Figure 2. Visualization of amphisomes in D. melanogaster, H. sapiens and C. elegans by colocalization between the autophagic protein Atg8p/LC3 and the endosomal protein Vps27p/HRS. Left: Vps27p/HRS localizes to Atg8p/LC3-positive autophagosomes. Confocal microscopic images of native Vps27p/HRS (red) and Atg8/pLC3-positive vesicles (green) in HeLa cells that stably expressed GFP-Atg8p/LC3, grown under nutrient-starvation conditions. Arrows indicate co-localization of both stainings. Scale bar is 10 µm. (From ref-29, with permission). Middle: Colocalization in wild-type cells was investigated in the fat body of D. melanogaster (L3 larval stage). A subset of GFP-Atg8a (green) structures in mid L3 fat body colocalizes with Vps27p/HRS (red, arrows). Scale bar is 5µm. (From ref-33, with permission). Right: Confocal images of VPS-27 (red) and GFP-Atg8p/LGG-1 (green) in C. elegans embryo. Because amphisomes are very rare in wild-type animals, a rab-7(RNAi) animal is shown, where amphisomes are easier to visualize. Scale bar is 10 µm. (From ref-25, with permission.)