Nesprin-2 drives bidirectional cargo transport along microtubules. (A) Inducible peroxisome trafficking assay in COS7 cells transfected with PEX-GFP-FKBP together with KIF5B-HA-FRB (top), HA-BicD2-FRB (middle), or HA-SR48ΔKASH-FRB (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. (B) Distribution of GFP fluorescence along the distance from cell center to periphery at different time points. Kif5B- or BicD2-expressing cells show rapid PEX-GFP displacement towards the cell edge or cell center, respectively. Nesprin-2-SR48-expressing cells show persistent fluctuation of GFP distribution, indicating continuous bidirectional cargo transport. (C) Quantification of the number of peroxisomes that moved 3 μm or more in respective time windows. The values are normalized to the number of moving peroxisomes in the first time window (0–5 min). n = 9 (Kif5B), 16 (BicD2), and 16 (SR48) cells from three independent experiments per group. (D–F) Representative time-lapse sequences (left) and kymographs (right) of rapalog-induced peroxisome transport by Kif5B (D), BicD2 (E), and Nesp2-SR48 (F). Images were taken at 0.1 s interval in MRC5 cells with microtubules oriented from minus (left) to plus (right) ends. Kymographs from different experiments are shown by different colors and the leftmost kymograph in each panel corresponds to the peroxisome shown in the time-lapse images. Data points and error bars show mean ± SEM. Scale bars, 10 μm in A; 2 μm in D–F. Related to Videos 4 and 6.