Exploring Autophagy in Drosophila

Abstract

Autophagy is a catabolic process in eukaryotic cells promoting bulk or selective degradation of cellular components within lysosomes. In recent decades, several model systems were utilized to dissect the molecular machinery of autophagy and to identify the impact of this cellular "self-eating" process on various physiological and pathological processes. Here we briefly discuss the advantages and limitations of using the fruit fly Drosophila melanogaster, a popular model in cell and developmental biology, to apprehend the main pathway of autophagy in a complete animal.

Keywords: Atg8a; Drosophila; Ref(2)P/p62; autophagy.

Figure 1

(A–C) Starved larval fat tissue samples from mosaic animals expressing RNAi constructs only in Green Fluorescent Protein (GFP)-positive cells. (A) GFP-marked Atg1 RNAi cells fail to induce autophagy as compared to surrounding control cells, based on lack of mCherry-Atg8a puncta. (B) As Rab7 is required for autophagosome-lysosome fusion, only faint and small mCherry-Atg8a dots (representing mostly autophagosomes) can be detected in GFP-positive cells depleted for Rab7, as opposed to the surrounding control cells where large bright structures (autolysosomes) are abundant. (C) LysoTracker staining also supports the detection of autolysosome formation seen in surrounding control cells, which is impaired in GFP-marked Rab7 RNAi cells. (D) The flux reporter mCherry-GFP-Atg8a shows that starvation-induced autophagic degradation proceeds normally in control cells, as GFP is quenched in autolysosomes while the mCherry signal remains prominent. (E) GFP fluorescence is retained and colocalizes with mCherry in Rab7-depleted cells, indicating a failure in autophagic flux. Note that mCherry-GFP double positive structures are also smaller than the structures seen in control cells, suggesting that these vesicles are most likely autophagosomes or small non-functioning autolysosomes.

Figure 1

(A–C) Starved larval fat tissue samples from mosaic animals expressing RNAi constructs only in Green Fluorescent Protein (GFP)-positive cells. (A) GFP-marked Atg1 RNAi cells fail to induce autophagy as compared to surrounding control cells, based on lack of mCherry-Atg8a puncta. (B) As Rab7 is required for autophagosome-lysosome fusion, only faint and small mCherry-Atg8a dots (representing mostly autophagosomes) can be detected in GFP-positive cells depleted for Rab7, as opposed to the surrounding control cells where large bright structures (autolysosomes) are abundant. (C) LysoTracker staining also supports the detection of autolysosome formation seen in surrounding control cells, which is impaired in GFP-marked Rab7 RNAi cells. (D) The flux reporter mCherry-GFP-Atg8a shows that starvation-induced autophagic degradation proceeds normally in control cells, as GFP is quenched in autolysosomes while the mCherry signal remains prominent. (E) GFP fluorescence is retained and colocalizes with mCherry in Rab7-depleted cells, indicating a failure in autophagic flux. Note that mCherry-GFP double positive structures are also smaller than the structures seen in control cells, suggesting that these vesicles are most likely autophagosomes or small non-functioning autolysosomes.