Maturity regulates the association and function of dynein effectors. (A) Quantification of autophagosomal maturity under BafA1 or vehicle only (DMSO) treatment. n = 11–14 neurons. Mann-Whitney U test of fraction with EGFP (P = 0.0050). (B and C) Example line scans and quantification showing HAP1 colocalization with LC3 in the proximal axon after BafA1 treatment. n = 10–12 neurons. Mann-Whitney U test (P = 0.0007). Scale bar, 2 µm. (D) Quantification of autophagosomal maturity under SNAP-Stx17^FL or dominant-negative mutant (Stx17^TM) expressing conditions. n = 12–13 neurons. Mann-Whitney U test of fraction with EGFP (P = 0.0013). (E and F) Example line scans and quantification showing HAP1 colocalization with LC3 in the proximal axon with Stx17 expression. n = 12–13 neurons. Mann-Whitney U test (P = 0.0020). Scale bar, 2 µm. (G and H) Example kymographs and quantification of LC3+ puncta motility in the proximal axon following JIP3 siRNA transfection and/or Stx17 expression. n = 19–20 neurons. Two-way ANOVA with Tukey’s multiple comparisons test for all LC3+ puncta (retrograde: mock Stx17^FL vs. mock Stx17^TM, P = 0.9372; mock Stx17^FL vs. KD Stx17^FL, P = 0.0037; mock Stx17^FL vs. KD Stx17^TM, P = 0.9954; KD Stx17^FL vs. KD Stx17^TM, P = 0.0019; stationary/bidirectional: mock Stx17^FL vs. mock Stx17^TM, P > 0.9999; mock Stx17^FL vs. KD Stx17^FL, P = 0.0003; mock Stx17^FL vs. KD Stx17^TM, P = 0.9018; KD Stx17^FL vs. KD Stx17^TM, P = 0.0044.) Bars throughout show mean ± SEM. **, P < 0.01; ***, P < 0.001 compared to mock.