Fig. 5
From: USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7
USP32 regulates the Rab7-positive endosome. a Effect of USP32 depletion on Rab7-positive and Rab5-positive endosomes. Top panels: representative confocal images of endogenous Rab7 (white). Boxed region perinuclear (PN) and peripheral (PP) zoom overlays of Rab7 (green) highlight colocalization with late endosome (LE) marker LAMP1 (magenta). Bottom panels: representative confocal images of endogenous Rab5 (white). Boxed PN and PP region overlays of Rab5 (green) highlight colocalization with EE marker EEA1 (magenta). Cell and nuclear boundaries depicted in dashed magenta and white lines, respectively. Scale bars = 10 µm. b Rab pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. c Alterations in LE morphology in response to USP32 depletion as visualized by correlative light and electron microscopy (CLEM). Overlays of GFP-Rab7 fluorescence (green) and transmission electron micrographs (TEM) are shown, scale bars = 0.25 µm. d Comparison of GFP-Rab7-positive LE profiles in control cells (siCtrl, black line) vs. those depleted of USP32 (siUSP32_2, red line), x-axis: LE diameter in µm, y-axis: number of LE profiles. See also Supplementary Fig. 6. e LE enlargement (white bars) and/or dispersion (gray bars) in MelJuSo cells depleted of the indicated GTPase (% cells), n = 4 or 5 independent experiments as indicated. See also Supplementary Fig. 7a. f Effects of USP32 depletion on cellular abundance of endogenous Rab7, as assessed by immunoblot. g Analysis of membrane-bound vs. cytosolic fractions of GFP-Rab7 stably expressed in MelJuSo cells with actin and transferrin receptor (TrfR) as loading controls for the cytosolic and membrane fractions, respectively. h Ratio of membrane-bound/cytosolic GFP-Rab7 in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars) normalized to siCtrl, n = 3 independent experiments. i Schematic illustration of consequences of USP32 depletion on Rab7 membrane-to-cytosol equilibrium. Bar graphs report mean of independent measurements (black circles), error bars reflect ±s.d. Where applicable, total number of cells analyzed per condition appears above each bar/scatter. All significant values were calculated using Student’s t test: *p < 0.05, ***p < 0.001, NS = not significant
