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How and why to study autophagy in Drosophila: it's more than just a garbage chute.

Nagy P, Varga Á, Kovács AL, Takáts S, Juhász G - Methods (2014)

Bottom Line: This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.

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Western blot analysis of autophagy. (A) The autophagic cargo p62 accumulates in starved larvae lacking the core autophagy gene Atg7, and also in animals that are mutant for Vps16A or Vps11, encoding subunits of the HOPS tethering complex. Autophagosome-associated, lipidated Atg8a-II is missing in Atg7 mutants but accumulates in HOPS loss-of-function (mutant for Vps16A or Vps11) larvae, as this tethering complex is required for the fusion of autophagosomes with lysosomes (compare Atg8a-II levels to tubulin in controls and mutants, respectively). (B) Various Atg mutants all accumulate p62, whereas the levels of Atg8a-II are increased in starved larvae lacking Atg1, Atg13, Atg18 and Atg2. Atg7 and Atg3 encode the E1 and E2 enzymes required for Atg8a lipidation, respectively, so the generation of Atg8a-II is blocked in these mutants. Both forms of Atg8a are missing in Atg8a mutants, as expected.
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f0020: Western blot analysis of autophagy. (A) The autophagic cargo p62 accumulates in starved larvae lacking the core autophagy gene Atg7, and also in animals that are mutant for Vps16A or Vps11, encoding subunits of the HOPS tethering complex. Autophagosome-associated, lipidated Atg8a-II is missing in Atg7 mutants but accumulates in HOPS loss-of-function (mutant for Vps16A or Vps11) larvae, as this tethering complex is required for the fusion of autophagosomes with lysosomes (compare Atg8a-II levels to tubulin in controls and mutants, respectively). (B) Various Atg mutants all accumulate p62, whereas the levels of Atg8a-II are increased in starved larvae lacking Atg1, Atg13, Atg18 and Atg2. Atg7 and Atg3 encode the E1 and E2 enzymes required for Atg8a lipidation, respectively, so the generation of Atg8a-II is blocked in these mutants. Both forms of Atg8a are missing in Atg8a mutants, as expected.

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 [57]. 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 [41].


How and why to study autophagy in Drosophila: it's more than just a garbage chute.

Nagy P, Varga Á, Kovács AL, Takáts S, Juhász G - Methods (2014)

Western blot analysis of autophagy. (A) The autophagic cargo p62 accumulates in starved larvae lacking the core autophagy gene Atg7, and also in animals that are mutant for Vps16A or Vps11, encoding subunits of the HOPS tethering complex. Autophagosome-associated, lipidated Atg8a-II is missing in Atg7 mutants but accumulates in HOPS loss-of-function (mutant for Vps16A or Vps11) larvae, as this tethering complex is required for the fusion of autophagosomes with lysosomes (compare Atg8a-II levels to tubulin in controls and mutants, respectively). (B) Various Atg mutants all accumulate p62, whereas the levels of Atg8a-II are increased in starved larvae lacking Atg1, Atg13, Atg18 and Atg2. Atg7 and Atg3 encode the E1 and E2 enzymes required for Atg8a lipidation, respectively, so the generation of Atg8a-II is blocked in these mutants. Both forms of Atg8a are missing in Atg8a mutants, as expected.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358840&req=5

f0020: Western blot analysis of autophagy. (A) The autophagic cargo p62 accumulates in starved larvae lacking the core autophagy gene Atg7, and also in animals that are mutant for Vps16A or Vps11, encoding subunits of the HOPS tethering complex. Autophagosome-associated, lipidated Atg8a-II is missing in Atg7 mutants but accumulates in HOPS loss-of-function (mutant for Vps16A or Vps11) larvae, as this tethering complex is required for the fusion of autophagosomes with lysosomes (compare Atg8a-II levels to tubulin in controls and mutants, respectively). (B) Various Atg mutants all accumulate p62, whereas the levels of Atg8a-II are increased in starved larvae lacking Atg1, Atg13, Atg18 and Atg2. Atg7 and Atg3 encode the E1 and E2 enzymes required for Atg8a lipidation, respectively, so the generation of Atg8a-II is blocked in these mutants. Both forms of Atg8a are missing in Atg8a mutants, as expected.
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 [57]. 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 [41].

Bottom Line: This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.

Show MeSH
Related in: MedlinePlus