Fig. 3: Syt1 and Syt7 synergistically regulate Ca²⁺-evoked fusion via competitive binding to the same SNARE complex.

a The inclusion of the Ca²⁺ binding deficient Syt7 mutant (Syt7^DA) in the bilayer inhibited Ca²⁺ (100 µM) evoked fusion of Syt1^WT/VAMP2 vesicles in a dose-dependent manner, without altering the overall fusion kinetics. b Syt7^WT from the bilayer rescued the Ca²⁺-evoked fusion of Syt1^DA /VAMP2 vesicles but the fusion events were desynchronized to the Ca²⁺ signal. Complexin (2 µM) in solution was included in all experiments. c Quantitative pull-down and Western-blot analysis with Syt7 as ‘bait’ and CPX-SNARE complex as ‘prey’ demonstrate that Syt1 disrupts Syt7-SNARE interaction in a concentration-dependent manner. Data (mean ± standard deviation) are from 5 independent experiments (N = 5) for each condition (~40–50 vesicles per experiment) in (a) and (b) and from 4 independent experiments (N = 4) in (c). One-way ANOVA revealed statistically significant difference in % Ca²⁺-evoked fusion of docked vesicles in the presence of Syt7^DA (***p < 0.001) or Syt7^WT (***p < 0.001) as compared to condition without Syt7 in the bilayer. The data from ANOVA and Tukey’s HSD post-hoc comparing specific groups are shown in Supplementary Tables 3 and 4 respectively. The source data is provided as a ‘Source Data’ file. Figure 3c (top) created in BioRender. Krishnakumar, S. (2023) BioRender.com/p26b995.