Vesicle Preparation.

Vesicles were prepared by drying the lipids (Avanti Polar Lipids), which were dissolved in chloroform, under nitrogen in a glass tube; submitting them to vacuum for more than 2 h; and finally resuspending them in a buffer solution (25 mM Hepes, 100 mM KCl, pH 7.4) to have a total lipid concentration of 18 mM. The solution was then frozen and thawed 5 times before being extruded 21 times through 50-nm polycarbonate filters (Avanti Polar Lipids). The final diameter of the vesicles was 63 ± 16 nm (Fig. S2). Vesicles were conserved overnight at 4 °C under argon for stabilization of the membranes before the experiment. The nonfluorescent vesicles contained only DOPC or POPC. The fluorescent vesicles contained DOPC (or POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-PE) in the molar ratio of 97:1.5:1.5.

Size histogram of the POPC vesicle after overnight incubation at 4 °C. The sizes have been determined by the measurement plugin of Image J of six micrographs (total 175 vesicles). We obtained a size of 63 ± 16 nm, error being the SD. This distribution ranges between 30 nm and 100 nm in diameter, which suggests that curvature effect may impact our measurements. Hence, it is critical to ensure the fusion reaction we observe is not only due to a subpopulation of the vesicles with specific diameters. This can be achieved by analyzing the slope variation in the fusion experiment. On average, we found that the slope decreases by ∼10% of the initial slope (at t = 0) after ∼5% of the vesicles have fused (close to the end of the experiment in the fastest cases). The slope is proportional to the number of fluorescent vesicles accessible for fusion. Hence, a 10% decrease of the slope after 5% of the vesicles have fused means that 50% are involved in the fusion process. Even though these percentages are not precise because the slope variation is difficult to accurately determine, they mean that a significant fraction (tens of percent) of the vesicles are involved in the fusion process. This result shows that the fusion-potent vesicles can not only correspond to a tiny subpopulation. Thus, it can be considered here that the measured activation energy is actually that of vesicles having 60 nm diameter. Because of the low percentage of fused vesicles (less than 5%; Fig. 1B legend), no significant change in the size distribution can be observed at the end of the experiment.