pp 1-9 | Cite as

Detection of Hypoxia-Induced and Iron Depletion-Induced Mitophagy in Mammalian Cells

Protocol
Part of the Methods in Molecular Biology book series

Abstract

Mitochondrial quality and quantity are not only regulated by mitochondrial fusion and fission but also by mitochondria degradation. Mitophagy, an autophagy specific for damaged or unnecessary mitochondria, is believed to be an important pathway for mitochondrial homeostasis. To date, several stimuli are known to induce mitophagy. Some of these stimuli, however, including hypoxia, iron depletion, and nitrogen starvation, induce mild mitophagy, which is difficult to detect through decreased mitochondrial mass. Recently, we have clearly detected mitophagy induced under these conditions using mito-Keima as a reporter. In this chapter, we describe the protocols for induction and detection of hypoxia-induced and iron depletion-induced mitophagy using mito-Keima-expressed cells.

Keywords:

Mitochondria Autophagy Mitophagy Hypoxia Iron depletion Deferiprone Keima 

References

  1. 1.
    Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS (2008) Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J 27(2):433–446. doi:10.1038/sj.emboj.7601963 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407. doi:10.1146/annurev.genet.39.110304.095751 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, Springer W (2010) PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 12(2):119–131. doi:10.1038/ncb2012 CrossRefPubMedGoogle Scholar
  4. 4.
    Matsuda N, Sato S, Shiba K, Okatsu K, Saisho K, Gautier CA, Sou YS, Saiki S, Kawajiri S, Sato F, Kimura M, Komatsu M, Hattori N, Tanaka K (2010) PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J Cell Biol 189(2):211–221. doi:10.1083/jcb.200910140 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Novak I, Kirkin V, McEwan DG, Zhang J, Wild P, Rozenknop A, Rogov V, Lohr F, Popovic D, Occhipinti A, Reichert AS, Terzic J, Dotsch V, Ney PA, Dikic I (2010) Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 11(1):45–51. doi:10.1038/embor.2009.256 CrossRefPubMedGoogle Scholar
  6. 6.
    Sandoval H, Thiagarajan P, Dasgupta SK, Schumacher A, Prchal JT, Chen M, Wang J (2008) Essential role for Nix in autophagic maturation of erythroid cells. Nature 454(7201):232–235. doi:10.1038/nature07006 ADSCrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Schweers RL, Zhang J, Randall MS, Loyd MR, Li W, Dorsey FC, Kundu M, Opferman JT, Cleveland JL, Miller JL, Ney PA (2007) NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Proc Natl Acad Sci U S A 104(49):19500–19505. doi:10.1073/pnas.0708818104 ADSCrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhang Y, Goldman S, Baerga R, Zhao Y, Komatsu M, Jin S (2009) Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proc Natl Acad Sci U S A 106(47):19860–19865. doi:10.1073/pnas.0906048106 ADSCrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hirota Y, S-i Y, Kurihara Y, Jin X, Aihara M, Saigusa T, Kang D, Kanki T (2015) Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways. Autophagy 11(2):332–343. doi:10.1080/15548627.2015.1023047 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Liu L, Feng D, Chen G, Chen M, Zheng Q, Song P, Ma Q, Zhu C, Wang R, Qi W, Huang L, Xue P, Li B, Wang X, Jin H, Wang J, Yang F, Liu P, Zhu Y, Sui S, Chen Q (2012) Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat Cell Biol 14(2):177–185. doi:10.1038/ncb2422 CrossRefPubMedGoogle Scholar
  11. 11.
    Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB, Gonzalez FJ, Semenza GL (2008) Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem 283(16):10892–10903. doi:10.1074/jbc.M800102200 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Allen GF, Toth R, James J, Ganley IG (2013) Loss of iron triggers PINK1/Parkin-independent mitophagy. EMBO Rep 14(12):1127–1135. doi:10.1038/embor.2013.168 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Narendra D, Kane LA, Hauser DN, Fearnley IM, Youle RJ (2010) p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both. Autophagy 6(8):1090–1106CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Narendra D, Tanaka A, Suen DF, Youle RJ (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 183(5):795–803. doi:10.1083/jcb.200809125 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Vives-Bauza C, Zhou C, Huang Y, Cui M, de Vries RL, Kim J, May J, Tocilescu MA, Liu W, Ko HS, Magrane J, Moore DJ, Dawson VL, Grailhe R, Dawson TM, Li C, Tieu K, Przedborski S (2010) PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci U S A 107(1):378–383. doi:10.1073/pnas.0911187107 ADSCrossRefPubMedGoogle Scholar
  16. 16.
    Lazarou M, Sliter DA, Kane LA, Sarraf SA, Wang C, Burman JL, Sideris DP, Fogel AI, Youle RJ (2015) The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524(7565):309–314. doi:10.1038/nature14893 ADSCrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A (2011) A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol 18(8):1042–1052. doi:10.1016/j.chembiol.2011.05.013 CrossRefPubMedGoogle Scholar
  18. 18.
    Kageyama Y, Hoshijima M, Seo K, Bedja D, Sysa-Shah P, Andrabi SA, Chen W, Hoke A, Dawson VL, Dawson TM, Gabrielson K, Kass DA, Iijima M, Sesaki H (2014) Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain. EMBO J 33(23):2798–2813. doi:10.15252/embj.201488658 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kogure T, Karasawa S, Araki T, Saito K, Kinjo M, Miyawaki A (2006) A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat Biotechnol 24(5):577–581. doi:10.1038/nbt1207 CrossRefPubMedGoogle Scholar
  20. 20.
    Violot S, Carpentier P, Blanchoin L, Bourgeois D (2009) Reverse pH-dependence of chromophore protonation explains the large stokes shift of the red fluorescent protein mKeima. J Am Chem Soc 131(30):10356–10357. doi:10.1021/ja903695n CrossRefPubMedGoogle Scholar
  21. 21.
    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(4):685–696. doi:10.1016/j.molcel.2015.10.009 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Cellular PhysiologyNiigata University Graduate School of Medical and Dental SciencesNiigataJapan

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