Nesprin-2 drives persistent bidirectional cargo transport in CGCs. (A–C) Inducible peroxisome trafficking assay in cultured CGCs transfected with PEX-mCherry-FKBP together with KIF5B-HA-FRB (left), HA-BicD2-FRB (middle), or HA-SR48ΔKASH-FRB (right). Images at 0, 30, and 60 min after rapalog treatment are shown. mCherry signals are shown in black and cell contours are outlined in blue. Lower graphs show the distribution of mCherry fluorescence along the distance from the trailing process to the leading process at different time points. Kif5B- and BicD2-expressing cells showed rapid PEX-mCherry displacement toward the tip of the leading process and the microtubule-organizing center, respectively. Nesprin-2-SR48-expressing cells showed persistent fluctuation of PEX-mCherry distribution, indicating continuous bidirectional cargo transport. (D–F) Representative dual-color time-lapse sequences showing peroxisomes (magenta) moving along DCX-GFP-labeled MT filaments (green) in CGCs. Colored arrowheads trace the movement of individual peroxisomes. Bottom, trajectories of individual peroxisomes. (G) Quantification of the number of peroxisomes that moved 3 μm or more in respective time windows. n = 9 (Kif5B), 11 (BicD2), and 11 (SR48) cells from three independent experiments per group. Data points and error bars show mean ± SEM. Scale bars, 5 μm. Related to Videos 8 and 9.