Figure 3.

Typical results obtained in our reconstitution experiments. Lipid mixing (a) between VSyt1- and T-liposomes is monitored from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing (b) is monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the VSyt1-liposomes. The assays were performed in the presence of NSF-αSNAP without other additional proteins (T+VSyt1) or in the presence of Munc18–1 (M18) and/or Munc13–1 C₁C₂BMUNC₂C (M13) as indicated. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca²⁺ (600 μM) was added after 300 s. The data were normalized as described in step 25. (c,d) Raw data (i.e. without normalization) obtained in analogous experiments performed with T- and VSyt1-liposomes in the presence of NSF-αSNAP, Munc18–1 and Munc13–1 C₁C₂BMUNC₂C under the same conditions as those used in (a,b) to illustrate the typical variability observed in analogous experiments performed with the same liposome preparations in the same day (day × repeats 1 and 2) or in different days (compare data from day y with data from day x).