Advertisement

Annals of Biomedical Engineering

, Volume 40, Issue 9, pp 1903–1916 | Cite as

Mitochondrial Dynamics and Motility Inside Living Vascular Endothelial Cells: Role of Bioenergetics

  • Randy J. Giedt
  • Douglas R. Pfeiffer
  • Anastasios Matzavinos
  • Chiu-Yen Kao
  • B. Rita Alevriadou
Article

Abstract

The mitochondrial network is dynamic with conformations that vary between a tubular continuum and a fragmented state. The equilibrium between mitochondrial fusion/fission, as well as the organelle motility, determine network morphology and ultimately mitochondrial/cell function. Network morphology has been linked with the energy state in different cell types. In this study, we examined how bioenergetic factors affect mitochondrial dynamics/motility in cultured vascular endothelial cells (ECs). ECs were transduced with mitochondria-targeted green fluorescent protein (mito-GFP) and exposed to inhibitors of oxidative phosphorylation (OXPHOS) or ATP synthesis. Time-lapse fluorescence videos were acquired and a mathematical program that calculates size and speed of each mitochondrial object at each time frame was developed. Our data showed that inner mitochondrial membrane potential (ΔΨm), ATP produced by glycolysis, and, to a lesser degree, ATP produced by mitochondria are critical for maintaining the mitochondrial network, and different metabolic stresses induce distinct morphological patterns (e.g., mitochondrial depolarization is necessary for “donut” formation). Mitochondrial movement, characterized by Brownian diffusion with occasional bursts in displacement magnitude, was inhibited under the same conditions that resulted in increased fission. Hence, imaging/mathematical analysis shed light on the relationship between bioenergetics and mitochondrial network morphology; the latter may determine EC survival under metabolic stress.

Keywords

Mitochondrial fusion/fission Mitochondrial motility Endothelial function Fluorescence microscopy Digital image processing Mathematical analysis Object tracking 

Notes

Acknowledgments

The authors would like to thank Mr. C. J. Lloyd, undergraduate researcher, for his assistance with data analysis. This work was supported by National Institutes of Health (NIH) grant HL106392 to B. R. Alevriadou and an American Heart (AHA) predoctoral fellowship to R. J. Giedt. D. R. Pfeiffer was supported by the Ellie Kovalck Charitable Trust. A. Matzavinos was supported in part by the Mathematical Biosciences Institute at the Ohio State University and National Science Foundation (NSF) grant DMS-093164. C.-Y. Kao was supported in part by NSF grant DMS-0811003 and an Alfred P. Sloan Fellowship.

Supplementary material

10439_2012_568_MOESM1_ESM.tif (18.5 mb)
Supplementary material 1 (TIFF 18981 kb)

Supplementary material 2 (MPG 982 kb)

