Figure 8. The stimulatory effects of the individual open syntaxin-1 and P335A Munc18-1 mutations on Ca²⁺-independent liposome fusion cancel each other in the double mutant.

(A, B) Lipid mixing (A) between V-liposomes and S-liposomes containing open syntaxin-1 mutant was monitored from the fluorescence de-quenching of Marina Blue lipids, and content mixing (B) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the S-liposomes upon liposome fusion. Assays were performed in the presence of NSF, αSNAP, Munc13-1C and WT, P335A or D326K Munc18-1 as indicated by the color code. Experiments were started in the presence of 100 mM EGTA and 5 mM streptavidin, and Ca²⁺ (600 mM) was added at 300s.

(C, D) Quantification of the fusion assays shown in panels A, B. Bars represent averages of the normalized fluorescence intensities observed for lipid mixing (C) or content mixing (D) at 300 s, performed in triplicates. Error bars represent standard deviations.