Panel A shows a western blot with two bands labeled Endophilin-A1 and Tubulin, comparing Control, EndA1-KD, EndA1-KD rescued with 3Flag-EndA1-WT-SR, and EndA1-KD rescued with 3Flag-EndA1-E264A-SR. Panel B includes a line graph showing the total recycling pool size over time for Control (black) and EndA1-KD (red) neurons, with a dot plot comparing the peak of 600 APs. Panel C features a line graph estimating vesicular pH over time for Control (black) and EndA1-KD (red) neurons, with a dot plot quantifying vesicular pH. Panel D illustrates the structure of the mouse Endophilin-A1-E264A mutant, highlighting the Glu264 to Ala change. Panel E presents a western blot and a line graph showing the binding ratio of MBP-CAST-1 and dose-dependent Myc-Dynamin-1-C to GST-Endophilin-A1 or GST-Endophilin-A1-E264A. Panel F includes line traces of activity-dependent SypHy fluorescence responses for Control (black), EndA1-KD (red), EndA1-KD with Endophilin-A1-WT-SR (blue), and EndA1-KD with Endophilin-A1-E264A-SR (green) neurons, with a dot plot comparing the peak of 200 APs.
An endophilin-A linker region mutant exhibits defective binding to CAST and SV responses. (A) The blot shows EndA1-KD efficiency and endophilin-A1–E264A mutant expression level in rat hippocampal neurons. Endophilin-A1 and tubulin were detected by the western blot analysis in control, EndA1-KD, EndA1-KD rescued with shRNA-resistant 3×Flag-EndA1-WT (3×Flag-EndA1-WT-SR; EndA1 driven by the CaMKII0.3 promoter), and EndA1-KD rescued with shRNA-resistant 3×Flag-EndA1-E264A (3×Flag-EndA1-E264A-SR). (B) TRP size in EndA1-KD neurons. Left traces show the TRP size in control (black) or EndA1-KD (red) rat hippocampal cultured neurons. Results are mean ± SEM. n = 14 images, each. Right dot plots show the peak of 600 APs. P values were obtained by nonparametric Mann–Whitney test, ****P < 0.0001. (C) Vesicular pH of SypHy-bearing vesicles in EndA1-KD rat hippocampal neurons. Left traces show the average for estimating the vesicular pH in control (black) or EndA1-KD (red) neurons. Results are mean ± SEM. n = 10 images, each. Right dot plots show quantification of the vesicular pH. P values were obtained by nonparametric Mann–Whitney test, ****P < 0.0001. (D) The image shows mouse endophilin-A1–E264A mutant in this study. Glu264 of endophilin-A1 (between the BAR domain and SH3 domain) was changed to Ala. (E) Endophilin-A1 mutants altered dynamin-1–dependent binding ability to CAST. Left representative Figures indicates MBP-CAST-1 and dose-dependent Myc-dynamin-1-C (residues 657–867) were precipitated with GST–endophilin-A1 or GST–endophilin-A1-E264A. MBP-CAST-1 and Myc-dynamin-1-C were detected by western blot analysis and GST-endophilin-A1-full was detected by CBB staining. Right line graph shows quantified results of the binding ratio (n = 3 independently prepared samples). Results are mean ± SD. (F) Effects of SV release in the endophilin-A1 mutant. Activity-dependent SypHy fluorescence response in control (black), EndA1-KD (red), EndA1-KD with endophilin-A1-WT-SR (blue), or EndA1-KD with endophilin-A1-E264A-SR mutant (green) in rat hippocampal cultured neurons. Traces show average fluorescence of individual SypHy upon 20 Hz, 200 APs stimulation. Results are mean ± SEM. n = 10 images, each. Right dot plots of the peak of 200 APs. P values were obtained by comparison with EndA1-KD neurons (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, *P < 0.05, ****P < 0.0001). Source data are available for this figure: SourceData F8.