In vitro assays to measure SNARE-mediated vesicle fusion

Membrane fusion is fundamental for a broad variety of physiological processes, such as synaptic transmission, fertilization, and viral entry. Intracellular fusion along the secretory and endocytic pathway is mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. When recombinant v- and t-SNAREs are reconstituted into distinct liposome populations, membrane fusion can be monitored by either lipid or content mixing. The in vitro assays use fluorescence dequenching to measure vesicle fusion. The lipid-mixing assay is based on fluorescence resonance energy transfer between the fluorophores 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) and rhodamine, which are covalently coupled to lipids. Fusion of labeled v-SNARE liposomes with unlabeled t-SNARE liposomes increases the distance between NBD and rhodamine, increasing the NBD fluorescence. In the content-mixing assay, the water-soluble fluorophore 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) (pyranine) and its quencher p-Xylene-bis-pyridinium bromide (DPX) are incorporated into v-SNARE vesicles. The fusion of labeled v-SNARE vesicles with unlabeled t-SNARE vesicles dilutes the quencher and thus increases HPTS fluorescence. By controlling the lipid and protein composition, these assays provide important tools to detect fusion intermediates (e.g., hemifusion), and to elucidate the molecular mechanisms that regulate membrane fusion.