Peroxisome motility was driven by Nesp2 mutants. (A) Schematic of rapalog-inducible peroxisome trafficking assay. (B) Temporal color-coded time-lapse images of GFP-EB3-expressing COS7 and MRC5 cells. Images were taken at 1-s intervals. Projection of 15 frames is shown. (C) Analysis of EB3 comet movements. ROIs were selected for generating kymographs (right). The directions of EB3 kymographs were analyzed using KymoButler. (D) In both COS7 and MRC5 cells, ∼90% of EB3 comets were moving toward the cell periphery. n = 15 (COS7) and 11 (MRC5) cells from three independent experiments, and over 100 trajectories were analyzed per cell. (E) Rapalog-induced peroxisome transport by Nesp2-SR48ΔKASH-LEAA (SR48-LEAA) (top) and Nesp2-SR48ΔKASH-5A-AAVV (SR48-5A-AAVV) (bottom). Images at 0, 30, and 60 min after rapalog treatment are shown. GFP signals are shown in black and cell contours are outlined in blue. Right: Representative trajectories of GFP signals are shown with lines by different colors, with time denoted by color gradient and ending points denoted by filled circles. (F) Distribution of GFP fluorescence along the distance from the cell center to the periphery at different time points. Each graph corresponds to each cell shown in E. SR48-LEAA drove limited movement of peroxisomes. SR48-5A-AAVV induced slow movement toward the cell periphery. (G–I) Representative time-lapse sequences (left) and kymographs (right) of rapalog-induced peroxisome transport by SR48-LEAA, SR48-5A-AAVV and DHC. Images were taken at 0.1 s interval in MRC5 cells with microtubules oriented from minus (left) to plus (right) ends. Bars show mean ± SEM. Scale bars, 10 μm in B, C, and E; 2 μm in G–I. Related to Video 5.