Panel A contains three sets of microscopy images and graphs. The images show the localization of clathrin light chain (CLC) and synaptophysin in control, CAST/ELKS-KD, and Endophilin-A1-KD neurons. The graphs display fluorescence intensity profiles measured along dotted lines in the images. The dot plot on the right shows the Pearson correlation coefficient between CLC and synaptophysin for each condition. Panel B contains similar microscopy images and graphs for Dynamin-1 and synaptophysin. The dot plot shows the Pearson correlation coefficient between Dynamin-1 and synaptophysin. Panel C contains microscopy images and graphs for CLC and synaptophysin in control, CAST/ELKS-cDKO, CAST/ELKS-cDKO with CAST-WT, and CAST/ELKS-cDKO with CAST-mt neurons. The dot plot shows the Pearson correlation coefficient between CLC and synaptophysin for each condition.
Binding of CAST to endophilin-A is required for the presynaptic localization of CLC. (A and B) Localization of endosomal proteins in CAST/ELKS or EndA1-KD neurons. Control, CAST/ELKS-KD, or EndA1-KD rat hippocampal cultured neurons stained with CLC (A) or dynamin-1 (B) and synaptophysin. Fluorescence intensity profiles were measured along the dotted lines shown in the images and are presented on the right. Right dot plots show the correlation co-efficiency between CLC vs. synaptophysin (A) and dynamin-1 vs. synaptophysin (B) (n = 10 images, each). P values were obtained by comparison with control neurons. Bar = 2 μm (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, **P < 0.01, ****P < 0.0001). (C) Localization of CLC in CAST/ELKS-cDKO neurons. control, CAST/ELKS-cDKO, CAST/ELKS-cDKO with CAST-WT, or CAST/ELKS-cDKO with CAST-mt (CAST-7RKA+6R9KA) in mouse hippocampal cultured neurons stained with CLC and synaptophysin. Right dot plots show the correlation co-efficiency between CLC and synaptophysin (n = 10 images, each). P values were obtained by comparison with cDKO neurons (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, **P < 0.01). Scale bars: 2 μm.