Hostname: page-component-76fb5796d-x4r87 Total loading time: 0 Render date: 2024-04-25T10:14:29.231Z Has data issue: false hasContentIssue false
  • English
  • Français

Altered Mitochondrial Structure and Motion Dynamics in Living Cells with Energy Metabolism Defects Revealed by Real Time Microscope Imaging

Published online by Cambridge University Press:  17 March 2004

Nhu-An Pham ,
Tim Richardson ,
Jessie Cameron ,
Bruno Chue  and
Brian H. Robinson
Nhu-An Pham
Affiliation:
Richardson Technologies Inc. at The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
Tim Richardson
Affiliation:
Richardson Technologies Inc. at The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
Jessie Cameron
Affiliation:
The Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
Bruno Chue
Affiliation:
Richardson Technologies Inc. at The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
Brian H. Robinson
Affiliation:
The Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
Get access

Abstract

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 μm wide and up to 10 μm long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.Abbreviations: EM: electron microscope; L/P: lactate/pyruvate; QT: QuickTime; CCCP: carbonyl cyanide 3-chloro-phenylhydrazone; CMXRos, chloromethyl-X-rosamine; Rh123, Rhodamine 123; RTM, real time microscope

Keywords

mitochondrialiveQuickTimerespiratory chainreal-time
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 10 , Issue 2 , April 2004 , pp. 247 - 260
Copyright
© 2004 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Alexander, C., Votruba, M., Pesch, U.E.A., Thiselton, D.L., Mayer, S., Moore, A., Rodriguez, M., Kellner, U., Leo-Kottler, B., Auburger, G., Bhattacharya, S.S., & Wissinger, B. (2000). OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Gen 26, 211–215.Google Scholar
Atlante, A., Passarella, S., Moreno, G., & Salet, C. (1992). Effects of rhodamine 123 in the dark and after irradiation on mitochondrial energy metabolism. Photochem Photobiol 56, 471–478.Google Scholar
Bereiter-Hahn, J. (1990). Behavior of mitochondria in the living cell. Int Rev Cytol 122, 1–63.Google Scholar
Bereiter-Hahn, J. & Vöth, M. (1994). Dynamics of mitochondria in living cells: Shape changes, dislocations, fusion, and fission of mitochondria. Microsc Res Tech 27, 198–219.Google Scholar
Bleazard, W., McCaffery, J.M., King, E.J., Bale, S., Mozdy, A., Tieu, Q., Nunnari, J., & Shaw, J.M. (1999). The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat Cell Biol 1, 298–303.Google Scholar
Boldogh, I., Vojitov, N., Karmon, S., & Pon, L.A. (1998). Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p. Cell Biol 141, 1371–1381.Google Scholar
Chen, L.B. (1989). Fluorescent labeling of mitochondria. Vol. 29. pp. 103–123. New York: Academic Press.
Christodoulou, J. (2000). Genetic defects causing mitochondrial respiratory chain disorders and disease. Hum Reprod 15, 28–43.Google Scholar
Collins, T.J., Berridge, M.J., Lipp, P., & Bootmann, M.D. (2002). Mitochondria are morphologically and functionally heterogenous within cells. EMBO J 21, 1616–1627.Google Scholar
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, 84–94.Google Scholar
Couchman, J.R. & Rees, D.A. (1982). Organelle-cytoskeleton relationships in fibroblasts: Mitochondria, Golgi apparatus, and endoplasmic reticulum in phases of movement and growth. Eur J Cell Biol 27, 47–54.Google Scholar
Crawford, J.M. & Braunwald, N.S. (1991). Toxicity in vital fluorescence microscopy: Effect of dimethylsulfoxide, rhodamine-123, and DiI-Low density lipoprotein on fibroblast growth in vitro. In vitro Cell Dev Biol 27, 633–638.Google Scholar
Dedov, N. & Roufogalis, D. (1999). Organisation of mitochondria in living sensory neurons. FEBS Lett 456, 171–174.Google Scholar
De Giorgi, F., Lartigue, L., & Ichas, F. (2000). Electrical coupling and plasticity of the mitochondrial network. Cell Calcium 28, 365–370.Google Scholar
