Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos

Life Sciences Institute and Departments of Molecular, Cellular and Developmental Biology, and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Autophagy (Impact Factor: 11.42). 06/2009; 5(4):520-6. DOI: 10.4161/auto.5.4.7768
Source: PubMed

ABSTRACT 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.

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Available from: Weibin Zhou, Aug 05, 2015
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    • "Wild-type embryos were collected and treated with the following drugs dissolved in DMSO. Rapamycin, which is a known inducer of autophagy, was added to a final concentration of 1 mM to 48 hpf embryos for 24 h [28] [29]. Chloroquine, a chemical that increases lysosomal pH thus inhibiting autophagy was added at a concentration of 50 mM to 56 hpf embryos for 16 h [25,30]. "
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    Experimental Cell Research 10/2014; 328(1). DOI:10.1016/j.yexcr.2014.07.014 · 3.37 Impact Factor
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    • "The zebrafish model has also been used to examine links among NADPH oxidase activity, autophagy and C. albicans infection. A transgenic line expressing the GFP- LC3 fusion protein has been found to report robustly on autophagic flux, and was used to demonstrate recruitment of LC3 to bacteria-containing phagosomes in vivo for the first time (He et al., 2009; Meijer et al., 2014). Remarkably , C. albicans-containing phagosomes only rarely recruit significant levels of GFP-LC3 in vivo, and this low level recruitment is not abolished by either the antioxidant vitamin E or the NADPH oxidase inhibitor diphenyleneiodonium (Brothers et al., 2013). "
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    • "Fluorescent vital dyes also allow for imaging subcellular structures in vivo. BODIPY, Alizarin Red, quantum dots and dyes, such as MitoTracker or LysoTracker, are versatile counterstains to visualize dynamic cellular processes in live fish (Cooper et al., 2005; He et al., 2009; Köster and Fraser, 2006; Rieger et al., 2005; Zhang et al., 2008). In combination, transgenics and dyes provide a microscopic view of intracellular processes and a macroscopic view of how disrupting basic cellular processes interrupt development or cause disease. "
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