Previously, fast fusion between v-SNARE-reconstituted vesicles (v-vesicles) and t-SNARE-reconstituted supported bilayers (t-SBL) was successfully demonstrated (Liu et al., 2005; Domanska et al., 2009; Karatekin et al., 2010; Kiessling et al., 2013; Stratton et al., 2016). This environment displayed the fusion kinetics that better mimicked what was observed in vivo. The results suggested that SNAREs alone, without the help of any auxiliary proteins, are capable of driving sub 25 ms membrane fusion.

To investigate SNARE-mediated single vesicle-to-SBL fusion in our hands, we prepare the t-SBL with PEGylated liposomes following the method previously reported by Karatekin et al. (2010). It has been shown that the PEGylated lipids provide a hydrated cushion of ~4 nm in thickness between the SBL and the imaging surface which allows for critical dynamic movement of transmembrane proteins (Karatekin and Rothman, 2012). Once the SBL is properly formed we place the slide under the total internal reflection fluorescence (TIRF) microscope and start video acquisition as we introduce the v-vesicles (Figure 1A). The v-vesicles contain lipophilic cationic fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (excitation at 549 nm, emission at 565 nm). Thus, we are able to monitor individual vesicle docking events by counting the immobilized fluorescent spots in the TIRF video (Figure 1B). The fusion of the docked v-vesicles onto the SBL is identified by the characteristic two-dimensional diffusion of the fluorescent lipid dyes (Figure 1C). With this setup, we are able to monitor the docking rate, the docking-to-fusion delay, and the fusion efficiency in real-time (Figures 1D–G).

We first examine the rate of v-vesicles docking onto the t-SBL. We find, under our experimental conditions, that ~20 vesicles dock in our viewing area (~55 × ~110 μm) during a 1 min video recording (Figure 1E). It is well-established that SNARE complex formation requires VAMP2, syntaxin 1A, and SNAP-25, and even a single missing component fails to elicit docking and fusion. Thus, as a control, we examine the v-vesicles docking onto t-SBLs prepared without SNAP-25 or without both SNAP-25 and syntaxin 1A. In either case, the vesicles fail to associate with the SBL. Furthermore, to test if vesicle docking is exclusively SNARE-mediated we pre-incubate t-SBL with soluble VAMP2 without the transmembrane domain (VpS). VpS has been frequently used as a competitive inhibitor for SNARE-mediated vesicle docking. We observe no docking in the presence of VpS as well, indicating that the v-vesicle docking to the SBL is SNARE-mediated. Because Ca²⁺-sensor Syt1 is not included at this time, we expect that Ca²⁺ should not affect either docking or membrane fusion. Indeed, when the v-vesicles premixed with 500 μM Ca²⁺ are injected into the flow chamber we do not observe any apparent changes in docking kinetics when compared with the results without Ca²⁺ (Figure 1E).

Next, we take a closer look into the individual docked v-vesicles to find out if membrane fusion happens. We analyze three independent recordings and count a total of 23 fusion events out of 62 docked v-vesicles (~35% fusion efficiency) and 20 fusion events out of 63 docked v-vesicles (~32% fusion efficiency) in the absence and presence of Ca²⁺, respectively (Figure 1F). Furthermore, approximately half of the fusion events occur within the first 20 ms after docking for both cases (Figure 1F and G). Thus, our results show that SNAREs alone are capable of mediating fast vesicle fusion within 20 ms.

Syt1 harbors tandem Ca²⁺-binding C2 domains that are tethered to the vesicle membrane via a transmembrane helix (Perin et al., 1991; Chapman, 2008). Previously, in their seminal study, Sudhof and coworkers used the gain of function and the loss of function mutants of Syt1 to demonstrate that the main function of Syt1 as a Ca²⁺-sensor is to increase the probability of fast membrane fusion in response to the Ca²⁺ signal (Fernández-Chacón et al., 2001).

Having observed that SNAREs alone can mediate fast membrane fusion with the SBL platform, we now ask if Syt1 and Ca²⁺ together enhance the membrane fusion probability as Sudhof and coworkers observed in the neuron. If so, how much enhancement does it have?

