Using enhanced-mitophagy to measure autophagic flux.
Bottom Line: As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations.As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process.We consider therefore that this assay will prove to be a valuable resource for investigations in which autophagy is considered important and is believed to be modulated.
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.Show MeSH
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Mentions: Since autophagy is the only cellular mechanism for mitochondrial removal, we were interested to know if enhanced-mitophagy could be used to reveal differences in autophagic flux between cells. Saos-2 osteosarcoma cells and SVEC endothelial cells were therefore infected with YFP-Parkin to enable mass elimination of mitochondria by autophagy and the depletion of mitochondrial proteins was measured in these and YFP-Parkin-expressing 3T3-SA cells following treatment with antimycin A and oligomycin. This revealed that not only were there significant differences in the rate of loss of mitochondrial proteins between different cell lines, but there were also differences in the rate of depletion of different mitochondrial proteins in each individual cell line Fig. 3A and B). Notably, cytochrome c and Porin (also known as VDAC1) are depleted very rapidly in SVEC and 3T3-SA after treatment with antimycin A and oligomycin (Fig. 3A and B). As a result, we did not consider them useful in determining differential rates of autophagy in these cells over this time frame.
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.