FIGURE 2.

Schematic diagram and representative traces of the single vesicle docking and lipid mixing assay. A, schematic diagram of the single vesicle docking and lipid mixing assay. t-vesicles reconstituted with SNAP-25/syntaxin-1a were immobilized on the surface of the flow cell. v-vesicles reconstituted with VAMP2 or VAMP2 together with Syt1 (VAMP2/Syt1 = 1:1) were flowed into the flow cell with or without 1 μm Munc18-1. B, representative imaging areas of DiI (green-framed, left) and DiD (red-framed, right) emission under 532-nm excitation for DiI-labeled t-vesicles. Each green circle on the left represents emission from an immobilized t-vesicle, and the fluorescence emission was analyzed as shown on the right. Three representative spots were chosen as shown in yellow circles and labeled a, b, and c. C, the trace of spot a represents immobilized t-vesicle only. D, the trace of spot b represents docking of v-vesicle onto the immobilized t-vesicle without significant lipid mixing. E, the trace of spot c represents docking of the v-vesicle onto the immobilized t-vesicle with significant lipid mixing. Green bar, 532-nm excitation for FRET; red bar, 635-nm excitation for checking the presence of docked vesicles.