Plus end–directed movement activates dynein-driven transport. (A) Schematic of K576–mCherry-Pex26 constructs. Dimeric Drosophila KHC includes amino acids 1–576 (head and first coiled-coil domains) cloned into the mCherry-Pex26 peroxisome–targeting vector. (top) K576–mCherry-Pex26 with wild-type neck linker. (bottom) Motility-deficient K576^ran10–mCherry-Pex26 with mutated neck linker (Case et al., 2000). (B) K576–mCherry-Pex26 recues bidirectional peroxisome motility in KHC-depleted cells, but a motility-deficient version does not. (top) Representative micrographs of S2 cells plated in the presence of CytoD and stably transfected with either K576–mCherry-Pex26 (left) or motility-deficient K576^ran10–mCherry-Pex26 (right) in endogenous KHC-depleted backgrounds. Boxed areas delineate regions selected for kymograph analysis (bottom). Arrowheads highlight location of a single peroxisome within a process. Arrow delineates time (30 s). DIC, differential interference contrast. Bars, 5 µm. (bottom) Kymographs showing peroxisome tracks over 1 min. Asterisks indicate the track of a single peroxisome. Bars, 1 µm. Video 5 corresponds to peroxisome motility in K576–mCherry-Pex26–expressing cells. (C) Graph showing the relative number of peroxisome vectors >0.2 µm in S2 cells expressing K576–mCherry-Pex26 in an endogenous KHC-depleted background. Data represent mean values ± SD from 30 cells per condition (from three separate dsRNA treatments). PLUS refers to those peroxisomes moving toward the tips of processes, whereas MINUS refers to those moving toward the cell center. (D) Motility-deficient K576^ran10–mCherry-Pex26 does not rescue dynein-driven peroxisome motility toward the minus ends of microtubules. Graph represents the percentage of processes containing peroxisomes after induction of replacement motors in KHC-depleted backgrounds. Data represent mean values ± SD from 120 cells (from three separate dsRNA treatments).