References

  1. 1.
    Benard, G., N. Bellance, D. James, P. Parrone, H. Fernandez, T. Letellier, and R. Rossignol. Mitochondrial bioenergetics and structural network organization. J. Cell Sci. 120:838–848, 2007.PubMedCrossRefGoogle Scholar
  2. 2.
    Bomzon, Z., M. M. Knight, D. L. Bader, and E. Kimmel. Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm. J. Biomech. Eng. 128:674–679, 2006.PubMedCrossRefGoogle Scholar
  3. 3.
    Brookes, P. S., Y. Yoon, J. L. Robotham, M. W. Anders, and S. S. Sheu. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol. Cell Physiol. 287:C817–C833, 2004.PubMedCrossRefGoogle Scholar
  4. 4.
    Cassidy-Stone, A., J. E. Chipuk, E. Ingerman, C. Song, C. Yoo, T. Kuwana, M. J. Kurth, J. T. Shaw, J. E. Hinshaw, D. R. Green, and J. Nunnari. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev. Cell 14:193–204, 2008.PubMedCrossRefGoogle Scholar
  5. 5.
    Chang, C. R., and C. Blackstone. Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1. Ann. N. Y. Acad. Sci. 1201:34–39, 2010.PubMedCrossRefGoogle Scholar
  6. 6.
    Chang, K. T., R. F. Niescier, and K. T. Min. Mitochondrial matrix Ca2+ as an intrinsic signal regulating mitochondrial motility in axons. Proc. Natl Acad. Sci. USA 108:15456–15461, 2011.PubMedCrossRefGoogle Scholar
  7. 7.
    Chen, H., and D. C. Chan. Emerging functions of mammalian mitochondrial fusion and fission. Hum. Mol. Genet. 14 Spec No. 2:R283–R289, 2005.Google Scholar
  8. 8.
    Chen, H., A. Chomyn, and D. C. Chan. Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J. Biol. Chem. 280:26185–26192, 2005.PubMedCrossRefGoogle Scholar
  9. 9.
    Collins, T. J. ImageJ for microscopy. Biotechniques 43:25–30, 2007.PubMedCrossRefGoogle Scholar
  10. 10.
    Culic, O., M. L. Gruwel, and J. Schrader. Energy turnover of vascular endothelial cells. Am. J. Physiol. 273:C205–C213, 1997.PubMedGoogle Scholar
  11. 11.
    De Vos, K. J., V. J. Allan, A. J. Grierson, and M. P. Sheetz. Mitochondrial function and actin regulate dynamin-related protein 1-dependent mitochondrial fission. Curr. Biol. 15:678–683, 2005.PubMedCrossRefGoogle Scholar
  12. 12.
    Demidenko, E. Mixed Models: Theory and Applications (Wiley’s Series in Probability and Statistics). Hoboken, NJ: Wiley, 2004.CrossRefGoogle Scholar
  13. 13.
    Duvezin-Caubet, S., R. Jagasia, J. Wagener, S. Hofmann, A. Trifunovic, A. Hansson, A. Chomyn, M. F. Bauer, G. Attardi, N. G. Larsson, W. Neupert, and A. S. Reichert. Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology. J. Biol. Chem. 281:37972–37979, 2006.PubMedCrossRefGoogle Scholar
  14. 14.
    Frank, S., B. Gaume, E. S. Bergmann-Leitner, W. W. Leitner, E. G. Robert, F. Catez, C. L. Smith, and R. J. Youle. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev. Cell 1:515–525, 2001.PubMedCrossRefGoogle Scholar
  15. 15.
    Frederick, R. L., and J. M. Shaw. Moving mitochondria: establishing distribution of an essential organelle. Traffic 8:1668–1675, 2007.PubMedCrossRefGoogle Scholar
  16. 16.
    Giedt, R. J., C. Yang, J. L. Zweier, A. Matzavinos, and B. R. Alevriadou. Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species. Free Radic. Biol. Med. 52:348–356, 2012.PubMedCrossRefGoogle Scholar
  17. 17.
    Gonzalez, R. C., and R. E. Woods. Digital Image Processing (3rd ed.). Upper Saddle River, NJ: Pearson Prentice Hall, 2008.Google Scholar
  18. 18.
    Gottlob, K., N. Majewski, S. Kennedy, E. Kandel, R. B. Robey, and N. Hay. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev. 15:1406–1418, 2001.PubMedCrossRefGoogle Scholar
  19. 19.
