Fig 2: Intracellular localization of Rab7a and vimentin filaments. HeLa cells overexpressing GFP-tagged Rab7a wt (A–B) or L129F (C), K157N (D), N161T (E) V162M and (F) mutant proteins were subjected to immunofluorescence analysis using an anti-vimentin antibody followed by a Cy3 secondary antibody. (G–H) Hela cells were subjected to immunofluorescence analysis using anti-Rab7a and anti-vimentin antibodies followed by Cy2 and a Cy3 conjugated secondary antibodies, respectively. The images represent maximum-intensity projections of Z stacks. Bar = 10 μm.

Fig 3: A model for the role of Rab7a in vimentin assembly. Rab7a induces vimentin phosphorylation at different sites and this, in turn, causes vimentin disassembly. Rab7a is able to bind both to the soluble and filamentous forms of vimentin.

Fig 4: Phosphorylation of Rab7A does not affect interaction with RILP.HEK293 cells were transiently transfected with the indicated plasmids. Note that the Kinase inactive (KI) mutant corresponds to LRRK1[D1409A]. Twenty-four hours post-transfection cells were lysed either GFP-RILP (upper panel) or HA-Rab7A (middle panel) immunoprecipitated with GFP or HA antibody, respectively. The immunoprecipitates as well as cell extracts (20 µg, lower panel) were subjected to immunoblot analysis with the indicated antibodies (all at 1 µg/ml). Each lane represents cell extract obtained from a different replicate. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system.

Fig 5: EGFR signalling is elevated in FLCN-/- mouse tumours and treatment with an EGFR inhibitor suppressed tumour growth.(a) Immunohistochemical staining of littermate mouse FLCN WT kidney (Flcn flox/flox) and mouse kidney tumours generated in the FLCN kidney KO (Sglt2-Cre;Flcnflox/flox) mouse model. The cysts and tumours in FLCN KO kidney tumours strongly express pAKT, pERK and pSTAT3 (best seen in the high magnification (× 40) images). The glomeruli (arrows) and collecting ducts (arrowheads) of normal (FLCN WT) mouse kidneys express pERK, but pAKT and pSTAT3 staining was almost absent, except for a few positive cells. Scale bars, 0.25 mm at × 10 magnification; 0.2 mm at × 20 magnification; 0.05 mm at × 40 magnification. (b) Immunohistochemical staining of a panel of human kidney tumours representing the clear cell, oncocytoma and chromophobe/oncocytoma histologies demonstrated increased pERK and pS6 signalling compared to normal kidney. The glomeruli and collecting ducts express pERK, but otherwise pERK and pS6 staining was almost absent in normal kidney tissue. Scale bars, 0.05 mm. (c) FLCN-/- FTC-133 cells were injected subcutaneously into nude mice and once tumours were established, the mice were treated daily with vehicle or Afatinib (20 mg kg-1 for 6 days followed by 15 mg kg-1 for 3 days). The tumour volume relative to the size of the tumour on day 1 of treatment is presented and the error bars are the mean±s.e.m. (n=9 mice in the vehicle group, and n=8 mice in the Afatinib group). The relative tumour volume at the end of the study (day 10) is significantly different between the vehicle and Afatinib treated mice, * indicates significance, t-test, P<0.01. (d) Active Rab7A accelerates the endocytic trafficking of internalized EGFR to the lysosome for degradation, resulting in reduced EGFR signalling (phosphorylated EGFR, ERK and S6). When cells lose the tumour suppressor function of FLCN due to a germline mutation in the FLCN gene (BHD disease), Rab7A GTP-to-GDP turnover is decreased. A decrease in Rab7A activity slows the endocytic trafficking of EGFR, resulting in prolonged and elevated phosphorylated EGFR and downstream signalling.