Fig. 2: Active RAB31 drives EGFR entry into CD63-positive MVEs to form ILVs and exosomes.
From: RAB31 marks and controls an ESCRT-independent exosome pathway
a Up panels, immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). Low panel left, the ratio of co-localization of EGFR-HA with CD63-GFP-positive LE/MVE in Vector (n = 6 fields), RAB31WT (n = 8 fields) and RAB31Q65L (n = 11 fields). Low panel middle, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in RAB31WT (n = 8 fields) and RAB31Q65L (n = 11 fields). Low panel right, the ratio of entry of Flag-RAB31 into CD63-GFP-positive LE/MVE in RAB31WT (n = 8 fields) and RAB31Q65L (n = 11 fields). b–d Immunofluorescence of the localization of EGFR-HA (red) with CD63-GFP (green) (b), Flag-RAB31Q65L (red) with CD63-GFP (green) (c), and Flag-RAB31Q65L (green) with EGFR-HA (red) (d) in Flag-RAB31Q65L stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under SS using three-dimensional structured illumination microscopy (3D-SIM). e Immunoelectron microscopy of the localization of EGFR-HA in Vector stable HeLa cells transiently expressing EGFR-HA under SS. NE, nuclear envelope; N, nucleus; C, cytoplasm. f Immunoelectron microscopy of the localization of EGFR-HA and Flag-RAB31WT in Flag-RAB31WT stable HeLa cells transiently expressing EGFR-HA under SS. NE, nuclear envelope; N, nucleus; C, cytoplasm. g Immunoelectron microscopy of the localization of EGFR-HA and Flag-RAB31Q65L in Flag-RAB31Q65L stable HeLa cells transiently expressing EGFR-HA under SS. h Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells under SS. i NanoSight nanoparticle tracking analysis of the concentrated conditional media from the indicated stable HeLa cells under SS. j Transmission electron microscopy analysis of the concentrated conditional media from Flag-RAB31Q65L stable HeLa cells under SS. All data are means ± SD. Unpaired t-test was used to analyze the difference between the two groups. ****P < 0.0001. Scale bars, 10 μm (a–d), 200 nm (e–g), 100 nm (j).
