Fig. 2: Contribution of Syt7 to the establishment of the fusion clamp.

The involvement of Syt7 in the fusion clamp was evaluated using vesicles containing low-copy VAMP2 (~15 copies) and a non-clamping Syt1 mutant, Syt1^Q (carrying R281A,E295A,Y338W,R398A,R399A mutations that disrupt the Syt1-SNARE primary interface) in the absence of CPX. a The time between docking and spontaneous fusion was measured for each docked vesicle and the ‘docking-to-fusion’ latency time was cumulatively expressed as the survival percentage. This ‘survival analysis’ provided the measure of the strength of the fusion clamp. In the absence of Syt7 (gray), the majority of the docked VAMP2^low/Syt1^Q vesicles proceed to fuse spontaneously with a half-time of ~1 s. The inclusion of Syt7 in the bilayer resulted in stably docked vesicles in an immobile state, with clamping efficiency correlating with the amount of Syt7 included. Approximately 40% of vesicles were clamped under low Syt7 concentration (1:800, light blue) and this increased to ~90% under high Syt7 concentration (1:200, dark blue). b, c Syt7 clamped VAMP2^low/Syt1^Q vesicles remained fusion competent and could be triggered to fuse by the addition of Ca²⁺ (100 µM) and the observed fusion was desynchronized to the Ca²⁺ signal. In the absence of Syt7, a very small percent of the docked VAMP2^low/Syt1^Q vesicles underwent fusion which precluded meaningful kinetic analysis. Data (mean ± standard deviation) are from 3 independent experiments (N = 3) for each condition (~40–50 vesicles per experiment). One-way ANOVA revealed statistically significant difference (***p < 0.001) in % Ca²⁺-evoked fusion of docked vesicles in the presence of Syt7 as compared to the condition without Syt7 in the bilayer. The data from ANOVA and Tukey’s HSD post-hoc comparing specific groups are shown in Supplementary Table 2. The source data is provided as a ‘Source Data’ file.