p150Glued-CBD disrupts JIP1-mediated KHC motility in vitro and anterograde APP-positive vesicle transport in DRGs. (A) Representative kymographs show that addition of p150Glued-CBD disrupts enhancement of KHC-Halo motility by JIP1. Lysate from COS7 cells transfected with myc-JIP1 were combined with FLAG-p150Glued-CBD lysate and immediately combined with KHC-Halo lysate. This lysate mixture was applied to flow chambers containing immobilized fluorescent microtubules and imaged. 100 total frames (∼33 s) are shown. (B) Addition of p150Glued-CBD decreases the number of motile KHC events mediated by JIP1. Motility measurements in the presence of FLAG-p150Glued-CBD were normalized to the condition containing only myc-JIP1 and KHC-Halo and represent three independent experiments (n = 60–100 microtubules and n = 23–214 runs). (C) p150Glued-CBD competitively inhibits JIP1-mediated KHC motility in vitro. At constant levels of myc-JIP1 lysate, addition of incrementally higher levels of p150Glued-CBD lysate leads to complementary decreases in relative KHC-Halo run frequency. Data represents three independent experiments (n = 6–52 microtubules). (D) Kymographs of APP-DsRed motility in DRGs transfected with a bicistronic construct coexpressing FLAG-p150Glued-CBD and GFP. Approximately 80 total frames (∼20 s) are shown. (E) Expression of p150Glued-CBD significantly decreases the percentage of anterograde APP-positive vesicles and correspondingly increases the percentage of arrested vesicles. (E–G) Data represent four independent experiments (n = 12–14 neurons). (F and G) Expression of p150Glued-CBD significantly decreases run length and speed of both anterograde and retrograde APP-positive vesicles. Means represent only vesicles categorized as motile (i.e., anterograde or retrograde in E). Error bars show the mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
