Limits...
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.

Show MeSH

Related in: MedlinePlus

Assays for autophagic flux. (A) and (B) The tandem tagged mCherry-GFP-Atg8a reporter labels autophagosomes as yellow (positive for both GFP and mCherry), while autolysosomes appear mostly red due to faster lysosomal quenching of GFP than mCherry. Knockdown of Vps16A (encoding a HOPS tethering complex subunit) prevents autophagosome-lysosome fusion, so all dots are double positive for GFP and mCherry in fat body cells of a starved larva (panel B), compared to the larger dots that are only positive for mCherry in control cells (panel A). Note that this change is also obvious in the pixel intensity correlation profiles calculated from these images (left panels) and in Pearson correlation coefficients (R values). (C) Overexpression of the transcription factor Myc promotes autophagic degradation both under well-fed and starved conditions, as shown here by the increased conversion of mCherry-GFP-Atg8a into free mCherry in larval fat body lysates. Note that the level of the full-length protein is reduced accordingly. Vice versa, inhibition of Myc activity by overexpression of Mad, or by silencing Myc using either of two independent RNAi lines, reduces the autophagic degradation-dependent conversion of mCherry-GFP-Atg8a into free mCherry. Bar in panel A equals 20 μm for A, B.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4358840&req=5

f0025: Assays for autophagic flux. (A) and (B) The tandem tagged mCherry-GFP-Atg8a reporter labels autophagosomes as yellow (positive for both GFP and mCherry), while autolysosomes appear mostly red due to faster lysosomal quenching of GFP than mCherry. Knockdown of Vps16A (encoding a HOPS tethering complex subunit) prevents autophagosome-lysosome fusion, so all dots are double positive for GFP and mCherry in fat body cells of a starved larva (panel B), compared to the larger dots that are only positive for mCherry in control cells (panel A). Note that this change is also obvious in the pixel intensity correlation profiles calculated from these images (left panels) and in Pearson correlation coefficients (R values). (C) Overexpression of the transcription factor Myc promotes autophagic degradation both under well-fed and starved conditions, as shown here by the increased conversion of mCherry-GFP-Atg8a into free mCherry in larval fat body lysates. Note that the level of the full-length protein is reduced accordingly. Vice versa, inhibition of Myc activity by overexpression of Mad, or by silencing Myc using either of two independent RNAi lines, reduces the autophagic degradation-dependent conversion of mCherry-GFP-Atg8a into free mCherry. Bar in panel A equals 20 μm for A, B.

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.


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)

Assays for autophagic flux. (A) and (B) The tandem tagged mCherry-GFP-Atg8a reporter labels autophagosomes as yellow (positive for both GFP and mCherry), while autolysosomes appear mostly red due to faster lysosomal quenching of GFP than mCherry. Knockdown of Vps16A (encoding a HOPS tethering complex subunit) prevents autophagosome-lysosome fusion, so all dots are double positive for GFP and mCherry in fat body cells of a starved larva (panel B), compared to the larger dots that are only positive for mCherry in control cells (panel A). Note that this change is also obvious in the pixel intensity correlation profiles calculated from these images (left panels) and in Pearson correlation coefficients (R values). (C) Overexpression of the transcription factor Myc promotes autophagic degradation both under well-fed and starved conditions, as shown here by the increased conversion of mCherry-GFP-Atg8a into free mCherry in larval fat body lysates. Note that the level of the full-length protein is reduced accordingly. Vice versa, inhibition of Myc activity by overexpression of Mad, or by silencing Myc using either of two independent RNAi lines, reduces the autophagic degradation-dependent conversion of mCherry-GFP-Atg8a into free mCherry. Bar in panel A equals 20 μm for A, B.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0025: Assays for autophagic flux. (A) and (B) The tandem tagged mCherry-GFP-Atg8a reporter labels autophagosomes as yellow (positive for both GFP and mCherry), while autolysosomes appear mostly red due to faster lysosomal quenching of GFP than mCherry. Knockdown of Vps16A (encoding a HOPS tethering complex subunit) prevents autophagosome-lysosome fusion, so all dots are double positive for GFP and mCherry in fat body cells of a starved larva (panel B), compared to the larger dots that are only positive for mCherry in control cells (panel A). Note that this change is also obvious in the pixel intensity correlation profiles calculated from these images (left panels) and in Pearson correlation coefficients (R values). (C) Overexpression of the transcription factor Myc promotes autophagic degradation both under well-fed and starved conditions, as shown here by the increased conversion of mCherry-GFP-Atg8a into free mCherry in larval fat body lysates. Note that the level of the full-length protein is reduced accordingly. Vice versa, inhibition of Myc activity by overexpression of Mad, or by silencing Myc using either of two independent RNAi lines, reduces the autophagic degradation-dependent conversion of mCherry-GFP-Atg8a into free mCherry. Bar in panel A equals 20 μm for A, B.
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.

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