To answer these questions, we reconstitute both VAMP2 and Syt1 into the vesicles in a 1:1 ratio. In order to follow through from docking to membrane fusion in a continuous time frame, the vesicles are premixed with Ca²⁺ and the mixture is injected into the flow cell prepared with t-SBL. The vesicle concentration here is set to be equal to that in the previous section, in the absence of Syt1, for the proper comparison (Figure 2A).

We first examine the effect of Syt1 and Ca²⁺ on docking. It is established that Syt1 assists docking either by its t-SNARE interaction (Rickman et al., 2004; Loewen et al., 2006) or by direct binding to negatively charged lipids (Schiavo et al., 1996; Kim et al., 2012). Remarkably, we observe more than ~540 docking events during our 1 min video recordings with 500 μM Ca²⁺, a ~25-fold enhancement over those in the absence of Syt1 (Figures 2B,C, Supplementary Video 1). As for membrane fusion, we find that ~70% of the docked v-vesicles exhibit membrane fusion, which is a 2-fold enhancement when compared with the percentage with SNARE only. Among those, ~55% of the fusion population display docking-to-fusion delay shorter than 20 ms, similar to the case with SNAREs only. In direct comparison with the fusion population observed with v-vesicles without Syt1, we find that Syt1 and 500 μM Ca²⁺ together elicit over a ~50-fold increase in both fast and total fusion population (Figure 2D). Intriguingly, however, most of the enhancement of fast membrane fusion by Syt1 and Ca²⁺ stems from the ~25-fold enhancement of docking while there is only a ~2-fold increase in the fusion-to-docking ratio.

It is often recognized that lipid mixing does not necessarily report the formation of a fusion pore. To make sure that the fusion pore is indeed formed, we prepare v-vesicles that are loaded with both lipid-reporter DiD (excitation at 644 nm, emission at 665 nm) and the content-reporter sulforhodamine B (SRB) (excitation 565 nm, emission at 586 nm) (Kiessling et al., 2013; Stratton et al., 2016) (Supplementary Figure 1A). We are able to monitor both lipid mixing and content release via simultaneous excitation with red (640 nm) and green (532 nm) lasers. We find that v-vesicles showing the lipid diffusion, characteristic to lipid mixing lose the SRB signal instantaneously at the onset of lipid mixing (Supplementary Figure 1B left), while the v-vesicles showing no lipid diffusion maintain their SRB signal (Supplementary Figure 1B right). This implies that lipid mixing and fusion pore opening occur simultaneously within our experimental time resolution of 20 ms. Furthermore, it appears that using DiD instead of DiI or the incorporation of SRB do not significantly affect the overall docking and fusion characteristics (Supplementary Figures 1C,D).

In the experiments presented in the previous section, we premixed Ca²⁺ with the v-vesicles. This experimental preparation mimics the pre-binding of Ca²⁺ to Syt1 prior to Syt1's binding to SNAREs and the target membrane. However, in reality, it is possible that Syt1 pre-binds to SNAREs in the absence of Ca²⁺ in preparation for the Ca²⁺ influx. In fact, it is shown that Syt1 has the capacity to bind the binary t-SNARE complex in the absence of Ca²⁺ (Rickman et al., 2004; Loewen et al., 2006). To explore the outcomes of this alternative situation, we examine docking and fusion in the absence of Ca²⁺ (Figure 3A) and investigate whether Ca²⁺ can trigger membrane fusion of a priorily docked vesicles (Figure 3B).

When we examine vesicle docking in the absence of Ca²⁺ we observe a ~6-fold increase in docking compared to the results from the vesicles with only VAMP2 (Figure 3C). This enhancement factor falls significantly short of those observed when both Ca²⁺ and Syt1 are present, indicating that Ca²⁺ plays a role in vesicle docking.

To our surprise, no apparent accumulation of fast membrane fusion is observed within the first 20 ms time period. This is in sharp contrast to that of which was observed for the experiments with only SNAREs. Membrane fusion events among docked vesicles are all scattered randomly over the period of our experimental time (1 min) (Figures 3D,E). Nevertheless, the results appear to be somewhat consistent with the proposal that Syt1 might function as a fusion clamp of spontaneous fusion (Chicka et al., 2008).

