Generation and localization of Syt 1 Ca²⁺-binding mutations. (A) Schematic diagrams of essential residues that coordinate Ca²⁺ binding to the Syt 1 C2A and C2B domain, rendered according to Ferandez et al. (18). The key Ca²⁺ binding D3 and D4 aspartate residues (highlighted in blue) were neutralized to asparagines in the C2A or C2B domain. (B) Immunostaining of hatching stage (21–24 h after fertilization) embryonic NMJs with anti-HRP (green), a neuronal membrane marker to highlight presynaptic terminals, and anti-Syt 1 (magenta). The merged signal is displayed in the bottom image. WT, as well as C2A and C2B mutant (indicated with D > N) proteins from transgenes, show similar levels and distribution of Syt 1 compared with endogenous protein in WT embryos (Left). Null mutants lack anti-Syt 1 immunoreactivity. (Scale bar, 5 μm.)

Altered release properties in Syt 1 C2A and C2B Ca²⁺ binding mutants. (A) Representative traces of synaptic currents at muscle fiber 6 of hatching stage embryos (21 h after fertilization) evoked by motor nerve stimulation from the genotypes as indicated in Fig. 1. Two representative traces are shown for each genotype. Stimulation artifacts signal the onset of nerve stimulation, indicated by arrows. Arrowheads indicate asynchronous release in null mutants and null animals rescued with C2A mutant Syt 1. (B) Synaptic charge transfer following simulation calculated from the synaptic current. Most synchronous release occurs within 10 ms of stimulation (9). The asynchronous component has an exponential distribution with a time constant of approximately 100 ms, with 95% of asynchronous release occurring within 300 msec after stimulation. We thus quantified the asynchronous component as release events occurring between 10 and 300 ms. (C) Expanded bar graphs quantifying the asynchronous release components for each genotype. (D) Total synaptic charge transfer, including the asynchronous and synchronous components, for each genotype. For C and D, the four groups were analyzed with the Kruskal-Wallis test using a one-way ANOVA by ranks, and significant differences between the groups were found (P < 0.01). **P < 0.01 by Dunn post-hoc multiple comparison test; n = 50 in each genotype. Error bars are SEM. In B–D, WT refers to null mutants rescued with WT Syt 1, and “C2A D > N” and “C2B D > N” refer to null mutants rescued with Ca²⁺ binding mutants described in Fig. 1. The physiological saline solution contained 4 mM Ca²⁺ to highlight the asynchronous release component (9).

Latency analyses of nerve-evoked synaptic currents. (A) Latencies of synaptic currents within 1 s following nerve stimulation were measured and plotted for each genotype as indicated in Fig. 1. All latencies were counted for each 10-ms bin and presented as the number of events per stimulation in each bin. Examples containing spontaneous bursting activity originating from the CNS were excluded from the analysis. A small number of events triggered by endogenous action potentials, as well as spontaneous miniature release, were observed as random fusion events unrelated to stimulation in the background in each panel. The physiological saline solution contained 4 mM Ca²⁺ to clearly demonstrate asynchronous release component (9). Bottom: Data shown with enlarged y axis to highlight asynchronous release component. Note that the C2A mutant rescue displays a clear asynchronous component, whereas the C2B mutant rescue does not. In null mutants (Left), the first bin does not show increased height compared with other bins, indicating no contribution of a synchronous component with the exponential distribution of asynchronous release component (9). The numbers analyzed for each genotype were: Syt 1^−/− (n = 1,100), WT rescue (n = 471), C2A mutant rescue (n = 429), and C2B mutant rescue (n = 395). (B) Quantification of asynchronous release. We counted release events occurring between 10 and 300 ms per stimulation as the asynchronous component in the same experiments for A. The numbers analyzed for each genotype were the same as those of A. The four groups were analyzed with the Kruskal-Wallis test using a one-way ANOVA by ranks, and significant difference between the groups was found (P < 0.01). **P < 0.01 and *P < 0.05 by Dunn post hoc multiple comparison test. Error bars are SEM. (C) Quantification of spontaneous release. Synaptic currents were recorded between 0.5 s and 1 s after stimulation in the same experiments for A when asynchronous release has subsided. Release frequency per second was calculated per animal. Given occasional sparse action potential firings in motor neurons, these values are an overestimate of miniature release occurring at presynaptic terminals. Results were averaged between animals. The numbers of animal for each genotype were: Syt 1^−/− (n = 8), WT rescue (n = 6), C2A mutant rescue (n = 4), and C2B mutant rescue (n = 4). The four groups were analyzed with one-way ANOVA, and significant difference between the groups was found (P < 0.01). **P < 0.01 by Tukey post hoc multiple-comparison test between the WT rescue and each mutant rescue strain. In B and C, WT refers to null mutants rescued with WT Syt 1, and “C2A D > N” and “C2B D > N” refer to null mutants rescued with Ca²⁺ binding mutants described in Fig. 1.

Comparison of the Ca²⁺ dependency of release between WT and C2A mutant rescues. (A) The synchronous release component measured as charge transfer within 10 ms following nerve stimulation is plotted. Note that release at 0.2 mM and 0.5 mM Ca²⁺ is significantly larger in the C2A mutant rescue that in WT rescue. (B) The asynchronous release component measured as charge transfer from 10 ms to 300 ms following stimulation is plotted. Note that the C2A mutant rescue shows a dramatic enhancement of asynchronous release at all Ca²⁺ concentrations tested. **P < 0.01 by Mann-Whitney U test. Asynchronous release at 0.2 mM in WT rescue was too rarely observed to allow statistical analysis; n = 50 for each Ca²⁺ concentration in each genotype. Error bars are SEM. WT refers to null mutants rescued with WT Syt 1 and “C2A D > N” refers to null mutants rescued with the C2A Ca²⁺ binding mutant described in Fig. 1.

Hypertonic-induced release reveals restoration of the readily releasable pool in the C2A and C2B mutant rescues. (A) Sample traces of hypertonic-induced release for each genotype. Hypertonic stimulated release was achieved by puffing 500 mM sucrose solution onto the NMJ for 3 s (bars) in the absence of Ca²⁺. (B) Quantification of hypertonic stimulated release for null mutants and WT, C2A, and C2B mutant rescues. Miniature synaptic currents occurring within 5 s after the onset of stimulation were counted. The four groups were analyzed with the Kruskal-Wallis test using a one-way ANOVA by ranks, and significant difference between the groups was found (P < 0.01). **P < 0.01 by Dunn post-hoc multiple comparison test between the null and each rescue strain. The numbers of samples analyzed for each genotype were: Syt 1^−/− (n = 9), WT rescue (n = 18), C2A mutant rescue (n = 25), and C2B mutant rescue (n = 20). Error bars are SEM.