Using enhanced-mitophagy to measure autophagic flux

Baudot, A., Haller, M., Mrschtik, M., Tait, S. and Ryan, K. (2015) Using enhanced-mitophagy to measure autophagic flux. Methods, 75, pp. 105-111. (doi:10.1016/j.ymeth.2014.11.014) (PMID:25498004) (PMCID:PMC4358839)

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Abstract

Macroautophagy (hereafter termed autophagy) is a cellular membrane-trafficking process that functions to deliver cytoplasmic constituents to lysosomes for degradation. Autophagy operates at basal levels to turn over damaged and misfolded proteins and it is the only process for the turnover of organelles. The process is therefore critically important for the preservation of cellular integrity and viability. Autophagy is also highly adaptable and the rate and cargoes of autophagy can be altered to bring about desired cellular responses to intracellular and environmental cues, disease states and a spectrum of pharmaceutical drugs. As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations. To date, the majority of assays to monitor autophagy either measure changes in a parameter of the process at a set point in time or use markers/tracers to monitor flow of membrane-bound proteins from one point in the process to another. As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process. We describe here an assay to measure autophagic cargo degradation by engineering cells to degrade mitochondria en masse. We show that this ‘enhanced-mitophagy’ assay can be used to measure differences in the rate of autophagy between different cells or in response to agents which are known to promote or inhibit autophagic flux. 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Baudot, Dr Alice and Ryan, Professor Kevin and Tait, Professor Stephen and Haller, Ms Martina
Authors: Baudot, A., Haller, M., Mrschtik, M., Tait, S., and Ryan, K.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Methods
Publisher:Elsevier
ISSN:1046-2023
ISSN (Online):1095-9130
Copyright Holders:Copyright © 2014 The Authors
First Published:First published in Methods 75:105-111
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
607521A new approach to understanding mitochondrial functions in cell death, autophagy and beyondStephen TaitBiotechnology and Biological Sciences Research Council (BBSRC)BB/K008374/1RI CANCER SCIENCES