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 two most commonly used endogenous proteins evaluated in western blots are again Atg8 and p62 in Drosophila. The increased amount of p62 may indicate a block of autophagy (Fig. 4A and B) (note that the fly protein is larger than its mammalian homologs and migrates near 100 kDa) [18,40,41,57,58,63]. The level of p62 that can be detected in western blots is strongly influenced by sample preparation. Serial detergent extraction experiments revealed that not all of the p62 pool is recovered in a non-ionic detergent (such as Triton X-100) fraction, since aggregates containing p62 and ubiquitinated proteins that accumulate upon inhibition of autophagy must be solubilized by ionic detergents such as SDS . We routinely boil the samples that we collect (such as fly heads or whole animals) in an SDS-containing Laemmli buffer for 3 min, which is followed by homogenization and another round of boiling to recover as much protein as possible. Even under these harsh extraction conditions, some of the p62 pool likely remains aggregated in autophagy mutant heads, based on p62 signal at a very high molecular weight in western blots .
Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.