Panel A shows line traces of SypHy fluorescence response in control, CAST/ELKS-cDKO, CAST/ELKS-cDKO with CAST-WT, and CAST/ELKS-cDKO with CAST-mt neurons. The x-axis represents time in seconds, and the y-axis represents fluorescence change. The right dot plot shows the peak fluorescence change for 200 action potentials, with significant differences indicated by asterisks. Panel B shows line traces of iGluSnFR3 fluorescence response under the same conditions, with the x-axis representing time in seconds and the y-axis representing fluorescence change. The right dot plot shows the fluorescence change at the 10th action potential, with significant differences indicated by asterisks. Panel C shows microscopy images of hippocampal neurons stained for Endophilin-A1 and Synaptophysin under the same conditions. The right graphs show fluorescence intensity profiles along dotted lines, and the right dot plot shows the correlation coefficients between Endophilin-A1 and Synaptophysin, with significant differences indicated by asterisks.
Interaction of CAST/ELKS and endophilin-A is required for the stimulation-evoked SV response. (A) Effects of SV release with a CAST mutant. Activity-dependent SypHy fluorescence response in control (black), CAST/ELKS-cDKO (red), CAST/ELKS-cDKO with CAST-WT (blue), or CAST/ELKS-cDKO with CAST-mt (CAST-7RKA+6R9KA) (green) in mouse hippocampal neurons. The traces show average fluorescence of individual SypHy upon 20 Hz stimulation with 200 APs. Right dot plots of the peak of 200 APs. Results are mean ± SEM. n = 10 images, each. P values were obtained by comparison with control (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, ***P < 0.001, ****P < 0.0001). (B) Activity-dependent iGluSnFR3 fluorescence response in control (black), CAST/ELKS-cDKO (red), CAST/ELKS-cDKO with CAST-WT (blue), or CAST/ELKS-cDKO with CAST-mt (CAST-7RKA+ 6R9KA) (green) in the hippocampal neurons. The left traces show average fluorescence of individual iGluSnFR3 upon 10 Hz, 10 APs stimulation. The arrowheads indicate 10th action potential points. Results are mean ± SEM. n = 15 images, each. The right dot plots show 10th AP data. P values are compared with the cDKO-neurons (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, **P < 0.01, ****P < 0.0001). (C) Localization of endophilin-A1 in cultured hippocampal neurons. The left panels show control, CAST/ELKS-cDKO, CAST/ELKS-cDKO with CAST-WT, and CAST/ELKS-cDKO with CAST-mt (CAST-7RKA + 6R9KA) neurons stained for endophilin-A1 and synaptophysin. Fluorescence intensity profiles were measured along the dotted lines shown in the images and are presented on the right. The right dot plots show the correlation coefficients between endophilin-A1 and synaptophysin (n = 10 images). Scale bar: 10 µm. P values were obtained by comparison with cDKO neurons using one-way ANOVA followed by Bonferroni’s multiple comparison test, *P < 0.05, ***P < 0.001.