Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos.
Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes. During embryonic development in animals, a dramatic degradation of yolk proteins and synthesis of zygotic proteins takes place, leading to intracellular remodeling and cellular differentiation. Zebrafish represents a unique system to study autophagy due in part to its rapid embryonic development relative to other vertebrates. The technical advantages of this organism make it uniquely suited to various studies including high-throughput drug screens. To study autophagy in zebrafish, we identified two zebrafish Atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. Similar to yeast Atg8 and mammalian LC3, zebrafish Lc3 undergoes post-translational modification starting at the pharyngula stage during embryonic development. We observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. In addition, zebrafish Gabarap accumulates within lysosomes upon autophagy induction. Thus, we established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.
He
Congcong
C
Life Sciences Institute and Departments of Molecular, Cellular and Developmental Biology, and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Bartholomew
Clinton R
CR
Zhou
Weibin
W
Klionsky
Daniel J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001343:Autophagy; D001681:Biological Assay / Q000379:methods*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D016415:Sequence Alignment; D020123:Sirolimus / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D004151:Dipeptides; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D029961:Zebrafish Proteins; C542261:gabarapa protein, zebrafish; C542260:map1lc3b protein, zebrafish; D049452:Green Fluorescent Proteins; C071482:calpeptin; D020123:Sirolimus