Journal of Bioenergetics and Biomembranes

, Volume 45, Issue 1–2, pp 87–99 | Cite as

Characterization of functionally distinct mitochondrial subpopulations

  • Janet E. Saunders
  • Craig C. Beeson
  • Rick G. Schnellmann


Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca2+ concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca2+-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30 % of the mitochondria showed changes in response to Ca2+, which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca2+ over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca2+-induced swelling.


Mitochondria Heterogeneity Flow cytometry Kidney Subpopulations 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arrington DD, Van Vleet TR, Schnellmann RG (2006) Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction. Am J Physiol Cell Physiol 291(6):C1159–C1171CrossRefGoogle Scholar
  2. Bizeau ME, Willis WT, Hazel JR (1998) Differential responses to endurance training in subsarcolemmal and intermyofibrillar mitochondria. J Appl Physiol 85(4):1279–1284Google Scholar
  3. Bouvier T et al (2001) Using light scatter signal to estimate bacterial biovolume by flow cytometry. Cytometry 44(3):188–194CrossRefGoogle Scholar
  4. Bowser DN et al (1998) Role of mitochondria in calcium regulation of spontaneously contracting cardiac muscle cells. Biophys J 75(4):2004–2014CrossRefGoogle Scholar
  5. Butler WH, Judah JD (1970) Ultrastructural studies on mitochondrial swelling. Biochem J 118(5):883–886Google Scholar
  6. Chen H, Chan DC (2009) Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases. Hum Mol Genet 18(R2):R169–R176CrossRefGoogle Scholar
  7. Cogswell AM, Stevens RJ, Hood DA (1993) Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions. Am J Physiol 264(2 Pt 1):C383–C389Google Scholar
  8. Cossarizza A, Ceccarelli D, Masini A (1996) Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level. Exp Cell Res 222(1):84–94CrossRefGoogle Scholar
  9. Detmer SA, Chan DC (2007) Functions and dysfunctions of mitochondrial dynamics. Nat Rev Mol Cell Biol 8(11):870–879CrossRefGoogle Scholar
  10. Fannin SW et al (1999) Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria. Arch Biochem Biophys 372(2):399–407CrossRefGoogle Scholar
  11. Foladori P, Quaranta A, Ziglio G (2008) Use of silica microspheres having refractive index similar to bacteria for conversion of flow cytometric forward light scatter into biovolume. Water Res 42(14):3757–3766CrossRefGoogle Scholar
  12. Goyer RA, Krall R (1969) Ultrastructural transformation in mitochondria isolated from kidneys of normal and lead-intoxicated rats. J Cell Biol 41(2):393–400CrossRefGoogle Scholar
  13. Halestrap AP, Davidson AM (1990) Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem J 268(1):153–160Google Scholar
  14. Hammes F, Egli T (2010) Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications. Anal Bioanal Chem 397(3):1083–1095Google Scholar
  15. Jacobson J, Duchen MR, Heales SJ (2002) Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass. J Neurochem 82(2):224–233CrossRefGoogle Scholar
  16. Jastroch M, et al (2010) Mitochondrial proton and electron leaks. Essays Biochem 47:53–67Google Scholar
  17. Jimenez M et al (2002) Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting. Eur J Biochem 269(12):2878–2884CrossRefGoogle Scholar
  18. Kinsey GR et al (2007) Role of Ca2 + -independent phospholipase A2gamma in Ca2 + -induced mitochondrial permeability transition. J Pharmacol Exp Ther 321(2):707–715CrossRefGoogle Scholar
  19. Krieger DA et al (1980) Populations of rat skeletal muscle mitochondria after exercise and immobilization. J Appl Physiol 48(1):23–28Google Scholar
  20. Kuznetsov AV, Margreiter R (2009) Heterogeneity of Mitochondria and Mitochondrial Function within Cells as Another Level of Mitochondrial Complexity. Int J Mol Sci 10(4):1911–1929CrossRefGoogle Scholar
  21. Kuznetsov AV et al (2006) Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: possible physiological role? Biochim Biophys Acta 1757(5–6):686–691Google Scholar
  22. Lecoeur H et al (2004) Real-time flow cytometry analysis of permeability transition in isolated mitochondria. Exp Cell Res 294(1):106–117CrossRefGoogle Scholar
