Abstract
Mitochondria are organelles with numerous vital roles in cellular metabolism. Impaired or damaged mitochondria are degraded in autophagolysosomes in a process known as mitophagy. Given the fundamental role of mitophagy in maintenance of cellular homeostasis, methods and techniques with which to study this process are constantly evolving and emerging. So far, mitophagy flux was mostly monitored using fluorescently labeled LC3 protein on autophagosomal membrane and any of the labeled outer mitochondrial membrane proteins. However, this method is labor intensive, time consuming, and difficult to quantitatively validate due to the rapid mitochondrial turnover. Here, we describe a flow cytometry as a novel and promising quantitative method to monitor Bnip3- and Bnip3L/Nix-mediated mitophagy.
References
Ney PA (2015) Mitochondrial autophagy: origins, significance, and role of BNIP3 and NIX. Biochim Biophys Acta 1853:2775–2783
Hamacher-Brady A, Brady NR (2015) Bax/Bak-dependent, Drp1-independent targeting of X-linked inhibitor of apoptosis protein (XIAP) into inner mitochondrial compartments counteracts Smac/DIABLO-dependent effector caspase activation. J Biol Chem 290(36):22005–22018
Novak I (2012) Mitophagy: a complex mechanism of mitochondrial removal. Antioxid Redox Signal 17(5):794–802
Lazarou M et al (2015) The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524(7565):309–314
Novak I et al (2010) Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 11(1):45–51
Zhu Y et al (2013) Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis. J Biol Chem 288(2):1099–1113
Murakawa T et al (2015) Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation. Nat Commun 6:7527
Wei H, Liu L, Chen Q (2015) Selective removal of mitochondria via mitophagy: distinct pathways for different mitochondrial stresses. Biochim Biophys Acta 1853:2784–2790
Mauro-Lizcano M et al (2015) New method to assess mitophagy flux by flow cytometry. Autophagy 11(5):833–843
Zhang J, Ney PA (2010) Reticulocyte mitophagy: monitoring mitochondrial clearance in a mammalian model. Autophagy 6(3):405–408
Puleston D et al (2015) Techniques for the detection of autophagy in primary mammalian cells. Cold Spring Harb Protoc 2015(9):070391
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Šprung, M., Dikic, I., Novak, I. (2017). Flow Cytometer Monitoring of Bnip3- and Bnip3L/Nix-Dependent Mitophagy. In: Hattori, N., Saiki, S. (eds) Mitophagy. Methods in Molecular Biology, vol 1759. Humana Press, New York, NY. https://doi.org/10.1007/7651_2017_14
Download citation
DOI: https://doi.org/10.1007/7651_2017_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7749-9
Online ISBN: 978-1-4939-7750-5
eBook Packages: Springer Protocols