Fig. 3: The trimer and lateral interface variants impair vesicle capture and fusion in vitro.

A Schematic of the single-vesicle docking/fusion assay using confocal microscopy. To reconstitute Munc13/Munc18-dependent vesicle priming and fusion, palmitoylated SNAP25, 1:1 Munc18-1/Syntaxin 1 complexin, and 1,2-hexanoyl-sn-glycerol (DHG) were incorporated into the suspended lipid bilayer (labeled with 1% ATTO465-DOPE). VAMP2 and Synaptotagmin 1 (Syt1) were incorporated into small-unilamellar vesicles (V) and labeled with 2% Atto647N-DOPE to visualize vesicles near the suspended bilayer. Munc13C and Complexin 1 (Cpx1) were added in solution. B Single vesicles were tracked by confocal microscopy and classified as docked or undocked based on their lateral mobility. Docked vesicles sometimes fused in the absence of Ca²⁺ (spontaneous fusion), whereas most vesicles fused upon addition of 100 µM Ca²⁺ (Ca²⁺-triggered fusion). C Munc13C variants harboring trimer interface mutations (trimer 2xR, 2xD, and 3xD, with N = 5, 4, and 4 replicates, respectively) were compared to wild-type Munc13C (WT, N = 12 replicates) by quantifying the docked vesicle fate in the absence of Ca²⁺ (D), and in response to 100 µM Ca²⁺ (E). F Lateral hexamer Munc13C variants (lateral FI/NN, Quad, 3xNA, and 2xE, with N = 5, 5, 4, and 4 replicates, respectively) were compared to wild-type Munc13 for vesicle fate (G) and response to 100 µM Ca²⁺ (H). After initial docking, vesicle fates were subdivided into three categories: vesicles remained docked (purple), spontaneously fused (green), or undocked (orange) (D, G). All data presented as mean ± SEM and each biological replicate comprised ~30 vesicles. Data were compared using one-way ANOVA and Tukey–Kramer analysis for multiple comparisons. **p < 0.01, *p < 0.05 compared to wild type and exact p values are indicated along with the biological replicate numbers in (E, H). The p values in (D, G) are given in the Methods section.