Fig. 3: Successful exo-endocytosis coupling requires two or more vesicles to fuse simultaneously or sequentially.

a Phase diagram showing the relationship between the number fusing vesicles and dimension of the active zone for successful formation of endocytic pits (dark blue). b Plot showing curvature of the resulting endocytic pits as a function of the number of fusing vesicles. The red dotted line indicated the threshold for endocytic proteins to recognize the curvature for further maturation of pits into vesicles. c Example micrographs showing wild-type synapses unstimulated (left) or stimulated with electric field for 1 ms and frozen 100 ms later (right). The external calcium concentration is 1.2 mM. Black arrow: endocytic pit. d Number of endocytic pits at 100 ms after stimulation. Mean and 95% confidential interval are shown. ****p < 0.0001. Two-sided Mann-Whitney U test. See Supplementary Table 2 for detailed statistical analysis. e Example micrographs showing wild-type and Doc2α knockout synapses unstimulated or stimulated with electric field for 1 ms and frozen 100 ms later. Black arrow: endocytic pit. f Number of endocytic pits at 100 ms after stimulation. **p < 0.01. Error bars are SEM. p-values are only shown for direct comparison between unstimulated and stimulated neurons of the same genotype. Kruskal-Wallis test, with Dunn’s multiple comparisons test. See Supplementary Table 2 for the detailed numbers for each sample. g Example micrographs showing DMSO- or EGTA-AM-treated synapses unstimulated or stimulated with electric field for 1 ms and frozen 100 ms later. Black arrow: endocytic pit. h Number of endocytic pits at 100 ms after stimulation. **p < 0.01. Error bars are SEM. p-values are only shown for direct comparison between unstimulated and stimulated neurons of the same genotype. Kruskal-Wallis test, with Dunn’s multiple comparisons test. See Supplementary Table 2 for the detailed numbers for each sample. i, j Snapshots from simulations, showing the evolution of membrane curvature within the active zone when the second vesicle is fused when the depth of first vesicle is at 10 nm (i) and 1 nm (j). k Snapshots from simulations, showing the evolution of membrane curvature within the active zone when the second vesicle is fused much later, after the first vesicle completely flattens out by relaxing the membrane area conservation. The membrane area conservation is resumed for the second fused vesicle. l Simulation result showing the mechanical energy evolution of the system over simulation timesteps, corresponding to (i). The energy is equilibrated within ~7 ms. Source data are provided as a Source Data file.