Even more surprising is that the strong enhancement of membrane fusion by Ca²⁺, observed when Ca²⁺ was pre-bound to Syt1, is completely lost. When we inject Ca²⁺ (Figure 3B), after any unbound vesicles are washed out with sufficient amount of buffer, we do not detect any synchronization of membrane fusion events in the first 20 ms window. Mere ~7% of docked vesicles fuses with SBL with 500 μM Ca²⁺ and fusion events are sporadically scattered in the time frame of our observation (1 min) (Figures 3F,G). Thus, the results show that a priori binding of Syt1 to SNARE complexes in the absence of Ca²⁺ leads to an irreversible off-pathway that does not bring about Ca²⁺-triggered synchronized membrane fusion.

As controls, we evaluate docking and fusion of vesicles reconstituted with both VAMP2 and Syt1, with only VAMP2, with only Syt1, onto SBLs with t-SNAREs, with only syntaxin 1A, with t-SNAREs disabled by VpS, and without any proteins (Figure 4A). Experiments with all possible combinations are separately carried out either in the absence or presence of 500 μM Ca²⁺ in Figures 4B,C, respectively. Even in the absence of VAMP2, we observe docking with v-vesicles reconstituted with only Syt1 (Figure 4B). This is due to Syt1 binding to either the binary t-SNARE complex, the ternary SNARE complex or phosphatidylinositol-4,5-bisphosphate (PIP2) (Rickman et al., 2004; Loewen et al., 2006). Furthermore, it is established that Ca²⁺ increases binding between Syt1 and PIP2 which is shown in Figure 4C (Schiavo et al., 1996; Kim et al., 2012). However, we note that these docking events did not lead to membrane fusion.

We prepared DNA sequences encoding rat syntaxin 1A (amino acids 1–288 with three native cysteines replaced by alanines), VAMP2 (amino acids 1–116 with C103 replaced by alanines), VpS (amino acids 1-94), SNAP-25 (amino acids 1–206 with four native cysteines replaced by alanines) and inserted them into pGEX-KG vector as N-terminal GST fusion proteins. We also prepared rat Syt1 (amino acids 50-421 with four native cysteines C74, C75, C77 and C79 replaced by alanines and another C82 replaced by serine) and inserted it into pET-28b vector as C-terminal His-tagged proteins. DNA sequences were confirmed by the Iowa State University DNA Sequencing Facility.

Escherichia coli BL21 Rosetta (DE3) pLysS (Novagen) was used to express the recombinant GST fusion proteins VAMP2, SNAP-25, syntaxin 1A and VpS. The cells were grown in LB medium at 37°C with ampicillin (100 μg/mL) until the ~0.6–0.8 absorbance at 600 nm. The cells were further grown for another 12 h at 16°C after induction with the addition of Isopropyl β-D-1-thiogalactopyranoside (0.3 mM final concentration). The cell pellets were then harvested via centrifugation at 6,000 × g for 10 min and resuspended in PBS at pH 7.4 containing 2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 2 mM EDTA, and 2 mM dithiothreitol. For the transmembrane proteins, in addition to the resuspension buffer we added 0.5% Triton X-100. The cells were lysed with sonication immersed in an ice bath. The lysed cells were centrifuged at 15,000 × g for 20 min and the supernatant was added onto columns with glutathione-agarose beads for 4°C for 2 h. The unbound proteins were thoroughly washed off and thrombin (Sigma-Aldrich) was added to cleave off the GST fusion proteins at 4°C for 16 h. We note that the thrombin cleavage buffer was 50 mM Tris-HCl, 150 mM NaCl, and pH 8.0 and additional 1% n-octyl glucoside was added for transmembrane proteins.

The C-terminal His-tagged Syt1 (amino acids 51–421) was also expressed in E. coli BL21 Rosetta (DE3) pLysS and was purified identically to the aforementioned protocol with the exception of using Ni-NTA beads (Qiagen) instead of GST beads. Also, the elution buffer was prepared with 25 mM HEPES, 400 mM KCl, 500 mM imidazole, and 0.8% OG. All purified proteins were examined with 15% SDS-PAGE and the purity was at least 90%.

We used the following lipid molecules to make v-vesicles: 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Invitrogen), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD, Invitrogen), and cholesterol. All lipids in this study were obtained from Avanti Polar Lipids if not otherwise specified.

