Detection of Autophagy in Caenorhabditis elegans Using GFP::LGG-1 as an Autophagy Marker

Abstract

In yeast and mammalian cells, the autophagy protein Atg8/LC3 (microtubule-associated proteins 1A/1B light chain 3B encoded by MAP1LC3B) has been the marker of choice to detect double-membraned autophagosomes that are produced during the process of autophagy. A lipid-conjugated form of Atg8/LC3B is localized to the inner and outer membrane of the early-forming structure known as the phagophore. During maturation of autophagosomes, Atg8/LC3 bound to the inner autophagosome membrane remains in situ as the autophagosomes fuse with lysosomes. The nematode Caenorhabditis elegans is thought to conduct a similar process, meaning that tagging the nematode ortholog of Atg8/LC3-known as LGG-1-with a fluorophore has become a widely accepted method to visualize autophagosomes. Under normal growth conditions, GFP-modified LGG-1 displays a diffuse expression pattern throughout a variety of tissues, whereas, when under conditions that induce autophagy, the GFP::LGG-1 tag labels positive punctate structures, and its overall level of expression increases. Here, we present a protocol for using fluorescent reporters of LGG-1 coupled to GFP to monitor autophagosomes in vivo. We also discuss the use of alternative fluorescent markers and the possible utility of the LGG-1 paralog LGG-2.

Figure 1

Autophagy in C. elegans A. The process of autophagy has been delineated by studies in yeast and mammalian cells. We presume that induction of autophagy begins with the activation of UNC-51. B. Autophagosome formation requires the integral protein ATG-9, thought to contribute membrane to the developing autophagosome. C. Nucleation requires the Class III PI3K complex, which recruits downstream autophagy proteins to the isolation membranes (IM) in mammals or pre-autophagosomal structure (PAS) in yeast, through the production of PI3P (light purple). D. Two conjugation complexes (LGG-1 and ATG-12) are required for elongation of the isolation membranes and completion of the developing autophagosome. LGG-1 conjugated to phospatidylethanolamine (PE, red) binds to both the inner and outer membranes of the autophagosome. LGG-1 also has the ability to bind to the autophagic adaptor proteins, such as SQST-1 which bind poly-ubiquitinated aggregates. E. The complete autophagosome eventually fuses with the lysosome leading to the degradation of cargo within the autophagosome.

Figure 2

GFP::LGG-1 expression in hypodermal seam cells of daf-2(e1370) mutants A. daf-2(e1370) mutants grown on OP50 E. coli, at 15°C, display a diffuse localization of GFP::LGG-1. B. daf-2(e1370) mutants grown on OP50 E. coli, at 25°C, display an increase in GFP::LGG-1 positive puncta (up to 12 GFP::LGG-1 positive puncta/seam cell) that represent early autophagic structures or autophagosomes. C. daf-2(e1370) mutants grown on control RNAi E. coli (transformed with empty vector, L4440), at 25°C, display the characteristic GFP::LGG-1 positive punctate structures. D. daf-2(e1370) mutants fed bec-1 RNAi, and raised at 25°C, display an increase in GFP::LGG-1 expression and large GFP::LGG-1 positive aggregates