The trans-Golgi–associated degradative compartment contributes to autophagic vesicle growth during developmental and starvation-induced autophagy. (A) Confocal image of L3 fat body (wandering stage) showing autophagic marker Atg8a-GFP (green) and trans-Golgi marker GalT-TagRFP (magenta), both driven by BM-40-SPARC-GAL4. During the wandering stage, adipocytes initiate programmed autophagy prior to metamorphosis. Examples of nascent autophagic vesicles are shown on the right side at higher magnification. (B) Atg8a-GFP (green) and GalT-TagRFP (magenta) in fat body starved for 3 h. Nascent autophagic vesicles on the right side. (C) Proximity between Atg8a-GFP and GalT-TagRFP in fat body initiating programmed autophagy, measured in images like those in A. The upper graph represents proximity in 10 individual cells. The lower graph represents average proximity values (n = 10, error bars indicate SD): 89.0% of Atg8a puncta are trans-Golgi–associated. (D) Proximity between Atg8a-GFP and GalT-TagRFP in fat body starved for 3 h, measured in images like those in B. The upper graph represents proximity in 10 cells. The lower graph represents average proximity values (n = 10, error bars indicate SD). (E) Fat body in advanced programmed autophagy phase showing localization of Atg8a-GFP (green) and GalT-TagRFP (magenta). Growing autophagic vesicles are shown on the right side. (F) Fat body starved for 6 h showing localization of Atg8a-GFP (green) and GalT-TagRFP (magenta). Growing autophagic vesicles are shown on the right side. (G) Fat body in initial (left) and advanced (right) phases of programmed autophagy showing localization of Atg8a-mCherry (magenta) and mid-Golgi marker ManII-GFP (green), both driven by BM-40-SPARC-GAL4. Growing autophagic vesicles are shown below at higher magnification. Notice that ManII, unlike GalT, does not ingress into autophagic vesicles. (H) Atg8a-mCherry (magenta) and ManII-GFP (green) in fat body starved for 3 h (left) and 6 h (right). Growing autophagic vesicles are shown below.