A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy

Abstract

Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans.

Keywords

• Biomarker
• high-content screening
• LC3
• LGG-1
• probe
• small molecule