For v-vesicles with just DiI, we first mixed lipids with molar ratios of 5:54:40:1 (DOPS: POPC: Cholesterol: DiI) and dried it into lipid film using gentle nitrogen gas in a glass tube. The lipid film was stored overnight in a desiccator under house vacuum. The lipid film was resuspended with buffer containing 25 mM HEPES 100 mM KCL pH 7.4. We made liposomes with 10 flash freeze-thaw cycles and large unilamellar vesicles (~100 nm in diameter) were prepared by extrusion through the polycarbonate filter (Avanti Polar lipids). VAMP2 and Syt1 was mixed with the vesicles such that the lipid to protein ratio was 200:1. The liposome/protein mixture was diluted by adding three times the volume of the protein lipid mixture and then dialyzed in 2 L dialysis buffer at 4°C overnight.

We also prepared v-vesicles with both lipid-reporter DiD and the content-reporter sulforhodamine B (SRB). These vesicles were prepared identical to the aforementioned v-vesicles with the exception of substituting DiD for DiI. Also, the SRB concentration was kept constant (20 mM) after resuspension of the dried lipid film. Free SRB was removed with the PD-10 desalting column (GE healthcare) after overnight dialysis.

The lipid film for the t-SBL was prepared identically to the case of v-vesicles with the exception of lipid composition. We used the following lipids: DOPS, POPC, phosphatidylinositol-4,5-bisphosphate (PIP2, from porcine brain), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2000). We mixed the lipids using the following molar ratio of 15:78:2:5 (DOPS: POPC: PIP2: PEG2000). The lipid film was dried and stored in a desiccator overnight. The lipid film was resuspended with buffer containing 25 mM HEPES 100 mM KCL 1% OG pH 7.4. We mixed the resuspended lipid with t-binary complex which was prepared by mixing syntaxin 1A and SNAP-25 (1:1.5 molar ratio) for 30 min at room temperature. The lipid to t-binary complex ratio was 2000:1. Then the liposome/protein mixture was diluted by adding three times the volume of the protein lipid mixture using 25 mM HEPES 100 mM KCL pH 7.4 and then dialyzed overnight at 4°C in a 2 L beaker.

We subjected the imaging quartz slide to piranha cleaning, boiling mixture of sulfuric and hydrogen peroxide, for 20 min. The slides were rinsed with de-ionized water and placed in a sonicator for 20 min to rid of any residual acid. We quickly assembled the slides and gently injected the t-proteoliposomes into the flow chamber. We let the t-SBL form for 2 h at room temperature, washed out excess liposomes and let the samples settle for 2 h.

Once the t-SBL was prepared, we mounted the imaging quartz slide on the total internal reflection (TIR) fluorescence microscope. The TIR angle of the laser (532 nm) was properly adjusted and then we initiated the real-time movie acquisition with an imaging area of ~55 × ~110 μm and 20 ms time resolution for 90 s. We then gently injected the v-vesicles (prepared according to the experiment) into the flow chamber until the buffer within the flow chamber was exchanged with the v-vesicle solution. The injection pump was promptly stopped in order to prevent any unwanted fusion events due to the flow effect. Once the 90 s movie was obtained, we analyzed a 60 s segment of the movie starting after the injection pump was stopped with our custom built program.

With our custom-built analysis program we monitored the fluorescent DiI signals (DiD and SRB signals for the experiments in Figures 2E,F) from the v-vesicles in order to determine docking and fusion. First, we scrolled through the entire 60 s movie clip and handpicked every immobilized spot which represents docked v-vesicles. By selecting the immobilized spot with our program we were able to record the x- and y-coordinates as well as the time at which the vesicle docked on to the t-SBL. Using the x- and y- coordinates, the selected spots were visually marked in order to avoid any recounting or omission.

We then re-examined the movie clip and looked for the characteristic two dimensional diffusion of the fluorescent lipids. The docking-to-fusion delay time was determined by counting the number of frames between immobilization and two dimensional expansion of the fluorescence signal. Thus, the fast fusion events (sub 20 ms fusion delay time) displayed expansion of the fluorescence signal in the absence of a distinct immobilization step. From the analysis process we were able to monitor individual v-vesicles and obtained the docking rate, the fusion percentage, and the fusion delay time between docking and fusion within a movie clip. This analysis process was executed on each movie recording. Three independent recordings were analyzed to obtain the statistics and the statistical significance for each data set.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.