Hook1 KD reduces TrkB–BDNF-signaling endosome motility. (A) Time series of the mid-axon of hippocampal neurons expressing GFP-Hook1 and TrkB-RFP. Arrows show retrograde comigration of Hook1 with TrkB vesicles. Bars, 5 µm. (B) Kymographs of TrkB-RFP in control or Hk1 KD neurons. Traced events shown below are color-coded for ease of interpretation. Arrows show pausing in retrograde events. Bars, 10 µm; 1 min total. (C) Colocalization images of BDNF-Qdots with GFP-Hook1 in the axon of a hippocampal neuron. Arrows show Hook1 puncta colocalized with BDNF. Bar, 2 µm. (D) Line scan through axon in C. Arrow points to colocalized Hook1 and BDNF. (E) Quantification of BDNF-Qdots motility. Bar graph shows mean ± SEM; Kruskal–Wallis one-way ANOVA (***, P < 0.0001; *, P = 0.030; ns, P = 0.7405). Mock: n = 103 neurons; Hk1 KD: n = 63 neurons; Hk1 KD + Hk1-Halo: n = 55 neurons. (F) Kymographs of BDNF-Qdots in control, Hk1 KD, and Hk1 KD + Hk1-Halo neurons. Traced events shown below are color-coded for ease of interpretation. Green arrows indicate retrograde events. Bars: 5 µm (horizontal); 5 s (vertical).