A single-vesicle content mixing assay for SNARE-mediated membrane fusion

Abstract

The in vitro studies of membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have primarily been conducted by following the mixing of lipids. However, the formation of a fusion pore and its expansion has been difficult to detect directly because of the leakiness of proteoliposomes, vesicle aggregation and rupture that often complicate the interpretation of ensemble fusion experiments. Fusion pore expansion is an essential step for full-collapse fusion and for recycling of fusion mechanisms. Here, we demonstrate a method to detect the inter-vesicular mixing of large cargoes at the single-molecule and -vesicle level. The change in fluorescence resonance energy transfer signal when a DNA hairpin encapsulated in a surface-tethered vesicle encounters a complementary DNA strand from another vesicle indicates content mixing. We found that the yeast SNARE complex alone without any accessory proteins can expand the fusion pore large enough to transmit ~11 kDa cargoes.

Figure 1

Stable encapsulation of DNA probes inside SNARE protein reconstituted vesicles. (a) Histograms of FRET efficiency, E, (left) and average numbers (right) of molecules per imaging area (25 μm X 50 μm) before and after a DNase treatment of DNA probes encapsulated inside surface immobilized v-SNARE (protein:lipid = 1:200) vesicles. For histograms of FRET efficiency, Y-axis is normalized population, where we divided the distribution by the total number of vesicles measured and X-axis is FRET efficiency value. (b) Average count of molecules per imaging area before and after a DNase treatment of DNA probes immobilized outside vesicles. (Corresponding representative images for (a) and (b) are shown in Supplementary Fig. S2.) (c) Relative count of molecules per imaging area for surface immobilized v-vesicle encapsulating DNA probes after incubating at 37°C for 60 min. The number of molecules at 0 min was set as 1. Error bars denote the standard error.

Figure 2

Single molecule content mixing assay for yeast SNARE-mediated fusion. (a) Schematics of the assay. Vesicles reconstituted with Snc2p proteins (v-vesicles) and encapsulating dual labeled DNA probes are immobilized on the surface of the flow cell. Vesicles reconstituted with Sso1pHT proteins (t-vesicles) and encapsulating poly-A DNA strands are flown in along with soluble Sec9c proteins, and the sample is incubated at 37 °C. (b) E distributions of v-vesicles before the reaction, and (c) after incubating for 30 min with t-vesicles and 1 μM Sec9c protein.

Figure 3

Various controls to establish the single vesicle content mixing assay. (a) DNA annealing take place inside vesicles. E distributions (left) and average numbers of molecules per imaging area (25 μm X 50 μm) (right) of fusion products before and after a DNase treatment. Error bars denote standard error. Corresponding representative images are shown in Supplementary Fig. S6. (b) E distribution of the fusion product with 1 μM Sec9c performed in the bulk solution and subsequently immobilized on the quartz surface for observation. DNase treatment was applied to both v- and t-vesicles to eliminate free DNA molecules before reaction. (c) E distribution after fusion reaction using non-complementary poly-T DNA inside t-vesicles.

Figure 4

Content mixing requires full SNARE complex with the wild type Sec9c. (a) E distributions of fusion products for different Sec9c concentrations. (b) E distributions of yeast SNARE-mediated fusion with different incubation times ranging from 0 min to 30 min with 1 μM Sec9c. (c) Pore expansion efficiency quantified by low FRET peak percentage (E = 0-0.4) for wild type and mutant Sec9c, L626P and L646P. 1 μM Sec9c and its mutants were used. Error bars denote the standard deviation of three independent experiments. 30 min incubation at 37 °C was applied for all experiments in (a) and (c).