Fig. 6

Deubiquitylation of Rab7 by USP32 promotes late endosome transport. a Late endosome (LE) organization and dynamics as a function of Rab7 ubiquitylation status. Top panels: representative confocal images of live MelJuSo cells stably expressing GFP-Rab7 or GFP-Rab7-2KR (GFP-2KR) (white) taken at the start of time-lapse, t = 0. Scale bars = 10 µm. Bottom panels: vesicle displacement rates depicted on a rainbow color scale (blue: immobile; red: maximum mobility per time interval) tracked over 250 s at 5 s per frame. Cell and nuclear boundaries are depicted in dashed white lines, boxed zoom-ins highlight select perinuclear (PN) and peripheral (PP) regions. b Vesicle dispersion expressed as fractional distance of GFP pixels along a straight line from center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. c Quantification of vesicle motility calculated using TrackMate for Fiji (for details see the Methods section), n = 3 independent experiments. Plots report mean velocities of Lysotracker-positive structures calculated from multicell time-lapses of control cells (siCtrl, open circles) vs. those depleted of USP32 (siUSP32_2, closed circles). See also Supplementary Fig. 7b, c and Movies 9–12. d–f Co-immunoprecipitation (Co-IP) of Rab7-interacting protein (RILP) with GFP-Rab7 as a function of Rab7 ubiquitination status. d Immunoblots of Co-IP from HEK293T cells transfected and treated as indicated. e Quantification of Co-IP for HA-RILP with GFP-Rab7 (white bars) vs. GFP-2KR (gray bars), n = 3 independent experiments, is shown along with a schematic summary. f Quantification of Co-IP for HA-RILP with GFP-Rab7 from control HEK293T cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars) normalized to control, n = 3 independent experiments. See also Supplementary Fig. 7d. 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 significance was calculated using Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant