Hook1 requires interaction with LIC1 for signaling endosome motility. (A) Diagram of Hook1 domain structure with mutated constructs below. Arrowheads indicate point mutations. (B) Hook3 (yellow)–LIC1 helix (green) structure (PDB: 6B9H) with residues that were mutated highlighted in magenta (Hook1 residue numbering). (C) Kymographs of Halo-Hook1 constructs from a single-molecule TIRF motility assay. Arrows indicate motile events. Bars, 5 µm; total length, 1 min. (D) Quantification of Hook1 motility in a TIRF motility assay. Scatter plot shows mean ± SEM; Kruskal–Wallis one-way ANOVA (***, P < 0.0001; **, P = 0.0012). n = 14–15 videos from three individual experiments. MT, microtubule. (E) Kymographs of BDNF-Qdots in axons of hippocampal neurons. Traced events below are color-coded for ease of interpretation. Green arrows indicate retrograde events. (F) Quantification of BDNF motility. Bar graph shows mean ± SEM; Mann–Whitney t test (*, P = 0.0262). Mock: n = 31 neurons; Hk1(Q149A, I156A): n = 46 neurons. (G) Kymographs of BDNF-Qdots in axons of hippocampal neurons in Hk1 KD-rescue experiments with C-terminal mutated Hook1 constructs. Traced events below are color-coded for ease of interpretation. (E and G) Bars: 5 µm (horizontal); 5 s (vertical). (H) Quantification of BDNF motility in KD-rescue experiments. Bar graph shows mean ± SEM; one-way ANOVA (***, P < 0.0001; ns, P = 0.58–0.98). Mock: n = 29 neurons; Hk1 KD: n = 22 neurons; Hk1 KD + Hk1(K672A,S673A): n = 13 neurons; Hk1 KD + Hk1(S693A,D694A): n = 24 neurons.