    Guillery, O., F. Malka, P. Frachon, D. Milea, M. Rojo, and A. Lombes. Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts. Neuromuscul. Disord. 18:319–330, 2008.PubMedCrossRefGoogle Scholar
  20. 20.
    Hahn-Windgassen, A., V. Nogueira, C. C. Chen, J. E. Skeen, N. Sonenberg, and N. Hay. Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity. J. Biol. Chem. 280:32081–32089, 2005.PubMedCrossRefGoogle Scholar
  21. 21.
    Hansen, C., J. G. Nagy, and D. P. O’Leary. Deblurring Images—Matrices, Spectra, and Filtering. Philadelphia, PA: Society for Industrial and Applied Mathematics (SIAM), 2006.CrossRefGoogle Scholar
  22. 22.
    Haralick, R. M., and L. G. Shapiro. Computer and Robot Vision, Vol. I. Reading, MA: Addison-Wesley, 1992.Google Scholar
  23. 23.
    Hollenbeck, P. J., and W. M. Saxton. The axonal transport of mitochondria. J. Cell Sci. 118:5411–5419, 2005.PubMedCrossRefGoogle Scholar
  24. 24.
    Jahani-Asl, A., M. Germain, and R. S. Slack. Mitochondria: joining forces to thwart cell death. Biochim. Biophys. Acta 1802:162–166, 2010.PubMedCrossRefGoogle Scholar
  25. 25.
    Jaqaman, K., D. Loerke, M. Mettlen, H. Kuwata, S. Grinstein, S. L. Schmid, and G. Danuser. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5:695–702, 2008.PubMedCrossRefGoogle Scholar
  26. 26.
    Jendrach, M., S. Mai, S. Pohl, M. Voth, and J. Bereiter-Hahn. Short- and long-term alterations of mitochondrial morphology, dynamics and mtDNA after transient oxidative stress. Mitochondrion 8:293–304, 2008.PubMedCrossRefGoogle Scholar
  27. 27.
    Kuznetsov, A. V., M. Hermann, V. Saks, P. Hengster, and R. Margreiter. The cell-type specificity of mitochondrial dynamics. Int. J. Biochem. Cell Biol. 41:1928–1939, 2009.PubMedCrossRefGoogle Scholar
  28. 28.
    Lee, Y. J., S. Y. Jeong, M. Karbowski, C. L. Smith, and R. J. Youle. Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. Mol. Biol. Cell 15:5001–5011, 2004.PubMedCrossRefGoogle Scholar
  29. 29.
    Liu, X., and G. Hajnoczky. Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress. Cell Death Differ. 18:1561–1572, 2011.PubMedCrossRefGoogle Scholar
  30. 30.
    McBride, H. M., M. Neuspiel, and S. Wasiak. Mitochondria: more than just a powerhouse. Curr. Biol. 16:R551–R560, 2006.PubMedCrossRefGoogle Scholar
  31. 31.
    Meeusen, S., J. M. McCaffery, and J. Nunnari. Mitochondrial fusion intermediates revealed in vitro. Science 305:1747–1752, 2004.PubMedCrossRefGoogle Scholar
  32. 32.
    Misko, A., S. Jiang, I. Wegorzewska, J. Milbrandt, and R. H. Baloh. Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J. Neurosci. 30:4232–4240, 2010.PubMedCrossRefGoogle Scholar
  33. 33.
    Mitra, K., C. Wunder, B. Roysam, G. Lin, and J. Lippincott-Schwartz. A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase. Proc. Natl Acad. Sci. USA 106:11960–11965, 2009.PubMedCrossRefGoogle Scholar
  34. 34.
    Muller, M., S. L. Mironov, M. V. Ivannikov, J. Schmidt, and D. W. Richter. Mitochondrial organization and motility probed by two-photon microscopy in cultured mouse brainstem neurons. Exp. Cell Res. 303:114–127, 2005.PubMedGoogle Scholar
  35. 35.
    Pletjushkina, O. Y., K. G. Lyamzaev, E. N. Popova, O. K. Nepryakhina, O. Y. Ivanova, L. V. Domnina, B. V. Chernyak, and V. P. Skulachev. Effect of oxidative stress on dynamics of mitochondrial reticulum. Biochim. Biophys. Acta 1757:518–524, 2006.PubMedCrossRefGoogle Scholar
  36. 36.
    Qian, H., M. P. Sheetz, and E. L. Elson. Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys. J. 60:910–921, 1991.PubMedCrossRefGoogle Scholar
  37. 37.
    Rambold, A. S., B. Kostelecky, N. Elia, and J. Lippincott-Schwartz. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proc. Natl Acad. Sci. USA 108(25):10190–10195, 2011.PubMedCrossRefGoogle Scholar
