title: 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.
authors.affiliation: Department of Biology, Queens College-CUNY, Flushing, New York 11367; The Graduate Center, The City University of New York, New York 10016.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Palmisano
authors.affiliation: Department of Biology, Queens College-CUNY, Flushing, New York 11367; The Graduate Center, The City University of New York, New York 10016.
authors.forename: Alicia
authors.initials: A
authors.lastname: Meléndez
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics / Q000502:physiology*; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000196:embryology*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029742:Caenorhabditis elegans Proteins; C587195:LGG-1 protein, C elegans; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins