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Preparation of “Functional” Mitochondria: A Challenging Business

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1264)

Abstract

As the powerhouse of the cell, mitochondria play a crucial role in many aspects of life, whereby mitochondrial dysfunctions are associated with pathogenesis of many diseases, like neurodegenerative diseases, obesity, cancer, and metabolic as well as cardiovascular disorders. Mitochondria analysis frequently starts with isolation and enrichment procedures potentially affecting mitochondrial morphology having impact on their function. Due to the complex mitochondrial morphology, the major task is to preserve their structural integrity. Here we critically review a commonly used isolation procedure for mitochondria utilizing differential (gradient) centrifugation and depict major challenges to achieve “functional” mitochondria as basis for comprehensive physiological studies.

Key words

Isolation of mitochondria Differential gradient centrifugation Mitochondrial integrity 
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References

  1. 1.
    Nunnari J, Suomalainen A (2012) Mitochondria: in sickness and in health. Cell 148:1145–1159PubMedCrossRefGoogle Scholar
  2. 2.
    Elliott HR, Samuels DC, Eden JA, Relton CL, Chinnery PF (2008) Pathogenic mitochondrial DNA mutations are common in the general population. Am J Hum Genet 83(1030):254–260PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Schon EA, DiMauro S, Hirano M (2012) Human mitochondrial DNA: roles of inherited and somatic mutations. Nat Rev Genet 13:878–890PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Patti ME, Corvera S (2010) The role of mitochondria in the pathogenesis of type 2 diabetes. Endocr Rev 31:364–395PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Wallace DC (2012) Mitochondria and cancer. Nat Rev Cancer 12:685–698PubMedCrossRefGoogle Scholar
  6. 6.
    Piaceri I, Rinnoci V, Bagnoli S, Failli Y, Sorbi S (2012) Mitochondria and Alzheimer’s disease. J Neurol Sci 322:31–34PubMedCrossRefGoogle Scholar
  7. 7.
    Hill BG (2013) Recent advances in mitochondrial research. Circ Res 113:107–110CrossRefGoogle Scholar
  8. 8.
    Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2:287–295PubMedCrossRefGoogle Scholar
  9. 9.
    Hartwig S, Feckler C, Lehr S, Wallbrecht K, Wolgast H, Müller-Wieland D, Kotzka J (2009) A critical comparison between two classical and a kit-based method for mitochondria isolation. J Proteomics 11:3209–3214CrossRefGoogle Scholar
  10. 10.
    Hogeboom GH, Schneider WC, Pallade GE (1948) Cytochemical studies of mammalian tissues. I. Isolation of intact mitochondria from rat liver; some biochemical properties of mitochondria and submicroscopic particulate material. J Biol Chem 172:619–635PubMedGoogle Scholar
  11. 11.
    Mitchell P, Moyle J (1967) Chemiosmotic hypothesis of oxidative phosphorylation. Nature 213:137–139PubMedCrossRefGoogle Scholar
  12. 12.
    Nass MM, Nass S (1963) Intramitochondrial fibers with DNA characteristics. I. Fixation and electron staining reactions. J Cell Biol 19:593–611PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Palade GE (1952) The fine structure of mitochondria. Anat Rec 114:427–451PubMedCrossRefGoogle Scholar
  14. 14.
    Graham JM (2001) Purification of a crude mitochondrial fraction by density-gradient centrifugation. Curr Protoc Cell Biol Chapter 3:Unit 3.4Google Scholar
  15. 15.
    Noguchi T, Fujiwara S (1984) Developmental profiles and properties of hepatic peroxisomal apo- and mitochondrial holoalanine: glyoxylate aminotransferase during chick embryogenesis. J Biol Chem 259:14498–14504PubMedGoogle Scholar
  16. 16.
    Liu Y, He J, Ji S, Wang Q et al (2008) A comparative study of early liver dysfunction in senescence-accelerated mouse using mitochondrial proteomics approaches. Mol Cell Proteomics 7:1737–1747PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Lehr S, Kotzka J, Avci H, Knebel B et al (2005) Effect of sterol regulatory element binding protein-1a on the mitochondrial protein pattern in human liver cells detected by 2D-DIGE. Biochemistry 44:5117–5128PubMedCrossRefGoogle Scholar
  18. 18.
    Zischka H, Weber G, Weber PJ, Posch A et al (2003) Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 3:906–916PubMedCrossRefGoogle Scholar
  19. 19.
    Stahl WL, Smith JC, Napolitano LM, Basford RE (1963) BRAIN MITOCHONDRIA: I. Isolation of bovine brain mitochondria. J Cell Biol 19:293–307PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Cannon B, Lindberg O (1979) Mitochondria from brown adipose tissue: isolation and properties. Methods Enzymol 55:65–78PubMedCrossRefGoogle Scholar
  21. 21.
    Mela L, Seitz S (1997) Isolation of mitochondria with emphasis on heart mitochondria from small amounts of tissue. Methods Enzymol 55:39–46CrossRefGoogle Scholar
  22. 22.
    Ogata T, Yamasaki Y (1997) Ultra-high-resolution scanning electron microscopy of mitochondria and sarcoplasmic reticulum arrangement in human red, white, and intermediate muscle fibres. Anat Rec 248:214–223PubMedCrossRefGoogle Scholar
  23. 23.
    Fang H, Chen M, Ding Y, Shang W, Xu J et al (2011) Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals. Cell Res 21:1295–1304PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Picard M, Taivassalo T, Ritchie D, Wright KJ, Thomas MT et al (2011) Mitochondrial structure and function are disrupted by standard isolation methods. PLoS One 6:e18317PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibres, tissues and cells. Nat Protoc 3:965–976PubMedCrossRefGoogle Scholar
  26. 26.
    Saks V, Guzun R, Timohhina N, Tepp K, Varikmaa M et al (2010) Structure-function relationships in feedback regulation of energy fluxes in vivo in health and disease: mitochondrial interactosome. Biochim Biophys Acta 1797:678–697PubMedCrossRefGoogle Scholar
  27. 27.
    Franko A, Baris OR, Bergschneider E, von Toerne C, Hauck SM et al (2013) Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads. PLoS One 8:e82392PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Schulman JD, Blass JP (1971) Measurement of citrate synthase activity in human fibroblasts. Clin Chim Acta 33:467–469PubMedCrossRefGoogle Scholar
  29. 29.
    Pennington RJ (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J 80:649–654PubMedCentralPubMedGoogle Scholar
  30. 30.
    Bergmeyer HU (1974) Methoden der enzymatischen analyse. Verlag Chemie, WeinheimGoogle Scholar
  31. 31.
    Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126PubMedCrossRefGoogle Scholar
  32. 32.
    Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, Chen LB (1995) Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 260:406–417PubMedCrossRefGoogle Scholar
  33. 33.
    Li Z, Graham BH (2012) Measurement of mitochondrial oxygen consumption using a Clark electrode. Methods Mol Biol 837:63–72PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Leibniz Center for Diabetes ResearchHeinrich-Heine-University DuesseldorfDuesseldorfGermany

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