  38. 38.
    Saotome, M., D. Safiulina, G. Szabadkai, S. Das, A. Fransson, P. Aspenstrom, R. Rizzuto, and G. Hajnoczky. Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase. Proc. Natl Acad. Sci. USA 105:20728–20733, 2008.PubMedCrossRefGoogle Scholar
  39. 39.
    Saunter, C. D., M. D. Perng, G. D. Love, and R. A. Quinlan. Stochastically determined directed movement explains the dominant small-scale mitochondrial movements within non-neuronal tissue culture cells. FEBS Lett. 583:1267–1273, 2009.PubMedCrossRefGoogle Scholar
  40. 40.
    Sauvanet, C., S. Duvezin-Caubet, J. P. di Rago, and M. Rojo. Energetic requirements and bioenergetic modulation of mitochondrial morphology and dynamics. Semin. Cell Dev. Biol. 21:558–565, 2010.PubMedCrossRefGoogle Scholar
  41. 41.
    Saxton, M. J., and K. Jacobson. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26:373–399, 1997.PubMedCrossRefGoogle Scholar
  42. 42.
    Sedgewick, R. Algorithms in C, Parts 1–4, 3rd ed. Reading, MA: Addison-Wesley, 1998.Google Scholar
  43. 43.
    Song, W., B. Bossy, O. J. Martin, A. Hicks, S. Lubitz, A. B. Knott, and E. Bossy-Wetzel. Assessing mitochondrial morphology and dynamics using fluorescence wide-field microscopy and 3D image processing. Methods 46:295–303, 2008.PubMedCrossRefGoogle Scholar
  44. 44.
    Soubannier, V., and H. M. McBride. Positioning mitochondrial plasticity within cellular signaling cascades. Biochim. Biophys. Acta 1793:154–170, 2009.PubMedCrossRefGoogle Scholar
  45. 45.
    Tondera, D., S. Grandemange, A. Jourdain, M. Karbowski, Y. Mattenberger, S. Herzig, S. Da Cruz, P. Clerc, I. Raschke, C. Merkwirth, S. Ehses, F. Krause, D. C. Chan, C. Alexander, C. Bauer, R. Youle, T. Langer, and J. C. Martinou. SLP-2 is required for stress-induced mitochondrial hyperfusion. EMBO J. 28:1589–1600, 2009.PubMedCrossRefGoogle Scholar
  46. 46.
    Widlansky, M. E., and D. D. Gutterman. Regulation of endothelial function by mitochondrial reactive oxygen species. Antioxid. Redox Signal. 15:1517–1530, 2011.PubMedCrossRefGoogle Scholar
  47. 47.
    Yi, M., D. Weaver, and G. Hajnoczky. Control of mitochondrial motility and distribution by the calcium signal: a homeostatic circuit. J. Cell Biol. 167:661–672, 2004.PubMedCrossRefGoogle Scholar
  48. 48.
    Yoon, Y. S., D. S. Yoon, I. K. Lim, S. H. Yoon, H. Y. Chung, M. Rojo, F. Malka, M. J. Jou, J. C. Martinou, and G. Yoon. Formation of elongated giant mitochondria in DFO-induced cellular senescence: involvement of enhanced fusion process through modulation of Fis1. J. Cell. Physiol. 209:468–480, 2006.PubMedCrossRefGoogle Scholar
  49. 49.
    Youle, R. J., and M. Karbowski. Mitochondrial fission in apoptosis. Nat. Rev. Mol. Cell Biol. 6:657–663, 2005.PubMedCrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2012

Authors and Affiliations

  • Randy J. Giedt
    • 1
    • 2
  • Douglas R. Pfeiffer
    • 3
  • Anastasios Matzavinos
    • 4
  • Chiu-Yen Kao
    • 5
    • 6
  • B. Rita Alevriadou
    • 1
    • 2
  1. 1.Department of Biomedical EngineeringThe Ohio State UniversityColumbusUSA
  2. 2.Division of Cardiovascular Medicine, Department of Internal Medicine, 607 Davis Heart & Lung Research InstituteThe Ohio State UniversityColumbusUSA
  3. 3.Department of Molecular and Cellular BiochemistryThe Ohio State UniversityColumbusUSA
  4. 4.Department of MathematicsIowa State UniversityAmesUSA
  5. 5.Department of MathematicsThe Ohio State UniversityColumbusUSA
  6. 6.Department of Mathematics and Computer ScienceClaremont McKenna CollegeClaremontUSA

Personalised recommendations