(A and B) Probability of fusion vs time upon 500 μM Ca2+-injection and spontaneous fusion, both in the presence of full-length synaptotagmin-1 and neuronal SNAREs, but with and without 2 μM complexin-1 (Cpx) as indicated. (A) The histograms of Ca2+-triggered fusion events (1 s time bin) were normalized by the number of associated SV vesicles and fit to an exponential decay function. The decay rate with and without complexin-1 is 0.43 s−1 and 0.11 s−1 respectively. (B) The histograms of spontaneous fusion events were normalized by the number of associated SV vesicles. (C) Domain structure of complexin-1. To probe the roles of the four domains of complexin-1 we used the truncation mutant Cpx26–134 for the N-terminal domain, the truncation mutant Cpx41–134 for the accessory α-helical domain, the Cpx4M mutant for the SNARE binding domain, and the truncation mutant Cpx1–88 for the C-terminal domain. We chose these particular truncations/mutations based on studies of cortical neuronal cultures (Maximov et al., 2009; Kaeser-Woo et al., 2012). (D–G) The bar graphs show the effects of complexin-1 and its mutants on SV-PM vesicle association (D), the number of spontaneous fusion events over the 1-min observation period divided by the number of associated SV vesicles (E), the decay rate of the histogram upon Ca2+-injection (F), and the amplitude of the first 1-sec time bin upon Ca2+-injection (G). Each value in panel G was normalized by the respective number of fusion events after Ca2+-injection. Moreover, only those single vesicle–vesicle fluorescence intensity traces that exhibited a distinct event during the association period were analyzed for fusion events during the subsequent observation periods. Error bars in D, E, G are standard deviations for 6–13 independent repeat experiments. Error bars in F are error estimates computed from the covariance matrix upon fitting the corresponding histograms with a single exponential decay function using a Levenberg-Marquardt technique.