  23. Lichtenberg D et al (1981) Effect of surface curvature on stability, thermodynamic behavior, and osmotic activity of dipalmitoylphosphatidylcholine single lamellar vesicles. Biochemistry 20(12):3462–3467CrossRefGoogle Scholar
  24. Lombardi A et al (2000) Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis. FEBS Lett 475(2):84–88CrossRefGoogle Scholar
  25. Lopez-Mediavilla C et al (1989) Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver. FEBS Lett 254(1–2):115–120CrossRefGoogle Scholar
  26. Mattiasson G (2004) Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death. Cytometry A 60(2):145–154CrossRefGoogle Scholar
  27. Medina JM, Lopez-Mediavilla C, Orfao A (2002) Flow cytometry of isolated mitochondria during development and under some pathological conditions. FEBS Lett 510(3):127–132CrossRefGoogle Scholar
  28. Mollica MP et al (2006) Heterogeneous bioenergetic behaviour of subsarcolemmal and intermyofibrillar mitochondria in fed and fasted rats. Cell Mol Life Sci 63(3):358–366CrossRefGoogle Scholar
  29. O’Toole JF, et al (2010) Decreased cytochrome c mediates an age-related decline of oxidative phosphorylation in rat kidney mitochondria. Biochem J 427(1): 105–112.Google Scholar
  30. Palmer JW, Tandler B, Hoppel CL (1977) Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252(23):8731–8739Google Scholar
  31. Palmer JW, Tandler B, Hoppel CL (1985) Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations. Arch Biochem Biophys 236(2):691–702CrossRefGoogle Scholar
  32. Palmer JW, Tandler B, Hoppel CL (1986) Heterogeneous response of subsarcolemmal heart mitochondria to calcium. Am J Physiol 250(5 Pt 2):H741–H748Google Scholar
  33. Petit PX et al (1990) Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur J Biochem 194(2):389–397CrossRefGoogle Scholar
  34. Poot M, Pierce RH (1999) Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry. Cytometry 35(4):311–317CrossRefGoogle Scholar
  35. Porter RK, Hulbert AJ, Brand MD (1996) Allometry of mitochondrial proton leak: influence of membrane surface area and fatty acid composition. Am J Physiol 271(6 Pt 2):R1550–R1560Google Scholar
  36. Robertson BR, Button DK, Koch AL (1998) Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry. Appl Environ Microbiol 64(10):3900–3909Google Scholar
  37. Rodriguez ME et al (2008) Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization. Mitochondrion 8(3):237–246CrossRefGoogle Scholar
  38. Roederer M et al (2001) Probability binning comparison: a metric for quantitating multivariate distribution differences. Cytometry 45(1):47–55CrossRefGoogle Scholar
  39. Schnellmann RG, Cross TJ, Lock EA (1989) Pentachlorobutadienyl-L-cysteine uncouples oxidative phosphorylation by dissipating the proton gradient. Toxicol Appl Pharmacol 100(3):498–505CrossRefGoogle Scholar
  40. Takahashi M, Hood DA (1996) Protein import into subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Differential import regulation in distinct subcellular regions. J Biol Chem 271(44):27285–27291CrossRefGoogle Scholar
  41. Twig G, Hyde B, Shirihai OS (2008) Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochim Biophys Acta 1777(9):1092–1097CrossRefGoogle Scholar
  42. Umegaki T et al (2008) Flow cytometric analysis of ca-induced membrane permeability transition of isolated rat liver mitochondria. J Clin Biochem Nutr 42:35–44CrossRefGoogle Scholar
  43. Wikstrom JD, Twig G, Shirihai OS (2009) What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy? Int J Biochem Cell Biol 41(10):1914–1927CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York (outside the USA) 2012

Authors and Affiliations

  • Janet E. Saunders
    • 1
  • Craig C. Beeson
    • 1
    • 2
  • Rick G. Schnellmann
    • 1
    • 3
  1. 1.Center for Cell Death, Injury, and Regeneration, Department of Pharmaceutical and Biomedical SciencesMedical University of South CarolinaCharlestonUSA
  2. 2.Department of Pharmaceutical and Biomedical SciencesMedical University of South CarolinaCharlestonUSA
  3. 3.Ralph Johnson VA Medical CenterCharlestonUSA

Personalised recommendations