HAP1 drives autophagosomal transport by binding to dynein and dynactin. (A–D) Representative micrographs showing HAP1^WT and the four HAP1 point mutants colocalize with LC3+ puncta in the axon. Scale bars, 5 µm. (E) Example kymograph illustrates the typical motility of EGFP-LC3+ puncta in the presence of overexpressed Halo-HAP1^WT. (F–I) Example kymographs and quantification showing dominant-negative effect of HAP1 CC1 box mutants (HAP1^AAVV and HAP1^ID) and HAP1 Glued motif mutant (HAP1^EEAA) on LC3+ puncta motility. n = 8–15 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde: AAVV, P = 0.0002; ID, P = 0.0013; EEAA, P < 0.0001; stationary/bidirectional [Stat/Bidir]: AAVV, P = 0.0009; ID, P = 0.0091; EEAA, P = 0.0002). (J and K) Example kymograph and quantification showing the HAP1 Spindly mutant (HAP1^TA) has no effect on LC3+ puncta motile behavior in the mid-axon. n = 16 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde, P = 0.4635; Stat/Bidir, P = 0.3500). (L and M) Example kymograph and quantification of LC3+ puncta in cells transfected with both HAP1 siRNA and HAP1^WT or HAP1^TA. n = 16 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (retrograde, P = 0.0071; Stat/Bidir, P = 0.0075). Bars throughout show mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to WT. All data are from the mid-axon.