 Solution

There is no evidence for substantial lipid mixing during the cold preincubation. The trace begins near its low-fluorescence baseline when warming starts; a substantial pre-existing dilution of the dyes would already have raised that baseline. Nevertheless, vesicle docking or trans-SNARE complex formation can accumulate while the later membrane-rearrangement step remains slow. Absence of dequenching does not mean that the vesicles have had no interaction.