Delettre, C., Lenaers, G., Griffoin, J.-M., Gigarel, N., Lorenzo, C., Belenguer, P., Pelloquin, L., Grosgeorge, J., Turc-Carel, C., Perret, E., Astarie-Dequeker, C., Lasquellec, L., Arnaud, B., Ducommun, B., Kaplan, J., & Hamel, C.P. (2000). Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat Gen 26, 207–210.Google Scholar
Formigli, L., Papucci, L., Tani, A., Schiavone, N., Tempestini, A., Orlandini, G.E., Capaccioli, S., & Orlandini, S.Z. (2000). Aponecrosis: Morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol 182, 41–49.Google Scholar
Foschini, M.P., Macchia, S., Losi, L., Dei Tos, A.P., Di Tomasso, L., Del Luca, S., Roncaroli, F., & Dal Monte, P. (1998). Identification of mitochondria in liver biopsies. A study by immunochemistry, immunogold and western blot analysis. Virchows Arch 433, 267–263.Google Scholar
Funk, R.H.W., Nagel, F., Wonka, F., Krinke, H.E., Gölfert, F., & Hofer, A. (1999). Effects of heat shock on the functional morphology of cell organelles observed by video-enhanced microscopy. Anat Rec 255, 455–464.Google Scholar
Gilkerson, R.W., Margineantu, D.H., Capaldi, R.A., & Selker, J.M.L. (2000). Mitochondrial DNA depletion causes morphological changes in the mitochondrial reticulum of cultures human cells. FEBS Lett 474, 1–4.Google Scholar
Gosslau, A., Wittrich, W., Willig, A., & Jaros, P.P. (2001). Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts. Comparative Biochem Physiol 128, 241–249.Google Scholar
Hackenbrock, C.R. (1966). Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol 30, 269–297.Google Scholar
Hackenbrock, C.R. (1968). Ultrastructural bases for metabolically linked mechanical activity in mitochondria. II. Electron transport-linked ultrastructural transformations in mitochondria. J Cell Biol 37, 345–369.Google Scholar
Jia, I., Dourmashkin, R.R., Newland, A.C., & Kelsey, S.M. (1997). Mitochondrial ultracondensation, but not swelling, is involved in TNF-alpha-induced apoptosis in human T-lymphoblastic leukaemic cells. Leukemia Res 21, 973–983.Google Scholar
Kanazawa, M., Yano, M., Namchai, C., Yamamoto, S., Ohtake, A., Takayanagi, M., Mori, M., & Niimi, H. (1997). Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases. Biochem Biophys Res Commun 239, 580–584.Google Scholar
Karasaki, S. (1969). The fine structure of proliferating cells in preneoplastic rat livers during azo-dye carcinogenesis. J Cell Biol 40, 322–335.Google Scholar
Krendel, M., Sgourdas, G., & Bonder, E.M. (1998). Disassembly of actin filaments leads to increased rate and frequency of mitochondrial movement along microtubules. Cell Motility and the Cytoskeleton 40, 368–378.Google Scholar
Labrousse, A.M., Zappaterra, M.D., Rube, D.A., & van der Bilek, A.M. (1999). C. elegans dynamin-related protien DRP-1 controls severing of the mitochondrial outer membrane. Mol Cell 4, 815–826.Google Scholar
Loew, L., Tuft, R.A., Carrington, W., & Fay, F.S. (1993). Imaging in five dimensions: Time-dependent membrane potentials in individual mitochondria. Biophys J 65, 2396–2407.Google Scholar
Margineantu, D., Capaldi, R.A., & Marcus, A.H. (2000). Dynamics of the mitochondrial reticulum in living cells using Fourier imaging correlation spectroscopy and digital video microscopy. Biophys J 79, 1833–1849.Google Scholar
Marusich, M.F., Robinson, B.H., Taanman, J.-W., Kim, S.J., Schillace, R., Smith, J.L., & Capaldi, R.A. (1997). Expression of mtDNA and nDNA encoded respiratory chain proteins in chemically and genetically-derived Rho0 human fibroblasts treated with ethidium bromide and fibroblasts from a patient with mtDNA depletion syndrome. Biochim Biophys Acta 1362, 145–159.Google Scholar
Mathur, A., Hong, Y., Kemp, B.K, Barrientos, A.A., & Erusalimsky, J.D. (2000). Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultures cardiomyocytes. Cardiovsc Res 46, 126–138.Google Scholar
