Abstract
Autophagy evolved as a mechanism to sustain cellular homeostasis during instances of nutrient deprivation. Mounting evidence has also clarified that under basal and stress conditions, selective autophagy pathways can target the destruction of specific organelles. Mitochondrial autophagy, or mitophagy, has emerged as a key quality control (QC) mechanism to sustain the integrity of eukaryotic mitochondrial networks. We recently reported the development of mito-QC, a novel reporter mouse model that enables the high-resolution study of mammalian mitophagy with precision, in fixed and live preparations. This model holds significant potential to transform our understanding of mammalian mitophagy pathways in vivo, in a variety of physiological contexts. We outline a detailed protocol for use of our recently described mito-QC mouse model, including tips and troubleshooting advice for those interested in monitoring mitophagy in vitro and in vivo.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nunnari J, Suomalainen A (2012) Mitochondria: in sickness and in health. Cell 148(6):1145–1159
McWilliams TG, Muqit MMK (2017) PINK1 and Parkin: emerging themes in mitochondrial homeostasis. Curr Opin Cell Biol 45:83–91
McWilliams TG, Ganley IG (2016) Life in lights: illuminating mitochondrial delivery to the lysosome in vivo. Autophagy 12(12):2506–2507
McWilliams TG, Prescott AR, Allen GFG, Tamjar J, Munson MJ, Muqit MMK, Ganley IG (2016) Mito-QC illuminates mitophagy and mitochondrial architecture in vivo. The Journal of Cell Biology 214(3):333–345
Sun N, Yun J, Liu J, Malide D, Liu C, Rovira II, Holmstrom KM, Fergusson MM, Yoo YH, Combs CA, Finkel T (2015) Measuring in vivo mitophagy. Mol Cell 60:685–696
Stotland A, Gottlieb RA (2016) α-MHC MitoTimer mouse: in vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart. J Mol Cell Cardiol 90:53–58
Nowotschin S et al (2009) Live-imaging fluorescent proteins in mouse embryos: multi-dimensional, multi-spectral perspectives. Trends Biotechnol 27(5):266–276
McWilliams TG, Prescott AR, Montava-Garriga L, Ball G, Singh F, Barini E, Muqit MMK, Brooks SP, Ganley IG (2018) Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand. Cell Metab 27(2):439–449.e5. https://doi.org/10.1016/j.cmet.2017.12.008. Epub 2018 Jan 11
Lee JJ, Sanchez-Martinez A, Zarate AM, Benincà C, Mayor U, Clague MJ, Whitworth AJ (2018) Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol 217(5):1613–1622. https://doi.org/10.1083/jcb.201801044. Epub 2018 Mar 2
Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, Alper S (1996) Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105(4):261–267
Singhal PK, Sassi S, Lan L, Au P, Halvorsen SC, Fukumura D, Jain R, Seed B (2016) Mouse embryonic fibroblasts exhibit extensive developmental and phenotypic diversity. Proc Natl Acad Sci 113:122–127
Allen GFG, Toth R, James J, Ganley IG (2013) Loss of iron triggers PINK1/Parkin-independent mitophagy. EMBO Rep 14(12):1127–1135
Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159:896–910
Acknowledgments
We gratefully acknowledge the outstanding support from our colleagues and collaborators who have enabled us to develop and refine our methods using mito-QC. In particular, we wish to thank Dr. Alan R. Prescott for his expertise in microscopy and Dr. Graeme Ball for his expertise in image analysis. We thank Dr. Ngaire Dennison for her guidance on animal experiments. We thank Lambert Montava-Garriga, Dr. Jin-Feng Zhao, and Dr. François Singh for their critical reading of the manuscript. This work was funded by Medical Research Council, UK (IGG; MC_UU_12016/4).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
McWilliams, T.G., Ganley, I.G. (2019). Investigating Mitophagy and Mitochondrial Morphology In Vivo Using mito-QC: A Comprehensive Guide. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_41
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8873-0_41
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8872-3
Online ISBN: 978-1-4939-8873-0
eBook Packages: Springer Protocols