SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport (2020)
Figure 6

Figure 6 - Depletion of Rab7 GAP TBC1D15 disrupts the bilateral architecture, morphology and dynamics of late compartments.

A-C. Identification of a Rab7 GAP that promotes endolysosomal system's bilateral architecture along the PN/PP axis. A. Graphical summary depicting the functions of 3 known TBC domain-containing GAPs for Rab7. B. Representative confocal images of fixed HeLa cells depleted of TBC1D2, 1D5, or 1D15 using siRNA oligo pools and immunolabeled against CD63 (white), scale bar: 10µm. C. Plot of CD63 pixel distribution as a function of TBC1D2/1D5/1D15 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells per condition analysed from 2 independent experiments. Significance: one-way ANOVA test (relative to siC), *** p<0.001 (see also Fig EV2A and B).

D/E. Rescue of TBC1D15 depletion phenotype. D. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC) or oligo #3 targeting TBC1D15 (siTBC1D15) and ectopically expressing either GFP-EV, GFP-TBC1D5 or siRNA-resistant GFP-TBC1D15res (green), immunolabeled for CD63 (magenta), scale bar: 10µm. E. Quantification of rescue expressed as % cells (average) in the population exhibiting one of 3 phenotypes: clustered, dispersed or intermediate; total numbers of cells analysed per condition appear above each bar, n=3 independent experiments. Significance (based on clustered phenotype): one-way ANOVA test relative to either siC/EV (top row) or si1D15/EV (bottom row), *** p<0.001, ns: not significant.

F-H. Effects of TBC1D15 depletion on the morphology and dynamics of late organelles. F. Representative electron micrographs of fixed HeLa cells transfected with either control siRNA (siC) or a pool of oligos targeting TBC1D15 (siTBC1D15) are shown, scale bars as indicated. G. Analysis of late compartment dynamics as a function of TBC1D15 depletion. Tracks followed by SiR-lysosome-positive vesicles during a time-lapse lasting 255 sec (5 s per frame), with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) regions, scale bar: 10µm (see also Movies EV9 and EV10). H. Quantification of high motility fraction (% tracks with displacement rates above 0.9µm/sec), n siC=27, n si1D15=37 images (2≥ cells per image) analysed from 2 independent experiments. Effectiveness of TBC1D15 (si1D15) depletion is confirmed by immunoblot (also appearing in Fig EV2A) against endogenous TBC1D15 (e1D15) following its immunoprecipitation (IP), TL: total lysate.

I. FRAP of GFP-Rab7 in live HeLa cells transfected with either control siRNA (siC, black line) or a pool of oligos targeting TBC1D15 (si1D15, red line). Plotted is average GFP-Rab7 signal recovery during 200 s following bleaching, expressed as % of pre-bleach signal, n=3 bleach regions per sample.

Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, as applicable. Graphs report mean (red line) of sample values (open circles), error bars reflect +/- SD. Unless stated otherwise, significance was assessed using two-tailed Student's t-test, *** p<0.001.