Figure 3. Influence of Munc18-1, Munc13-1 C₁C₂BMUNC₂C, complexin-1 and synaptotagmin-1 on trans-SNARE complex assembly-disassembly in the presence of NSF-αSNAP.

(A,B) Kinetic assays monitoring trans-SNARE complex assembly between V- and T-liposomes (1:4 ratio) in the presence of NSF-αSNAP from the decrease in the donor fluorescence emission intensity. The experiments were performed in the absence of other proteins (T + V) or in the presence of different combinations of Munc18-1 (M18), Munc13-1 C₁C₂BMUNC₂C (M13), complexin-1 (Cpx), synaptotagmin-1 C₂AB and Syb49-93, as indicated by the colors. Experiments were started in 100 μM EGTA and Ca²⁺ (600 μM) was added after 750 s. (C) Analogous kinetic assays performed in the presence of Munc18-1, Munc13-1 C₁C₂BMUNC₂C, NSF-αSNAP and 100 μM EGTA, but adding 240 μM Ca²⁺ at 2 min to stimulate trans-SNARE complex assembly and adding 500 μM EGTA at different times to chelate the Ca²⁺ and interrogate whether there is trans-SNARE complex disassembly. An experiment that was also started in 100 μM EGTA but without addition of Ca²⁺ or EGTA at later times (gray trace) is shown for comparison. (D) Experiments analogous to those of (C), with addition of 240 μM Ca²⁺ at 2 min and 500 μM EGTA at 17 min, performed in the absence or presence of complexin-1 and/or synaptotagmin-1 C₂AB. (E) Kinetic assays monitoring trans-SNARE complex assembly between VSyt1- and T-liposomes (1:4 ratio) in the presence of NSF-αSNAP and different combinations of Munc18-1, Munc13-1 C₁C₂BMUNC₂C, complexin-1 and Syb49-93, as indicated by the colors. Experiments were started in 100 μM EGTA and Ca²⁺ (600 μM) was added after 750 s. (F) Kinetic assays analogous to those of (E) performed in the presence of Munc18-1, Munc13-1 C₁C₂BMUNC₂C, NSF-αSNAP and 100 μM EGTA, but adding 240 μM Ca²⁺ at 2 min to stimulate trans-SNARE complex assembly and adding 500 μM EGTA at different times to chelate the Ca²⁺ and interrogate whether there is trans-SNARE complex disassembly. An experiment that was also started in 100 μM EGTA but without addition of Ca²⁺ or EGTA at later times (gray trace) is shown for comparison. The light blue trace shows an additional experiment started in 100 μM EGTA in the presence of complexin-1, with addition of 240 μM Ca²⁺ at 2 min and 500 μM EGTA at 17 min. All experiments were performed in the presence of Mg²⁺ and ATP. For all traces shown in (A–F), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents.

Figure 3—figure supplement 1. Complexin-1 increases the efficiency of Ca²⁺-independent trans-SNARE complex assembly between VSyt1- and T-liposomes in the presence of NSF-αSNAP.

The traces show kinetic assays monitoring trans-SNARE complex assembly between VSyt1- and T-liposomes (1:4 ratio) in the presence of Munc18-1, Munc13-1 C₁C₂BMUNC₂C, NSF-αSNAP and 100 μM EGTA, without (red trace) or with complexin-1 (Cpx) (blue trace). The experiments are analogous to those of Figure 3E (red and blue traces), but without addition of Ca²⁺.

Figure 3—figure supplement 2. Ca²⁺-dependent fusion between VSyt1- and T-liposomes.

(A,B) Lipid mixing (A) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) 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. The assays were performed in the presence of NSF-αSNAP, and Munc18-1 (M18), Munc13-1 C₁C₂BMUNC₂C (M13) or both. Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca²⁺ (600 μM) was added at 300 s.