Fig. 5: Minor endocytic defects in the absence of endophilin in chromaffin cells.
From: Endophilin-A coordinates priming and fusion of neurosecretory vesicles via intersectin
a Immunofluorescence for clathrin heavy chain (HC), adaptor protein 2 (AP2) and dynamin-1 in WT and endophilin TKO cells. Scale bar 3 µm. Fluorescence quantification revealed small but significant increase in clathrin-HC intensity (N = 3, WT 3 mice (25 cells) and TKO 3 mice (24 cells), whereas intensities of AP2 (N = 3, WT 3 mice (20 cells) and TKO 3 mice (23 cells)) and dynamin-1 (N = 3, WT 3 mice (21 cells) and TKO 3 mice (21 cells)) were unaltered in endophilin TKO cells. b Protein levels of the main endocytic factors—clathrin, adaptor protein 180 (AP180) and dynamins 1–3 (inspected by western blotting) were not altered in adrenal gland homogenates. Below: Quantification from clathrin-HC (N = 3, 3 samples/genotype), AP180 (N = 3, 3 samples/genotype) and dynamin (N = 4, 4 samples/genotype)—note that each sample on the blot originates from 6 to 8 glands from 3 to 4 mice of the same genotype pooled together. c, d Transferrin (conjugated with Alexa Fluor-546) uptake in the endophilin TKO cells compared to the littermate control and WT (N = 3, 3 mice per genotype WT (41 cells), KOWTKO (40 cells), and TKO (55 cells); note that uptake was analyzed in the whole cell, no difference in uptake between WT and endophilin TKO cells was observed). Scale bar 3 µm. e, f mCLING-Atto647 uptake (example cells shown in Supplementary Movies 1, 3, and 4) by chromaffin cells of indicated genotypes showed no significant difference in the number of endocytosed vesicles (p-value = 0.44, N = 3, 3 mice per genotype: WT (44 cells), KOWTKO (56 cells), and TKO (42 cells)). Note that uptake was analyzed in only one cell plane, and the bright-field image was acquired before recording the mCLING fluorescent signals so the focal planes are not always identical. The specificity of mCLING-Atto647 uptake was tested in the stimulated cells in the presence of Pitstop-2 inhibitor (Supplementary Movie 2 and Suppl. Fig. 5b). Error bars denote SEM. N number of independent replicates. One-way ANOVA after Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ns not significant.
