Axonal amphisomes are the predominant AVs moving retrogradely. (A–C) Majority of autophagosomes in DRG axons target LEs after 3-h starvation. DRG neurons were cotransfected with GFP-LC3 and mRFP-Rab7 at DIV0 and imaged at DIV3 after incubation with serum (control; A) or serum-free medium (starvation; B) for 3 h. Images were taken from the middle segment of axons. Arrows indicate amphisomes colabeled with LC3 and Rab7, whereas arrowheads point out AV or LE alone. (D and E) Dual-channel kymographs showing comigration of GFP-LC3 and mRFP-Rab7 during 5-min time-lapse imaging. Vertical lines represent stationary organelles; slanted lines or curves to the right (negative slope) represent anterograde movement; to the left (positive slope), they indicate retrograde movement. An organelle is considered stationary if it remains immotile (displacement ≤ 10 µm). Under control conditions, GFP-LC3 was diffused, whereas LEs predominantly transported toward the soma (D). Under starvation, amphisomes (LC3 and Rab7) moved retrogradely (white lines in E). (F) Quantitative analysis showing that amphisomes and LEs share the similar predominant retrograde motility in DRG axons. Data were quantified from the total number of vesicles (V) in the total number of neurons (N) from greater than three experiments. Error bars: SEM. Student’s t test. Bars: (A and B) 5 µm; (D and E) 10 µm.