Two conserved Hook3 residues are critical for the assembly and motility of dynein–dynactin. (A) Single-point mutations Q147A, M151A, and I154A in sfGFP-tagged Hook3_1–552 were compared with WT and tested for binding to human GST-LIC1_389–523 as in Fig. 3 B (representative of triplicate experiments). Negative control lacks LIC1 on the beads. (B) Ratio of band intensity to the WT Hook3_1–552 signal in A; mean and SD from n = 3 independent experiments. (C) StrepII-Hook3 constructs, bound to Strep-Tactin resin, were incubated with porcine brain lysate; the beads were centrifuged; and the resin analyzed by immunoblotting for the dynein intermediate chain (IC) and the p150 subunit of dynactin. Negative control lacks Hook3 on the beads. The amount of each Hook3 construct was assessed by Coomassie stain. (D) Ratio of band intensity to the WT Hook3_1–552 signal in C; mean and SD from n = 3 independent experiments. (E) WT and single-point mutants were incubated with affinity-purified human dynein–dynactin and 1 mM ATP. SfGFP-tagged Hook3_1–552 was visualized by TIRF microscopy and classified as processive if it moved unidirectionally for >1 µm along microtubules. All constructs were normalized by dividing the total number of processive motors by the total length of microtubules in the field of view and the time of the movie (movements/µm per min). The ratios of the mutants to WT were calculated from side-by-side experiments performed on the same day. Shown are the mean ± SD of the ratios from three independent experiments performed on different days. The mean number of motile WT Hook3 molecules/µm per min was 0.039 ± 0.016. (F) Representative kymographs are shown for each construct that displayed motility. The kymographs are displayed using the same brightness and contrast.