Figure 9.
Incorporation of ER marker Sec61β into autophagic vesicles is a late event that depends on Atg2. (A) Confocal image of L3 fat body starved for 3 h showing autophagic marker Atg8a-GFP (green) and ER marker Sec61β-tdTomato (magenta), both driven by BM-40-SPARC-GAL4. Magnified inset in the lower right corner, with separate channels below. The graphs below quantify proximity between Atg8a-GFP puncta and Sec61β-tdTomato concentrations (yellow circle) measured in 10 different cells, as well as average proximity values (n = 10, error bars indicate SD). (B) Atg8a-GFP (green) and Sec61β-tdTomato (magenta) in L3 fat body initiating programmed autophagy. No Sec61β concentrations are observed. (C) Atg8a-GFP (green) and Sec61β-tdTomato (magenta) in L3 fat body during advanced programmed autophagy. (D) Confocal images of Lamp1-YFP (green) and Sec61β-tdTomato (magenta) in fat body from wild-type (upper image) and BM-40-SPARC>Atg2i (lower image) white pupae. Magnified insets show presence of Sec61β in autophagic vesicles in the wild type and complete absence upon Atg2 knockdown, with the Sec61β channel separated on the right (examples marked with asterisks). (E) Lamp1-YFP (green) and trans-Golgi GalT-TagRFP (magenta) in fat body from wild-type and BM-40-SPARC>Atg2i white pupae. Magnified insets show presence of GalT in autophagic vesicles unaffected upon Atg2 knockdown, with the GalT channel separated on the right. (F) TEM micrographs of autophagic vesicles (autolysosomes, marked with asterisks) in fat body from wild-type (upper row) and BM-40-SPARC>Atg2i (lower row) white pupae.