How and why to study autophagy in Drosophila: it's more than just a garbage chute.
Bottom Line: During the catabolic process of autophagy, cytoplasmic material is transported to the lysosome for degradation and recycling.This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.
Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.Show MeSH
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Mentions: The tandem tagged version of Atg8a is very frequently used for estimating autophagic flux in mammalian cells. This assay is based on the fact that GFP is quenched more rapidly in autolysosomes than mCherry, which made it possible to study autophagic degradation, first in cultured human cells and later in Drosophila[15,39]. Phagophores and autophagosomes appear yellow (both green and red) in merged images, whereas autolysosomes are labeled mostly red by this reporter (Fig. 5A and B) [15,18,39,40,63,74]. Thus, this assay is simple and easy to carry out, even though it is not as widely used in Drosophila as in cultured mammalian cells. Potential reasons for why it is not so popular in flies may be that it needs to be crossed into the genotype of interest, and that in mosaic analysis, one needs to mark mutant/RNAi clone cells which usually requires the use of GFP or RFP. An alternative solution to this latter problem is to use the flux reporter itself to identify cell clones (although in this case the surrounding tissue is not expressing the reporter, so it cannot be used as a built-in control). Nevertheless, we think that the tandem tagged Atg8a reporter is a very useful tool for estimating autophagic flux in the fly.
Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.