavonoid myricetin arrests soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated vesicle fusion at hemifusion, but that the hemifused vesicles spontaneously convert to full fusion when the myricetin clamp is removed by the enzyme laccase. In the present study, we visualized SNARE-mediated hemifusion between two SNARE-reconstituted giant unilamellar vesicles (GUVs) arrested by myricetin. The large size of the GUVs enabled us to directly image the hemifusion between them. When two merging GUVs were labeled with different fluorescent dyes, GUV pairs showed asymmetric fluorescence intensities depending on the position on the GUV pair consistent with what is expected for hemifusion. The flow of lipids from one vesicle to the other was revealed with fluorescence recovery after photobleaching (FRAP), indicating that the two membranes had hemifused. These results support the hypothesis that hemifusion may be the molecular status that primes Ca2+-triggered millisecond exocytosis. This study represents the first imaging of SNARE-driven hemifusion between GUVs.

A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay

Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds’ inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus.

N-terminal domain of complexin independently activates calcium-triggered fusion

Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release.

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

New In Vitro Hemagglutinin Inhibitor Screening System Based on Single Vesicle Fusion Assay

Hemagglutinins of influenza virus plays a pivotal role in the infection to host mammalian cell so is coming up as druggable target like neuraminidases of this virus. However, the researches regarding to influenza virus should be strictly restricted within the facilities guaranteed over BioSafety Level 3 because of the potential threats of the contagion of influenza virus. In order to develop new hemagglutinin inhibitor screening system without the contagion of influenza virus, we introduced single vesicle fusion assay with recombinant hemagglutinin. In this study, we show that recombinant hemagglutinin can exert the membrane fusion in single vesicle fusion assay and this single vesicle fusion assay using recombinant hemagglutinin can be applied as an in vitro inhibitor screening system against hemagglutinin of influenza virus.

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events.

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays.

Collective Action of SNAREpins Exerts Forces between Membranes that Activate Fusion

SNARE proteins mediate most intracellular membrane fusion processes such as exocytosis. It has been established that vesicle associated v-SNAREs and target-membrane associated t-SNAREs, assemble into a parallel four-helix bundle (SNAREpin) in a zipper like fashion, which brings the membranes close together. However, the detailed mechanisms whereby SNAREs drive fusion remains unclear. It is also unknown whether several SNAREpins cooperate to induce fusion, and widely varying SNARE requirements for fusion are reported, 2-15 in vivo (Montecucco et al., Trends Biochem Sci, 2005) and 1-11 in vitro (van den Bogaart et al., NSMB, 2010; Karatekin et al., PNAS, 2010). Here, we developed a mathematical model of SNAREpins connecting a vesicle to a planar membrane, quantifying inter-membrane, inter-SNAREpin and membrane-SNAREpin interactions, and taking account of the zippering/unzipping of the SNAREpins. Monte Carlo simulations showed that SNAREpins assemble through the SNARE motifs and self-organize into a ring. The ring tends to expand, driven by inter-SNAREpin and SNAREpin-membrane interactions, reducing membrane separation by geometrical coupling. Assuming an energy criterion for fusion, we determined the waiting times for fusion from the distributions of inter-membrane energies. Our data show that although one SNAREpin can induce membrane fusion, fusion waiting times decrease rapidly with the number of SNAREpins. Applying the model to single-vesicle fusion assays, we predict that the dependency of docking-to-fusion delay times on the number of v-SNAREs reaches a plateau, in agreement with experiments (Karatekin et al., PNAS, 2010). We also find that waiting times increase with vesicle size and with insertion of flexible segments into the SNARE linker domains, in qualitative agreement with experiments (McNew et al., Mol Cell, 1999). Our results suggest that fully zippered SNAREs work in concert to trigger fusion, and explain the wide range of reported SNARE requirements for fusion.

Role of Mechanical Properties of Cell Mediated Vesicles in Membrane Fusion

Cell mediated vesicles (CMVs) are endogenous particles involved in cell to cell communication. CMVs transport proteins and RNA and offer multiple advantages over conventional drug delivery systems. For example they are naturally stable in the body and could have natural targeting properties. Synthetic generation of liposomes with similar characteristics to CMVs could result in cost-effective drug carriers including all advantages CMVs offer. Interestingly, most CMVs contain large amounts of cytoskeletal proteins: actin and tubulin, but also related cross linkers and motor proteins. We hypothesize that the cytoskeletal elements present in these vesicles contribute to their efficient fusion with cells. In our study we compare three kinds of vesicles: natural vesicles excreted by blood cells (both erythrocytes and dendritic cells), empty liposomes and liposomes containing actin filaments. First we visualize the structure of these three types with fluorescent imaging and AFM. Furthermore, we indent the vesicles using AFM to learn about the mechanical properties of these small vesicles. These results, in combination with single vesicle fusion assays, will reveal the role of the cytoskeleton in vesicle fusion.

Single-Vesicle Fluorescence Study Reveals Dynamic Ca 2+-Dependent Activity of Membrane-Anchored Synaptotagmin 1

In Ca2+ evoked neurotransmitter release, synaptotagmin 1 (Syt1) has been thought as Ca2+ sensor that regulates synaptic vesicle fusion with plasma membrane. However, in vitro studies largely failed to recapitulate this stimulatory function. Using the single-vesicle fusion assay that tracks fusion between two 50-nm sized vesicles [1-2], we studied the activity of membrane-anchored Syt1 in unprecedented detail [3]. Our result shows that membrane-anchored Syt1 is able to accelerate both docking and fusion kinetics.