Fig. 7
Endosomal clathrin is required for EGFR degradation, cargo sorting and efficient ILV formation. a mCherry-HRS770 does not rescue EGFR degradation upon depletion of endogenous HRS. Experiment was done side by side with Supplementary Fig. 3E,F. Mean ± SD from three experiments. t-test, ***p < 0.001. b Stably expressing HeLa cells as indicated were depleted for endogenous HRS as shown in Fig. 5b and processed for EM as described in Supplementary Fig. 4A. The 10 nm gold particles mark internalized EGFR after 60 min of EGF stimulation. Note that in HRSwt-expressing cells gold particles can be found as clusters inside degradative organelles, indicating degradation of gold-labeled EGFR. In contrast, in HRS770-expressing cells the gold-labeled EGFR is still found in the limiting membrane, where it covers a large portion of the surface, indicating impaired cargo sorting. Scale bar, 200 nm and 50 nm (inset). c Stably expressing HeLa cells as indicated were depleted for endogenous HRS as shown in Fig. 5b and processed for EM as described in Supplementary Fig. 4A. The 10 nm gold particles mark newly internalized EGFR after 15 min of EGF stimulation. Asterisks denote ILVs (40–60 nm diameter), arrowheads the boundaries of an endosomal EGFR/HRS/clathrin (HRSwt) or EGFR/HRS (HRS770) coat. Note the complete absence of this electron density in the absence of HRS (left panel). Scale bar, 500 nm and 100 nm (inset). d Quantification of the number of ILVs per endosome section. Data represented as dot plots with mean ± SD. Kruskal–Wallis test: p < 0.0001; Dunn’s multiple comparison test: ***p < 0.001, n.s. not statistically significant. More than 100 gold-labeled endosomes were analyzed per condition from at least 3 different samples per condition
