Fig 1: EGFR phosphorylates RAB31 to drive EGFR into exosomes and the exosomes promoted by RAB31 mediate resistance to erlotinib.a Western blotting analyses of whole-cell lysates (WCL) from the indicated NCI-H1975 cells stably expressing shNC, shRAB31 or shFLOT1 and shFLOT2. b Western blotting analyses of the concentrated conditional media from the indicated stable NCI-H1975 cells used in a under serum starvation (SS). c Immunofluorescence of endogenous EGFR (green) and CD63 (red) in the indicated stable NCI-H1975 cells used in a under SS. d Western blotting analyses of the concentrated conditional media from the indicated stable NCI-H1975 cells under serum starvation (SS). e Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells under serum starvation (SS). f Western blotting analyses of WCL and immunoprecipitates (IP) from NCI-H1975 cells treated with afatinib or erlotinib under SS. g Western blotting analyses of the concentrated conditional media from the indicated stable NCI-H1975 cells under SS. h Representative clone images of PC9-GFP cells treated with the concentrated conditional media derived from the indicated stable NCI-H1975 cells without or with erlotinib. i Quantification of the numbers of each clone for h. Data are means ± SD of cell numbers in each clone with PBS (n = 75), Vector (n = 82), RAB31WT (n = 62), RAB31Q65L (n = 62), RAB31R77Q (n = 65) or RAB313YF (n = 84). j Representative clone images of PC9-GFP cells treated with the pure small EV (sEV) derived from the indicated stable NCI-H1975 cells without or with erlotinib. k Quantification of the numbers of each clone for j. Data are means ± SD of cell numbers in each clone with Vector (n = 71), RAB31WT (n = 80) or RAB313YF (n = 80). Unpaired t-test was used to analyze the difference between the two groups. ****P < 0.0001, NS, no statistical significance. Scale bars, 10 µm (c) and 100 µm (h and j).
Fig 2: Tyrosine phosphorylation of RAB31 by active EGFR acts similarly to its active form.a Immunofluorescence of EGFR-HA (red) and Flag-RAB31WT (magenta) with CD63-GFP (green) in Flag-RAB31WT stable HeLa cells transiently expressing EGFR-HA and CD63-GFP stimulated with EGF for 30 min as indicated. b Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells treated with 100 ng/mL of EGF at the indicated times. c Western blotting analyses of whole-cell lysates (WCL) and immunoprecipitates (IP) from HEK-293T cells co-expressing the indicated plasmids treated with 100 ng/mL of EGF at the indicated times. p-Tyr, anti-phosphotyrosine antibody. d Immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated Flag-RAB31 stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). e–g Immunofluorescence of EGFRM2-HA (red) with CD63-GFP (green) (e), Flag-RAB31WT (red) with CD63-GFP (green) (f) and Flag-RAB31WT (green) with EGFRM2-HA (red) (g) in Flag-RAB31WT stable HeLa cells transiently expressing EGFRM2-HA and CD63-GFP under SS using 3D-SIM. h Western blotting analyses of WCL and IP from HEK-293T cells co-expressing EGFRM2-HA and Flag-RAB31WT treated with the indicated EGFR-tyrosine kinase inhibitors under SS. i Immunofluorescence of EGFRM2-HA (red) and Flag-RAB31WT (magenta) with CD63-GFP (green) in Flag-RAB31WT stable HeLa cells transiently expressing EGFRM2-HA and CD63-GFP treated with the indicated inhibitors under SS. j, k Western blotting analyses of WCL and IP from HEK-293T cells co-expressing the indicated plasmids under SS. l Western blotting analyses of WCL and IP from HEK-293T cells co-expressing the indicated plasmids treated with EGF for the indicated times. m Western blotting (WB) and Coomassie brilliant blue (CBB) analyses of the purified different EGFR and RAB31 forms as indicated after in vitro kinase assay, as described in Materials and Methods section. n Mass spectrometry analysis of the phosphorylated tyrosine sites in RAB31 purified from in vitro kinase assay. Scale bars, 10 µm.
Fig 3: Active RAB31 drives EGFR entry into CD63-positive MVEs to form ILVs and exosomes.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).
Fig 4: The proposed model for the functions of RAB31 in exosome pathway.EGFR are endocytosed into cells to form signaling endosomes (SE) and early endosomes (EE) regulated by RAB5, and then are transported from early to late endosomes (LE) regulated by transition from RAB5 to RAB7. a At this time, ESCRT machinery sorts the ubiquitylated EGFR into intraluminal vesicles (ILVs) that are destined to lysosomes for degradation by the fusion of multivesicular endosomes (MVEs) with lysosomes regulated by RAB7. b However, high RAB31, guarding on the late endosomes, encounters active EGFR and can be activated via tyrosine phosphorylation by EGFR, and then active RAB31 engages FLOTs in lipid rafts to drive EGFR entry into MVEs to form ILVs. Meanwhile, RAB31 recruits TBC1D2B to inactivate RAB7 preventing the fusion of MVEs with lysosomes, thereby enabling that the sequestered EGFR ILVs are secreted as exosomes. c Representative image of MVE membrane budding to form ILVs driven by the active RAB31 in NCI-H1975 cells. The white triangles indicate the budding moments of MVE membrane. Immunofluorescence of endogenous RAB31 (green) and CD63 (red) in NCI-H1975 cells. Scale bar, 5 µm.
Fig 5: RAB31 recruits TBC1D2B to inactivate RAB7 suppressing the fusion of MVEs with lysosomes.a Immunofluorescence of endogenous RAB7 (red) and endogenous CD63 (magenta) with GFP-RAB31WT (green) in the indicated stable NCI-H1975 cells. b Western blotting analyses of whole-cell lysates (WCL) and streptavidin pull-down (PD) from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. c Immunofluorescence of endogenous RAB7 (red) and HA-RILP (magenta) with GFP-RAB31 (green) in the indicated stable HeLa cells transiently expressing HA-RILP. d Western blotting analyses of WCL and streptavidin PD from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. e Immunofluorescence of endogenous RAB7 (red) and endogenous TBC1D2B (magenta) with GFP-RAB31 (green) in the indicated stable NCI-H1975 cells. f Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB31 (green) in NCI-H1975 cells. g Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. h Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB7 (green) in NCI-H1975 cells stably expressing shNC or shRAB31. i Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. j Western blotting analyses of WCL and GTP agarose PD at their endogenous levels from NCI-H1975 and MDA-MB231 cells stably expressing shNC or shRAB31. Scale bars, 10 µm.
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