Fig. 4
From: Munc18-1 is crucial to overcome the inhibition of synaptic vesicle fusion by αSNAP

NSF rescues the inhibition of fusion between V- and T-liposomes caused by αSNAP. a, b Lipid mixing (a) between V- and T-liposomes 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 T-liposomes upon liposome fusion. Assays were performed in the presence of 0.8 µM NSF and 2 µM αSNAP without or with 1 µM Munc18-1 (M18) and/or 0.5 µM M13C1C2BMUNC2C (M13) as indicated by the color-coded labels. All traces were acquired with NSF in the presence of ATP, except the orange traces, where ATP was replaced by ATPγS. Experiments were started in the presence of 100 μM EGTA, 1 µM excess of SNAP-25, and 5 μM streptavidin, and Ca2+ (600 μM) was then added at 300 s. c, d Analogous assays where reagents were added sequentially. Experiments were started in the presence (black trace) or absence (green trace) of 1 µM Munc18-1; 0.5 µM M13C1C2BMUNC2C, 2 µM αSNAP, 600 µM Ca2+, and 0.8 µM NSF were added at the indicated time points. Note that, in the presence of Munc18-1, M13C1C2BMUNC2C strongly stimulated fusion, αSNAP immediately stopped fusion, Ca2+ induced a small amount of fusion that varied in different experiments, and NSF sharply restored fusion. No such recovery was observed in the absence of Munc18-1. Source data are provided as a Source Data file