Figure 3
Example of asymmetry in lipid and content mixing between V and T liposomes in the presence of C2AB or Syb49-93, but not in the presence of NSF, αSNAP, Munc18-1 and Munc13-1 C1C2BMUNC2C. Lipid mixing (A,C) between V1 and T1 liposomes, or between V2 and T2 liposomes, was measured from the fluorescence de-quenching of Marina Blue-labeled lipids present in the V1 or T2 liposomes, and content mixing (B,D) was monitored from the development of FRET between PhycoE-Biotin trapped in the T1 or V2 liposomes and Cy5-SA trapped in the V1 or T2 liposomes. In (A,B), the assays were performed in the absence (gray and blue traces) or presence (black and red traces) of synaptotagmin-1 C2AB. In (C,D), the assays were performed in the presence of Syb49-93 (gray and blue traces) or of NSF, αSNAP, Munc18-1 (M18) and Munc13-1 C1C2BMUNC2C (M13) (black and red traces). All experiments were started in the presence of 100 μM EGTA and 5 mM streptavidin, and Ca2+ (600 μM) was added after 300 s. Note that four reactions were performed in parallel and recording was stopped for about 60 s during the addition of Ca2+ to the four reactions [represented by a break (//) in the x axis]. The concentration of Cy5-SA used to prepare the V1 and T2 liposomes in all these experiments was 4 μM, and 4 μM PhycoE-biotin was used to prepare the T1 and V2 liposomes. (E,F) Quantification of the lipid mixing (E) or content mixing (F) observed after 1000 s in reconstitution assays in the presence of Syb49-93, synaptotagmin-1 C2AB or NSF, αSNAP, Munc18-1 and Munc13-1 C1C2BMUNC2C. Bars represent averages of the normalized fluorescence intensities observed after 1000 s in experiments performed in triplicate. Error bars represent standard deviations. Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Holm-Sidak test (**P < 0.01; ***P < 0.001).
