Abstract
Mitochondria are almost ubiquitous organelles in Eukaryota. They are highly dynamic and often complex structures in the cell. The mammalian mitochondrial proteome is predicted to comprise as many as 2000–2500 different proteins. Determination of the subcellular localization of any newly identified protein is one of the first steps toward unraveling its biological function. For most mitochondrial proteins, this can now be done relatively easily by cloning a complementary deoxyribonucleic acid of interest in frame with an additional sequence for a fluorescent or nonfluorescent protein tag. Transfection and subsequent visualization, either by direct fluorescence microscopy or by indirect immunofluorescence microscopy, will give the first clue to mitochondrial localization. In combination with a fluorescent “marker” dye, the mitochondrial localization can be confirmed. This chapter describes some of the methods used in determining mitochondrial protein localization, which can also be used to study dynamics of mitochondria or individual mitochondrial proteins or protein complexes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Scott, S. V., Cassidy-Stone, A., Meeusen, S. L., and Nunnari, J. (2003) Staying in aerobic shape: how the structural integrity of mitochondria and mitochondrial DNA is maintained. Curr. Opin. Cell Biol. 15, 482–488.
Margineantu, D. H., Brown, R. M., Brown, G. K., Marcus, A. H., and Capaldi, R. A. (2002) Heterogeneous distribution of pyruvate dehydrogenase in the matrix of mitochondria. Mitochondrion 1, 327–338.
Garrido, N., Griparic, L., Jokitalo, E., Wartiovaara, J., Van Der Bliek, A. M., and Spelbrink, J. N. (2003) Composition and dynamics of human mitochondrial nucleoids. Mol. Biol. Cell 14, 1583–1596.
Legros, F., Malka, F., Frachon, P., Lombes, A., and Rojo, M. (2004) Organization and dynamics of human mitochondrial DNA. J. Cell Sci. 117, 2653–2662.
Satoh, M., and Kuroiwa, T. (1991) Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp. Cell Res. 196, 137–140.
Iborra, F. J., Kimura, H., and Cook, P. R. (2004) The functional organization of mitochondrial genomes in human cells. BMC Biol. 2, 9.
Legros, F., Lombes, A., Frachon, P., and Rojo, M. (2002) Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol. Biol. Cell 13, 4343–4354.
Patterson, G. H., and Lippincott-Schwartz, J. (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877.
Chudakov, D. M., Verkhusha, V. V., Staroverov, D. B., Souslova, E. A., Lukyanov, S., and Lukyanov, K. A. (2004) Photoswitchable cyan fluorescent protein for protein tracking Nat. Biotechnol. 22, 1435–1439.
Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. U. S. A. 97, 11,984–11,989.
Lauf, U., Lopez, P., and Falk, M. M. (2001) Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11–15.
Campbell, R. E., Tour, O., Palmer, A. E., et al. (2002) A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U. S. A. 99, 7877–7882.
Koehler, C. M. (2004) New developments in mitochondrial assembly. Annu. Rev. Cell Dev. Biol. 20, 309–335.
Poot, M., Zhang, Y., Kramer, J., et al. (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains J. Histochem. Cytochem. 44, 1363–1372.
Ashley, N., Harris, D., and Poulton, J. (2005) Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining. Exp. Cell Res. 303, 432–446.
Pendergrass, W., Wolf, N., and Poot, M. (2004) Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A 61, 162–169.
Spelbrink, J. N., Li, F. Y., Tiranti, V., et al. (2001) Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat. Genet. 28, 223–231.
van de Corput, M. P., van den Ouweland, J. M., Dirks, R. W., et al. (1997) Detection of mitochondrial DNA deletions in human skin fibroblasts of patients with Pearson’s syndrome by two-color fluorescence in situ hybridization. J. Histochem. Cytochem. 45, 55–61.
Margineantu, D. H., Cox, W. G., Sundell, L., et al. (2002) Cell cycle dependent morphology changes and associated mitochondrial DNA redistribution in mitochondria of human cell lines. Mitochondrion 1, 425–435.
Sanes, J. R., Rubenstein, J. L., and Nicolas, J. F. (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 5, 3133–3142.
Minamikawa, T., Sriratana, A., Williams, D. A., Bowser, D. N., Hill, J. S., and Nagley, P. (1999) Chloromethyl-X-rosamine (MitoTracker Red) photosensitizes mitochondria and induces apoptosis in intact human cells. J. Cell Sci. 112, 2419–2430.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Goffart, S., Martinsson, P., Malka, F., Rojo, M., Spelbrink, J.N. (2007). The Mitochondria of Cultured Mammalian Cells. In: Leister, D., Herrmann, J.M. (eds) Mitochondria. Methods in Molecular Biology™, vol 372. Humana Press. https://doi.org/10.1007/978-1-59745-365-3_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-365-3_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-667-2
Online ISBN: 978-1-59745-365-3
eBook Packages: Springer Protocols