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Mechanism of SNARE-Mediated Membrane Fusion

Figure 1. Giant unilammellar proteoliposomes (GUVs) containing t-SNAREs probed with cognate fluorescent v-SNAREs.

For the 15 years or so, we have developed a series of reconstitution platforms to investigate the molecular mechanism of SNARE-mediated membrane fusion, and the regulation of this process by regulatory factors. In 1998, we established the central function of the SNARE proteins as fusogens when we reconstituted these proteins into small unilamellar vesicle (SUV) and measured lipid mixing between liposomes containing cognate SNARE proteins.

We have since demonstrated SNARE-mediated fusion by reconstituting SNAREs into giant unilamellar vesicle (GUV, shown in Figure 1) and supported bilayer (SBL, shown in Figure 2). Both of the systems provided flat, and therefore, more physiologically relevant membrane environment on the t-SNARE side. In the SUV-SBL fusion system, we have measured single fusion event in millisecond time-scales.

Figure 2. 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. (B) Schematic view of the setup. (C) Photograph of two flow channels in parallel, in the same PDMS block.

Recently, we developed nanodisc reconstitution platform (shown in Figure 4), an ideal model for the studies of fusion pore. The small amount of lipid in the Nanodiscs is sufficient to allow pores to open but not expand beyond their nascent, physiologically relevant state for neurotransmitter release.

This allowed us to identify that although only one SNARE per nanodisc is required for maximum rates of bilayer fusion, efficient release of content on the physiologically relevant time scale of synaptic transmission apparently requires three or more SNARE complexes.

Figure 3. Cartoon showing the Nanodiscs containing v-SNAREs (VAMP2). The nanodisc is a small piece of lipid bilayer wrapped by two MSPs (blue). VAMP2 (green) can insert into a nanodisc to form a v-disc.

Besides demonstrating the core function of SNAREs during membrane fusion, we also applied various biophysical approaches to evaluate the energy landscape of the fusion process in a quantitative way. As measured by Surface Forces Apparatus (SFA) (Figure 4), partial zippering of the SNARE core domain generates about 35 kBT energy, corresponding closely to the energy needed to fuse outer but not inner leaflets (hemi-fusion) of outer lipid bilayer.

Figure 4. Interaction energy–versus–distance profile of cognate SNAREs in apposing bilayers (squares, approach; circles, separation). Cytoplasmic domains of syntaxin-1A and SNAP-25 (red, with the Habc domain of syntaxin-1A in purple) and VAMP-2 (blue) proteins were chemically anchored to maleimide-containing (green) bilayers via the single cysteine residue introduced at their C-terminal ends. The force, F, normalized to the radius of curvature of the surfaces, R, is proportional to the corresponding interaction free energy per unit area, E, between two equivalent planar surfaces.

Currently, we are developing and applying novel tools, using DNA nanotechnology, microfluidic devices and single-molecule optical tweezers, to further elucidate the molecular mechanism and the energy evolved in the SNARE-mediate membrane fusion.