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. 2019 Jul 18;218(9):2982–3001. doi: 10.1083/jcb.201812170

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© 2019 Kendrick et al.

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Figure 5. — Hook3 is a scaffold for opposite-polarity motors. (A) Schematic of the experimental set up for three-color single-molecule motility assays. Four different species are detectable using three-color imaging: (1) KIF1C-TMR (KIF1C), (2) KIF1C-TMR with Hook3-488 (KIF1C/Hook3), (3) dynein-647 with dynactin and Hook3-488 (DDH), and (4) dynein-647 with dynactin, Hook3-488, and KIF1C-TMR (DDHK). (B) Representative kymographs from single-molecule motility assays with purified dynein-647, unlabeled dynactin, KIF1C-TMR, and Hook3-488. A three-color colocalized minus end–directed run (top panel) and three-color colocalized plus end–directed run (bottom panel) are marked with white arrows on each single-channel image and in the merge. The yellow signal in the merge highlights the colocalized run. Microtubule polarity is marked with minus (–) and plus (+). (C) Velocity analysis of the indicated complexes (KIF1C, n = 345; KIF1C/Hook3, n = 136; DDHK+, n = 18; DDH, n = 304; DDHK−, n = 53). Statistical significance was calculated with a one-way ANOVA with Tukey post-test; *, P = 0.0121. Combined data from four independent experiments is shown. (D) Percent processive events (mean ± SEM) reported in C. Combined data from four independent experiments is shown. (E) Percent processive events (mean ± SEM) in the two-color assay with purified dynein-647, unlabeled dynactin, Hook3-488, and unlabeled KIF1C. Increasing concentrations of unlabeled KIF1C are used as indicated by the dynein:KIF1C ratio. Combined data from three independent experiments is shown.