Figure 2
Example of asymmetry in lipid and content mixing between V and T liposomes in the presence of C2AB, but not in the presence of NSF, αSNAP, Munc18-1 and Munc13-1 C1C2BMUNC2C. (A) Diagrams illustrating the results expected from fusion reactions between T1 and V1 liposomes (above) or between T2 and V2 liposomes (below). The fluorescent probes used for lipid and content mixing are represented as in Fig. 1A. Upon fusion between a T and a V liposome, their bilayers merge and the ensuing dilution of the fluorescent lipids leads to de-quenching of the MB-PE fluorescence (represented in dark blue), while binding of Cy5-SA to PhycoE-Biotin due to content mixing leads to FRET between the probes and thus an increase in the emission fluorescence of Cy5-SA upon excitation of PhycoE-Biotin. (B–E) Lipid mixing (B,D) 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 (C,E) 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 (B,C), the assays were performed in the absence (gray and blue traces) or presence (black and red traces) of synaptotagmin-1 C2AB. In (D,E), the assays were performed in the presence of NSF, αSNAP, Munc18-1 (M18) and Munc13-1 C1C2BMUNC2C (M13). 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 8 μM, and 4 μM PhycoE-biotin was used to prepare the T1 and V2 liposomes.
