Figure 6. TOM1L1 specifically drives long-range trafficking of RAB-7/MT1-MMP-positive late endosomes.

(a) Endosome tracking assay. About 48 h after mCherry–MT1-MMP transfection with Ctrl or mouse TOLLIP siRNA, 3T3 cells infected as indicated were plated on gelatin-coated glass bottom dishes. Time-lapse imaging was performed with one acquisition every 260 ms for 1 min to visualize endosome movements (see also Supplementary Movie 4). The track pattern of randomly selected endosomes is seen as coloured lines. Insets show the localization of the cell areas (boxed) showed at higher magnification. Scale bar, 10 μm. (b) Analysis of endosome tracking. Average speed (μm s⁻¹), total distance (μm) and maximum distance from the point of origin (μm) were recorded. Histograms show mean±s.e.m. (n=101–202 tracked endosomes). **P≤0.01; ***P≤0.001 (Mann–Whitney test). (c) Co-localization of MT1-MMP with RAB-7. 48 h after GFP–RAB-7 and mCherry–MT1-MMP transfection, 3T3-neu cells infected with viruses expressing the indicated constructs were plated on gelatin-coated coverslips for 3 h to visualize RAB-7/MT1-MMP co-localization by confocal imaging. Boxed areas on the left panels are shown at higher magnification on the right panels. Scale bar, 20 μm (see also Supplementary Movie 5). (d) TOM1L1 does not affect lysosome trafficking. 3T3-neu cells infected as indicates were seeded on gelatin-coated glass bottom dishes and lysosomes were labelled with 50 nM Lysotracker-Red 30 min before imaging. Time-lapse imaging was performed with one acquisition every 260 ms for 1 min to visualize lysosome trafficking. The track pattern of randomly selected lysosomes is seen as coloured lines (see also Supplementary Movie 6). Scale bar, 20 μm. (e) Analysis of lysosome tracking. Average speed (μm s⁻¹), total distance (μm) and maximum distance from the point of origin (μm) were recorded (n=160 lysosomes per condition). Histograms show mean±s.e.m/NS, no significant/(Mann–Whitney Test).