A fast, single-vesicle fusion assay mimics physiological SNARE requirements

Abstract

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.

Fig. 1.

The SUV-SBL fusion assay and detection of single fusion events. (A) Schematic of a v-SUV reconstituted with Syb and a t-SBL reconstituted with the t-SNARE complex Syx·SNAP25. The v-SUV is doped with 2 mol % LR-PE (red) to detect single-vesicle docking and fusion events. The SBL is labeled with 2 mol % NBD-PE (green) to check its homogeneity and fluidity before every fusion test with v-SUVs (Materials and Methods). (B) Schematic view of the setup. A PDMS block containing microfabricated grooves is attached to a glass coverslip to form the microfluidic channels. A solution of SUVs (or buffer) is aspirated into the channel using a syringe pump. (C) Photograph of two flow channels in parallel, in the same PDMS block. (D) A single fusion event. The vesicle docked in frame 2 (marked D) remained docked for two more frames, then fused in frame 5 (marked F). The signature of fusion is the radial spread of fluorescence as LR-PE is transferred from the v-SUV into the t-SBL. Frames are 100 ms apart. Each square is 40 × 40 pixels (11 μm by 11 μm). (E) Two-dimensional Gaussian fits to frames 4–7 in D. (F) Variance, , peak amplitude (ampl.), and total integrated intensity (int.) versus time for Gaussian fits shown in E. Time 0 corresponds to frame 5 in D. Linear fit to the postfusion portion of (t) (red) was used to extract the diffusion coefficient of LR-PE in the SBL (see text).

Fig. 2.

Fusion rates. (A) Cumulative number of fusions as a function of time for t-SBLs (t-L:P = 10K) and v-SUVs (4.9 pM, v-L:P = 120) shown in blue for various individual acquisitions. Data for identically prepared v-SUVs over protein-free SBLs are shown in red. (B) Mean fusion rates normalized by detection area and SUV concentration for various conditions as indicated. TeNT, 50 nM tetanus neurotoxin; Syb_1–92, cytoplasmic domain of Syb. Numbers of experiments and total numbers of detected fusion events are indicated. All data were obtained at 27 °C.

Fig. 3.

Delays between individual docking and fusion events and SNAP25 dependence of the overall fusion rate. (A) Distribution of delay times, normalized to integrate to unity (t-LP = 10K, v-LP = 150, 32 °C, 175 delays from 8 acquisitions; bin width = 100 ms). (B) The same delays as in A, presented as a survivor function (SI Text). Inset shows the full span of the distribution, including a small fraction of delays (<15–20%) which occur on >1 s timescales. (C) Effect of the limited time resolution on the sampling of the true delays. The mean delay for fast fusions (delays s) versus acquisition period T_acq. The mean delay extrapolated to T_acq = 0 ms is ms. (D) Comparison of fusion rates between SBLs reconstituted with Syx·SNAP25 (190 fusions, 7 acquisitions, t-LP = 10K) and with Syx alone (60 fusions in 19 acquisitions, t-LP = 10K). SBLs were formed from t-SUVs reconstituted side by side. Identical v-SUV preparations were used. Omission of SNAP25 resulted in a 12-fold reduction in the normalized fusion rate (v-LP = 200, T = 30–32 °C).

Fig. 4.

The normalized fusion rate, , as a function of the number of Sybs per v-SUV, , plotted on a semilogarithmic scale. Note the precipitous drop in spanning approximately two orders of magnitude as is decreased from 20 to 10. (Inset) Blow-up of small region, linear scale (72 experiments, 1609 fusion events total, 32 °C). At least 6 experiments per data point for , 2–14 experiments per data point for . Error bars are ±SEM.