Figure 5: ALIX is required for sorting into ILVs and retention in MVBs of stress-internalized, but not EGF-bound EGFR.

(a, left) Co-staining of EGFR (green) with LBPA (red) showed little co-localization in serum-starved HeLa cells treated with EGF for 30 min, but considerable overlap in cells 1 h after UVC exposure. Note that, in serum-free conditions, LBPA does not accumulate in lysosomes, facilitating the detection of MVB-specific labelling. (a, right) Quantification of co-localization of EGFR with LBPA in EGF-treated versus UVC-exposed serum-starved HeLa cells. Data are mean±s.e.m., ***P<0.001 (Student’s t-test). (b) Lysates from HeLa cells transfected with control or ALIX siRNA were immunoblotted after 72 h for ALIX and Rab11 (as a loading control) to assess knockdown efficiency. (c) ALIX siRNA-treated cells were stimulated with EGF for 30 min, or exposed to UVC and incubated for 1 h, in the presence of anti-EGFR-gold, before EM processing. Ultrathin sections show gold (arrows) on ILVs of densely packed MVBs after EGF stimulation, but mainly on the limiting membrane of enlarged MVBs containing few ILVs in UVC-exposed cells. (d) Control, Tsg101, ALIX or Tgs101+ALIX siRNA-treated HeLa cells were exposed to UVC, incubated for 1 h and fixed, or further treated with PITSTOPII for 1 h. Immunostaining for EGFR (green) shows that depletion of Tsg101 or ALIX inhibits perinuclear EGFR accumulation, whereas EGFR redistributes to the plasma membrane upon PITSTOPII treatment, indicating that Tsg101 and ALIX are required for intracellular retention of EGFR. EGFR is found in very large vacuoles in double Tsg101+ALIX knocked-down cells after UVC exposure, before redistribution to the plasma membrane upon PITSTOPII treatment. Scale bars, 10 μm for confocal and 100 nm for EM pictures; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.