Fig. 5.

Triggered fusion between GABA SVs and PM vesicles. (a) Cartoon schematic of fusion triggering experiments in the hybrid in vitro system. (b) A representative example of Ca²⁺-triggered immediate fusion at 500 μM Ca²⁺ concentration, with the green trace representing SRB fluorescence intensity (PM vesicle) and the red trace representing Alexa-647 fluorescence intensity (Ca²⁺ entry). (c) A representative example of Ca²⁺-triggered delayed fusion at 500 μM Ca²⁺ concentration (same color scheme as in b). (d) Bar graph plotting the total number of observed fusion events for each Ca²⁺ and Mg²⁺ condition tested. See Supplementary Table 3 for details. (e–h) Frequency distribution histograms of fusion event times (all normalized to a Ca²⁺/Mg²⁺ injection time of 7 s). Each histogram was fit to a two-phase exponential decay function using GraphPad with the following R Squared values: 0.927 for 500 μM Ca²⁺, 0.811 for 250 μM Ca²⁺, 0.542 for 50 μM Ca²⁺, and 0.114 for 500 μM Mg²⁺. (i) Cumulative distribution plot of fusion event times for each condition. (j) Fusion synchronization for each Ca²⁺/Mg²⁺ condition calculated as the number of fusion events in the first second (5 acquisition frames) after triggering (statistical details are in Supplementary Table 3). Due to the small number of fusion events for the low Ca²⁺ concentration condition and the Mg²⁺ condition, determining the statistical significance on a per repeat-experiment basis is not reliable. We therefore used bootstrapping methods (one with data replacement and one without data replacement) to determine the mean, standard deviation, and statistical significance. Both bootstrapping methods produced very similar results. Shown is the bootstrapping analysis without replacement, with 10 random subsets extracted from each condition with a subset size of 20 “data items” (number of fusion events in the first second) for 500 μM Ca²⁺, 250 μM Ca²⁺, and 50 μM Ca²⁺, and 10 data items for 500 μM Mg²⁺. The mean of the 10 subsets for each condition is plotted as a bar graph and the error bars are standard errors of the mean. Unpaired, two-tailed t-tests were done to compare fusion synchronization between 500 μM Ca²⁺ vs. 250 μM Ca²⁺ (P = 0.3800), 500 μM Ca²⁺ vs. 50 μM Ca²⁺ (P < 0.0001), 250 μM Ca²⁺ vs. 50 μM Ca²⁺ (P < 0.0001), 500 μM Ca²⁺ vs. 500 μM Mg²⁺ (P < 0.0001), 250 μM Ca²⁺ vs. 500 μM Mg²⁺ (P < 0.0001), and 50 μM Ca²⁺ vs. 500 μM Mg²⁺ (P = 0.0002). All fusion data are available in the data repository associated with this work. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)