Limits...
Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

Show MeSH

Related in: MedlinePlus

Parkin overexpression promotes mitophagy upon mitochondrial depolarization. In normal conditions the ser/thr kinase PINK1 is constitutively localized at the mitochondria. After overexpression of exogenous YFP-Parkin and treatment with antimycin A and oligomycin, mitochondrial proteins are depleted due to elimination of mitochondria en masse.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4358839&req=5

f0005: Parkin overexpression promotes mitophagy upon mitochondrial depolarization. In normal conditions the ser/thr kinase PINK1 is constitutively localized at the mitochondria. After overexpression of exogenous YFP-Parkin and treatment with antimycin A and oligomycin, mitochondrial proteins are depleted due to elimination of mitochondria en masse.

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.


Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

Parkin overexpression promotes mitophagy upon mitochondrial depolarization. In normal conditions the ser/thr kinase PINK1 is constitutively localized at the mitochondria. After overexpression of exogenous YFP-Parkin and treatment with antimycin A and oligomycin, mitochondrial proteins are depleted due to elimination of mitochondria en masse.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358839&req=5

f0005: Parkin overexpression promotes mitophagy upon mitochondrial depolarization. In normal conditions the ser/thr kinase PINK1 is constitutively localized at the mitochondria. After overexpression of exogenous YFP-Parkin and treatment with antimycin A and oligomycin, mitochondrial proteins are depleted due to elimination of mitochondria en masse.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

Show MeSH
Related in: MedlinePlus