Abstract
Mitochondria are key players in cell death following intrinsic and, in some cell types, extrinsic stimuli. The recruitment of the mitochondrial pathway results in mitochondrial dysfunction and release of intermembrane space proteins like cytochrome-c that are required in the cytosol for complete activation of effector caspases. Apoptotic shape changes of this organelle and the role of “mitochondria-shaping” proteins in cell death has attracted considerable attention. We present protocols to investigate how morphological changes of the mitochondrial reticulum regulate release of cytochrome-c, as evaluated quantitatively by an in situ approach, and changes in mitochondrial membrane potential measured in real time.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hengartner, M. O. (2000) The biochemistry of apoptosis. Nature 407, 770–776.
Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456–1462.
Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899–1911.
Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413.
Frank, S., Gaume, B., Bergmann-Leitner, E. S., et al. (2001) The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev. Cell 1, 515–525.
Scorrano, L., Ashiya, M., Buttle, K., et al. (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell 2, 55–67.
Breckenridge, D. G., Stojanovic, M., Marcellus, R. C., and Shore, G. C. (2003) Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol. J. Cell Biol. 160, 1115–1127.
Scorrano, L. (2003) Divide et impera: Ca2+ signals, mitochondrial fission and sensitization to apoptosis. Cell Death. Differ. 10, 1287–1289.
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.
Terman, A., Dalen, H., Eaton, J. W., Neuzil, J., and Brunk, U. T. (2003) Mitochondrial recycling and aging of cardiac myocytes: the role of autophagocytosis. Exp. Gerontol. 38, 863–876.
Yaffe, M. P. (1999) The machinery of mitochondrial inheritance and behavior. Science 283, 1493–1497.
Yoon, Y., Krueger, E. W., Oswald, B. J., and McNiven, M. A. (2003) The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1. Mol. Cell Biol. 23, 5409–5420.
James, D. I., Parone, P. A., Mattenberger, Y., and Martinou, J. C. (2003) hFis1, a novel component of the mammalian mitochondrial fission machinery. J. Biol. Chem. 278, 36,373–36,379.
Chen, H., Detmer, S. A., Ewald, A. J., Griffin, E. E., Fraser, S. E., and Chan, D. C. (2003) Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. J. Cell Biol. 160, 189–200.
Koshiba, T., Detmer, S. A., Kaiser, J. T., Chen, H., McCaffery, J. M., and Chan, D. C. (2004) Structural basis of mitochondrial tethering by mitofusin complexes. Science 305, 858–862.
Ishihara, N., Eura, Y., and Mihara, K. (2004) Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity. J. Cell Sci. 117, 6535–6546.
Cipolat, S., de Brito, O. M., Dal Zilio, B., and Scorrano, L. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U. S. A. 101, 15,927–15,932.
Jagasia, R., Grote, P., Westermann, B., and Conradt, B. (2005) DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans. Nature 433, 754–760.
Germain, M., Mathai, J. P., McBride, H. M., and Shore, G. C. (2005) Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis. EMBO J. 24, 1546–1556.
Bernardi, P., Scorrano, L., Colonna, R., Petronilli, V., and Di Lisa F. (1999) Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur. J. Biochem. 264, 687–701.
Adachi, S., Gottlieb, R. A., and Babior, B. M. (1998) Lack of release of cytochrome c from mitochondria into cytosol early in the course of Fas-mediated apoptosis of Jurkat cells. J. Biol. Chem. 273, 19,892–19,894.
Petronilli, V., Penzo, D., Scorrano, L., Bernardi, P., and Di Lisa, F. (2001) The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J. Biol. Chem. 276, 12,030–12,034.
Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L., and Korsmeyer, S. J. (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241–251.
Cornwell, M. M., Pastan, I., and Gottesman, M. M. (1987) Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. J. Biol. Chem. 262, 2166–2170.
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
Frezza, C., Cipolat, S., Scorrano, L. (2007). Measuring Mitochondrial Shape Changes and Their Consequences on Mitochondrial Involvement During Apoptosis. 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_29
Download citation
DOI: https://doi.org/10.1007/978-1-59745-365-3_29
Publisher Name: Humana Press
Print ISBN: 978-1-58829-667-2
Online ISBN: 978-1-59745-365-3
eBook Packages: Springer Protocols