Figure 4: ELKS associates and cooperates with RAB6 during the biogenesis of melanosome.

(a) WB of MNT-1 cell lysates depleted for RAB6, ELKS or both probed with respective antibodies and tubulin as loading control. (b) Melanin content estimation of cells treated with indicated siRNAs. Data are normalized to control and presented as means±s.e.m. (six independent experiments). (c) Conventional EM analysis of control, ELKS or RAB6+ELKS-depleted MNT-1 cells (stages-I/-II, arrowsheads; stages-III/-IV, arrows). (d) Quantification of immature (stages-I/-II) and mature (stages-III/-IV) melanosomes (n) presented as mean percentage (normalized to total n)±s.e.m. (8–10 cells per condition, three independent experiments. siControl, n=572; siELKS, n=342; siRAB6+siELKS, n=347). (e) IFM on siControl (top) and siELKS (bottom) MNT-1 cells stained for endogenous ELKS using pre- (left) or -immune (right) sera that label specific structures associated to CD63-positive pigmented melanosomes (arrows). (f) Quantification of the total ELKS fluorescent intensity measured at the melanosomal plane in control and ELKS-depleted MNT-1 cells. (g) Ultrathin cryosections of GFP-ELKS expressing MNT-1 cells co-stained for TYRP1 (PAG-15 nm) and GFP (PAG-10 nm), showed ELKS localization to the limiting membranes of melanosomes (M), (arrows) and associated vesicle (arrowhead). (h) WB of PNS (1%) and melanosomes-enriched fraction (25%, Mel) isolated from MNT-1 cells and probed for RAB6 and ELKS (*), TYRP1, MART-1, RAB27, GM130 or GAPDH. (i) Live imaging frames of MNT-1 co-expressing GFP-ELKS or GFP-ELKS-ΔCt with mCh-RAB6. GFP-ELKS, but not GFP-ELKS-ΔCt, co-distributed with mChRAB6-positive vesicles close to the Golgi area (arrows, × 5 zoomed area, and Supplementary Movie 7). (j) Quantification of the number of cells (n) harbouring GFP-RAB6 to melanosomes (GFP-ELKS, n=117; GFP-ELKS-ΔCt, n=84). Data are normalized to control and presented as mean±s.e.m. (3 independent experiments). Scale bars: 1 μm (c); 10 μm (e,i); 500 nm (g); M.W. in KDa; two tailed, Unpaired t test (b,f), two way ANOVA multiple comparisons (d); **P<0.01; ***P<0.001; ****P<0.0001; n.s., non significant.