Fig. 6
Lack of clathrin recruitment to endosomes increases PtdIns3P levels. a Experimental setup as in Fig. 2a, but with addition of DMSO or SAR405. b Representative images from live-cell imaging show that SAR405 treatment impairs HRS and CHMP4B localization to endosomes (arrowheads). Scale bars, 1 µm. c Frame-by-frame co-occurrence analysis (see also Fig. 2b) shows a drastically reduced overlap between HRS or CHMP4B with EGF upon SAR405 treatment. Data from 6 (DMSO) and 5 (SAR405) independent experiments. Co-occurrence normalized to the maximum of the DMSO control for HRS and CHMP4B. Note that CHMP4B shows a very transient localization to endosomes and thus only few spots can be observed at any time point, and therefore the reduction in co-occurrence upon SAR405 treatment appears not as clear as for HRS, which has a larger “dynamic range” due to its more stable association to endosomes. d Fluorescence intensity profiles over time show normal ESCRT waves in the presence of DMSO and lack of recruitment of HRS or CHMP4B in the presence of SAR405. e Quantitative WB analysis shows severely impaired EGFR degradation upon SAR405 treatment. Mean ± SD of three independent experiments. t-test, **p < 0.01. f SAR405 treatment leads to a drastic reduction in ILV formation. Electron micrographs depict representative endosomes 15 min after EGF stimulation. Asterisks: ILVs; arrow: forming ILV bud. Number of ILVs per endosome section from ≥100 gold-labeled endosomes per condition. Dot plot: mean ± SD. t-test, ***p < 0.001. Scale bar, 500 nm and 100 nm (inset). g Quantitative high-content microscopy shows an almost complete loss of mCherry-HRSwt from endosomes upon SAR405 treatment. Also the majority of mCherry-HRS770 is lost upon SAR405 treatment, but a stable pool remains. Numbers: remaining intensity of endosomal HRS (% of control ± SD). Data from ≥1600 cells per condition from 4 individual image series. Scale bar, 20 µm. h Cells stably co-expressing mCherry-HRS770 show increased levels of GFP-2xFYVE on endosomes compared to mCherry-HRSwt. Total fluorescence intensities ± SD. Data from >4000 cells per condition from 4 independent experiments. t-test, **p < 0.01. Scale bar, 20 µm and 5 µm (inset)
