Single Molecule Observation of Liposome-Bilayer Fusion Thermally Induced by Soluble N-Ethyl Maleimide Sensitive-Factor Attachment Protein Receptors (SNAREs)

Abstract

A single molecule fluorescence assay is presented for studying the mechanism of soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs)-mediated liposome fusion to supported lipid bilayers. The three neuronal SNAREs syntaxin-1A, synaptobrevin-II (VAMP), and SNAP-25A were expressed separately, and various dye-labeled combinations of the SNAREs were tested for their ability to dock liposomes and induce fusion. Syntaxin and synaptobrevin in opposing membranes were both necessary and sufficient to dock liposomes to supported bilayers and to induce thermally activated fusion. As little as one SNARE interaction was sufficient for liposome docking. Fusion of docked liposomes with the supported bilayer was monitored by the dequenching of soluble fluorophores entrapped within the liposomes. Fusion was stimulated by illumination with laser light, and the fusion probability was enhanced by raising the ambient temperature from 22 to 37°C, suggesting a thermally activated process. Surprisingly, SNAP-25 had little effect on docking efficiency or the probability of thermally induced fusion. Interprotein fluorescence resonance energy transfer experiments suggest the presence of other conformational states of the syntaxin•synaptobrevin interaction in addition to those observed in the crystal structure of the SNARE complex. Furthermore, although SNARE complexes involved in liposome docking preferentially assemble into a parallel configuration, both parallel and antiparallel configurations were observed.