Moreadith, R.W., Batshaw, M.L, Ohnishi, T., Kerr, D., Knox, B., Jackson, D., Hruban, R., Olson, J., Reynafarje, B., & Lehninger, A.L. (1984). Deficiency of the iron-sulfur clusters of mitochondrial reduced nicatinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis. J Clin Invest 74, 685–697.Google Scholar
Naviaux, R.K. & McGowan, K.A. (2000). Organismal effects of mitochondrial dysfunction. Hum Reprod 15, 44–56.Google Scholar
Ord, M.J. & Smith, R.A. (1982). Correlation of mitochondrial form and function in vivo: Microinjection of substrate and nucleotides. Cell Tissue Res 227, 129–137.Google Scholar
Partikian, A., Olveczky, B., Swaminathan, R., Li, Y., & Verkman, A.S. (1998). Rapid diffusion of green flourescent protein in the mitochondrial matrix. J Cell Biol 140, 821–829.Google Scholar
Pelloquin, L., Belenguer, P., Menon, Y., Gas, N., & Ducommun, B. (1999). Fission yeast msp1 is a mitochondrial dynamin-related protein. J Cell Sci 112, 4151–4161.Google Scholar
Peskin, C.S., Odell, G.M., & Oster, G.F. (1993). Cellular motions and thermal fluctations: The Brownian ratchet. Biophys J 65, 316–324.Google Scholar
Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., & Gougeon, M. (1995). Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J Cell Biol 130, 157–167.Google Scholar
Pitts, K.R., Yoon, Y., Krueger, E.W., & McNiven, M.A. (1999). The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells. Mol Biol Cell 10, 4403–4417.Google Scholar
Reipert, S., Berry, J., Hughes, M.F., Hickman, J.A., & Allen, T.D. (1995). Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-Mix after treatment with etoposide: A correlative study by multiparameter flow cytometry and confocal and electron microscopy. Exp Cell Res 221, 281–288.Google Scholar
Richardson, T.M. (1998). Test slides: Diatoms to divisions—What are you looking at? Proc Roy Microsc Soc 33, 3–9.Google Scholar
Robinson, B.H., De Meirleir, L., Glerum, M., Sherwood, G., & Becker, L. (1987). Clinical presentation of mitochondrial respiratory chain defects in NADH-coenzyme Q reductase and cytochrome oxidase: Clues to pathogenesis of Leigh disease. Pediatrics 110, 216–222.Google Scholar
Salmeen, I., Zacmanidis, P., Jesion, G., & Feldkamp, L.A. (1985). Motion of mitochondria in cultured cells quantified by analysis of digitized images. Biophys J 48, 681–686.Google Scholar
Santel, A.F. & Fuller, M.T. (2001). Control of mitochondrial morphology by a human mitofusin. Cell Sci 114, 867–874.Google Scholar
Scaduto, J.R.C. & Grotyohann, L.W. (1999). Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76, 469–477.Google Scholar
Sesaki, H.J. & Jensen, R.E. (1999). Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J Cell Biol 147, 699–706.Google Scholar
Skulachev, V.P. (2001). Mitochondrial filaments and clusters as intracellular power-transmitting cables. Trends Biochem Sci 26, 23–29.Google Scholar
Smirnova, E., Griparic, L., Shurland, D.L., & van der Bliek, A.M. (2001). Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol Biol Cell 12, 2245–2256.Google Scholar
Stolz, B. & Bereiter-Hahn, J. (1987). Sequestration of iontophoretically injected calcium by living endothelial cells. Cell Calcium 8, 103–121.Google Scholar
Tanaka, Y.Y.K., Okada, Y., Nonaka, S., Takeda, S., Harada, A., & Hirokawa, N. (1998). Targeted distribution of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93, 1147–1158.Google Scholar
Thorburn, D.R. (2000). Practical problems in detecting abnormal mitochondrial function and genomes. Hum Reprod 15, 57–67.Google Scholar
Triepels, R.H., Heuvel, V.D., Trubels, J.M., & Smeitink, J.A. (2001). Respiratory chain complex I deficiency. Med Gen 106, 37–45.Google Scholar
Yang, X., Borg, L.A.H., Simán, C.M., & Eriksson, U.J. (1998). Maternal antioxidant treatments prevent diabetes-induced alterations of mitochondrial morphology in rat embryos. Anat Rec 251, 303–315.Google Scholar