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: Parkin-dependent mitophagy involves PTEN-induced putative kinase 1 (PINK1) and a ubiquitin ligase complex containing Parkin (Fig. 1). Studies have shown that the ectopic expression of Parkin in cells followed by treatment with agents that perturb mitochondrial function results in the complete removal of mitochondria over time [21,19]. We reasoned that by providing the cell with an excess endogenous cargo (damaged mitochondria), then the rate of loss of mitochondria would be directly related to the autophagic rate/flux within the cell. As a result, we considered that that this process of ‘enhanced-mitophagy’ could be used as an assay to measure differences in autophagic flux between cells and in response to external stimuli.
Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.