Figure 5.

Single vesicle–vesicle content/lipid-mixing assays. (A) Setup of the single vesicle–vesicle assay that simultaneously monitors content and lipid mixing and that allows studies of Ca²⁺ triggered fusion (Diao et al., 2012; Kyoung et al., 2011, 2013; Lai et al., 2014). “Acceptor” vesicles containing syntaxin (red) and SNAP-25 (green) are immobilized via biotinylated lipids (yellow) to a glass surface that has been passivated with biotin-PEG and coated with neutravidin (dark purple). “Donor” vesicles containing high concentrations (and thus self-quenched) of sulforhodamine B in the lumen and DiD lipids are reconstituted with synaptobrevin (blue) and synaptogamin (light purple) molecules. The donor vesicles are incubated with the immobilized acceptor vesicles. Fusion is initiated by flowing buffer containing Ca²⁺ into the imaging chamber. (B) Docking, hemifusion, and complete fusion are monitored by measuring the fluorescence dequenching of the lipid and content dyes that result from dilution. (C) Representative fluorescence intensity time traces that result from fusion of a single donor vesicle to a single acceptor vesicle illustrating the detection of the individual fusion states shown in panel B. (D) A single vesicle content mixing assay with a large content probe, dye-labeled DNA hairpins (Diao et al., 2010). Upon fusion unlabeled DNA molecules complimentary to the FRET labeled hairpin bind and destabilize the hairpin leading to a decrease in FRET.