Advertisement

Methods for Mitochondria and Mitophagy Flux Analyses in Stem Cells of Resting and Regenerating Skeletal Muscle

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1460)

Abstract

Mitochondria generate most of the cell’s supply of ATP as a source of energy. They are also implicated in the control of cell’s growth and death. Because of these critical functions, mitochondrial fitness is key for cellular homeostasis. Often, however, mitochondria become defective following damage or stress. To prevent accumulation of damaged mitochondria, the cells clear them through mitophagy, which is defined as the selective degradation of mitochondria by autophagy (the process for degradation of long-lived proteins and damaged organelles in lysosomes). Recently, constitutive mitophagic activity has been reported in quiescent muscle stem cells (satellite cells), which sustain regeneration of skeletal muscle. In response to muscle damage, these cells activate, expand, and differentiate to repair damaged myofibers. Mitophagy was shown to be required for maintenance of satellite cells in their healthy quiescent state. Conversely, damaged mitochondria accumulated in satellite cells with aging and this was attributed to defective mitophagy. This caused increased levels of reactive oxygen species (ROS) and loss of muscle stem cell regenerative capacity at old age. In this chapter, we describe different experimental strategies to evaluate mitochondria status and mitophagy in muscle stem cells from mice. They should improve our ability to study muscle stem homeostasis in adult life, and their loss of function in aging and disease.

Key words

Skeletal muscle regeneration Stem cell Satellite cell Mitochondria Mitophagy Autophagy Mitocondria membrane potential Reactive oxygen species (ROS) 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

Work in the authors’ laboratories has been supported by: MINECO, Spain SAF2012-38547, AFM, E-Rare/Eranet, Fundació Marató-TV3, MDA, EU-FP7 (Myoage, Optistem and Endostem) and DuchennePP-NL, to PM-C; and ISCIII, Spain (FIS-PS09/01267, FIS-PI13/02512, CP09/00184, PI14/01529) and CIBERNED to MM-V. L.G.-P. was supported by a Predoctoral Fellowship from Programa de Formación de Personal Investigador (Spain).

References

  1. 1.
    Mauro-Lizcano M et al (2015) New method to assess mitophagy flux by flow cytometry. Autophagy 11(5):833–843CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Dolman NJ et al (2013) Tools and techniques to measure mitophagy using fluorescence microscopy. Autophagy 9(11):1653–1662CrossRefPubMedGoogle Scholar
  3. 3.
    Cuervo AM (2004) Autophagy: many paths to the same end. Mol Cell Biochem 263(1–2):55–72CrossRefPubMedGoogle Scholar
  4. 4.
    Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6(4):463–477CrossRefPubMedGoogle Scholar
  5. 5.
    Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306(5698):990–995CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Klionsky DJ (2005) Autophagy. Curr Biol 15(8):R282–R283CrossRefPubMedGoogle Scholar
  7. 7.
    Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40(2):280–293CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ashrafi G, Schwarz TL (2013) The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ 20(1):31–42CrossRefPubMedGoogle Scholar
  9. 9.
    Clark SL Jr (1957) Cellular differentiation in the kidneys of newborn mice studies with the electron microscope. J Biophys Biochem Cytol 3(3):349–362CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wei H, Liu L, Chen Q (2015) Selective removal of mitochondria via mitophagy: distinct pathways for different mitochondrial stresses. Biochim Biophys Acta 1853(10 Pt B):2784–2790CrossRefPubMedGoogle Scholar
  11. 11.
    Yoshii SR, Mizushima N (2015) Autophagy machinery in the context of mammalian mitophagy. Biochim Biophys Acta 1853(10 Pt B):2797–2801CrossRefPubMedGoogle Scholar
  12. 12.
    Youle RJ, Narendra DP (2011) Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12(1):9–14CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Novak I et al (2010) Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 11(1):45–51CrossRefPubMedGoogle Scholar
  14. 14.
    Rikka S et al (2011) Bnip3 impairs mitochondrial bioenergetics and stimulates mitochondrial turnover. Cell Death Differ 18(4):721–731CrossRefPubMedGoogle Scholar
  15. 15.
    Hanna RA et al (2012) Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy. J Biol Chem 287(23):19094–19104CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Liu L et al (2012) Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat Cell Biol 14(2):177–185CrossRefPubMedGoogle Scholar
  17. 17.
    Geisler S et al (2010) PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol 12(2):119–131CrossRefPubMedGoogle Scholar
  18. 18.
    Kirkin V et al (2009) A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol Cell 33(4):505–516CrossRefPubMedGoogle Scholar
  19. 19.
    Lazarou M et al (2015) The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524(7565):309–314CrossRefPubMedGoogle Scholar
  20. 20.
    Ying H, Yue BY (2016) Optineurin: the autophagy connection. Exp Eye Res 144:73–80CrossRefPubMedGoogle Scholar
  21. 21.
    Johansen T, Lamark T (2011) Selective autophagy mediated by autophagic adapter proteins. Autophagy 7(3):279–296CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kraft C, Peter M, Hofmann K (2010) Selective autophagy: ubiquitin-mediated recognition and beyond. Nat Cell Biol 12(9):836–841CrossRefPubMedGoogle Scholar
  23. 23.
    Wild P, McEwan DG, Dikic I (2014) The LC3 interactome at a glance. J Cell Sci 127(Pt 1):3–9CrossRefPubMedGoogle Scholar
  24. 24.
    Sandoval H et al (2008) Essential role for Nix in autophagic maturation of erythroid cells. Nature 454(7201):232–235CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zhang J, Ney PA (2009) Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ 16(7):939–946CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Chu CT et al (2013) Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol 15(10):1197–1205CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Bertolin G et al (2013) The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance. Autophagy 9(11):1801–1817CrossRefPubMedGoogle Scholar
  28. 28.
    Orvedahl A et al (2011) Image-based genome-wide siRNA screen identifies selective autophagy factors. Nature 480(7375):113–117CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Fu M et al (2013) Regulation of mitophagy by the Gp78 E3 ubiquitin ligase. Mol Biol Cell 24(8):1153–1162CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Van Humbeeck C et al (2011) Parkin interacts with Ambra1 to induce mitophagy. J Neurosci 31(28):10249–10261CrossRefPubMedGoogle Scholar
  31. 31.
    Melser S et al (2013) Rheb regulates mitophagy induced by mitochondrial energetic status. Cell Metab 17(5):719–730CrossRefPubMedGoogle Scholar
  32. 32.
    Garcia-Prat L, Sousa-Victor P, Munoz-Canoves P (2013) Functional dysregulation of stem cells during aging: a focus on skeletal muscle stem cells. FEBS J 280(17):4051–4062CrossRefPubMedGoogle Scholar
  33. 33.
    Garcia-Prat L et al (2016) Autophagy maintains stemness by preventing senescence. Nature 529(7584): 37–42Google Scholar
  34. 34.
    Warnes G (2015) Flow cytometric assays for the study of autophagy. Methods 82:21–28CrossRefPubMedGoogle Scholar
  35. 35.
    Cottet-Rousselle C et al (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79(6):405–425CrossRefPubMedGoogle Scholar
  36. 36.
    Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224(Pt 3):213–232CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF)CIBER on Neurodegenerative diseases (CIBERNED)BarcelonaSpain
  2. 2.Neurodegenerative Diseases Research GroupVall d’Hebron Research Institute-CIBERNEDBarcelonaSpain
  3. 3.Institució Catalana de Recerca i Estudis Avançats (ICREA)BarcelonaSpain

Personalised recommendations

Cite protocol

Buy options