Autophagosomes in Drosophila motoneurons in situ concentrate at synaptic terminals and are transported by dynein. mCh-GFP-Atg8a protein was expressed in motoneurons using the Ok6-Gal4 driver to observe the distribution of autophagosomes. (A) Diagram of the larval prep with the approximate location of motoneuron cell bodies, the NMJ at muscle 4, and the axon connecting the cell body and NMJ. (B–B’’) Autophagosomes (double labeled with mCh and GFP) are observed in synaptic terminals of control larvae. (C–C’’) When lysosomal acidification is blocked (Vha26 RNAi), autophagosomes and defective autolysosomes are observed in synaptic terminals. The regions boxed in B’’ and C’’ are shown magnified to the right of the images. (D and E) Autophagosomes were rarely observed in cell bodies of motoneurons (D–D’’), but defective autolysosomes were observed in cell bodies when lysosomal acidification is blocked (E–E’’). (F and G) Synaptic terminals of controls did not accumulate autophagosomes within terminal boutons (F and F’), whereas autophagosomes accumulated when dynein was depleted (G and G’). The accumulation of autophagosomes is indicated by the arrow in the inset in G. Terminal boutons within the synaptic terminal are indicated by white arrowheads in F and G. (H and H’) Disruption of the autophagy initiator kinase Atg1 largely suppressed the accumulation of autophagosomes in terminal boutons when dynein was depleted. (I and I’) Autophagosome punctae are rarely present when Atg1 function is disrupted. The boxes in F’, G’, H’, and I’ are shown magnified as insets. (J) Percentage of terminal bouton volume occupied by autophagosomes. Data represent the mean and SEM. n, number of terminal boutons scored. Unpaired, two-tailed t test result: ***, P < 0.0001. Bars in magnified images and insets are 2 µm; all others are 10 µm.