HAP1 regulates autophagosomal motility specifically in the mid-axon. (A and B) Example kymographs and micrographs from the mid-axon of a mock-transfected (control) neuron and a neuron transfected with HAP1 siRNA. Kymographs depict distance on the x-axis and time on the y-axis. Annotated kymographs mirror the above kymographs with the LC3+ puncta paths pseudo-colored for visualization. (C–E) Quantification of LC3+ puncta motility in the distal, mid-, and proximal axon. n = 8–21 neurons; two-way ANOVA with Sidak’s multiple comparisons test (mid retrograde mock vs. HAP1 KD, P = 0.0033; mid stationary/bidirectional [Stat/Bidir] mock vs. HAP1 KD, P = 0.0009). (F) Example kymograph and micrograph from the mid-axon of a neuron transfected with HAP1 siRNA and siRNA-resistant Halo-HAP1^WT. (G) Quantification of LC3+ puncta motility in the mid-axon. Two-way ANOVA with Tukey’s multiple comparisons test (retrograde mock vs. HAP1 KD, P = 0.0005; Stat/Bidir mock vs. HAP1 KD, P = 0.0020). Bars throughout show mean ± SEM. **, P < 0.01; ***, P < 0.001 compared to mock. Scale bar, 5 µm.