Monitoring Autophagy in Drosophila Using Fluorescent Reporters in the UAS-GAL4 System

: Following autophagy induction, the autophagy-related protein Atg8 undergoes ubiquitin-like conjugation to phosphatidylethanolamine and inserts into the autophagosome membrane. Transgenic Drosophila lines expressing Drosophila Atg8 (DrAtg8a) fused to green fluorescent protein (GFP), mCherry, or dual-tagged GFP-mCherry have been constructed and are extremely useful for monitoring autophagy. The use of GFP-mCherry-Atg8a is particularly advantageous because it allows for the assessment of autophagy induction as well as autophagy flux. GFP-mCherry-Atg8a fluoresces yellow in nonacidic structures including the autophagosome. When autophagosomes fuse with lysosomes to form autolysosomes, GFP fluorescence is quenched by the acidic hydrolases and the resulting autolysosome will fluoresce red. The upstream activating sequence (UAS)-GAL4 system allows for the ectopic expression of the gene of interest (in this case, DrAtg8a) in a tissue-specific manner. Here we provide a protocol for monitoring autophagic flux by fluorescence microscopy by expressing UASp-GFP-mCherry-DrAtg8a in the Drosophila germline.