Abstract
Electron microscopy (EM) has been a central tool in delineating the subcellular organization and function of the eukaryotic cell. It has provided valuable information on the organization of the Golgi complex; the polarized distribution of proteins on the plasma membrane; and fundamental insights into the essential structure and function of mitochondria beginning with the first EM observations of Claude and Fullam on isolated mitochondria in 1944. Most significant for this volume is the contribution immunoelectron microscopy (IEM) has made in the study of mitochondrial dynamics and in demonstrating the localizations of key mitochondrial proteins in yeast, including, though not limited to, Dnm1p, Fiz1p, and Mgm1p. This chapter is not intended to provide a comprehensive review of all EM and IEM methods as there are a number of excellent books and reviews already available on these topics. Rather, this chapter provides detailed protocols of conventional EM and IEM methods successfully utilized in our center for the examination and analysis of mitochondria in yeast and mammalian cells.
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References
Geuze, H. J. (1999) A future for EM in cell biology? Trends Cell Biol. 9, 92–93.
Griffiths, G. (2001) Bringing electron microscopy back into focus for cell biology. Trends Cell Biol. 11, 153–154.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.
Wang, L., Jackson, W. C., Steinbach, P. A., and Tsien, R. Y. (2004) Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc. Natl. Acad. Sci. U. S. A. 101, 16,745–16,749.
Geuze, H. J., Slot, J. W., Strous, G. J., Lodish, H. F., and Schwartz, A. L. (1983) Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell 32, 277–287.
Wall, D. A., Wilson, G., and Hubbard, A. L. (1980). The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell 21, 79–93.
Willingham, M. C. and Pastan, I. (1980) The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts. Cell 21, 67–77.
Brown, W. J., Goodhouse, J., and Farqubar, M. G. (1986). Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes. J. Cell Biol. 103, 1235–1247.
Roth, J. and Berger, E. G. (1982). Immunocytochemical localization of galactosyl-transferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J. Cell Biol. 92, 223–229.
Roth, J., Taatjes, D. J., Lucocq, J. M., Weinstein, J., and Paulson, J. C. (1985). Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell 43, 287–295.
Farquhar, M. G., Hendricks, L. H., Noda, T., and Velasco, A. (1992). in Electron Microscopic Cytochemistry and Immunocytochemistry in Biomedicine (Ogawa, K. and Barka, T., eds.), CRC Press, Boca Raton, FL, p. 441–479.
Stow, J. L., de Almeida, J. B., Narula, F. J., Holtzman, E. J., Ercolani, L., and Ausiello, D. A. (1991). A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells. J. Cell Biol. 114, 1113–1124.
Nelson, J. (1992) Regulation of cell surface polarity from bacteria to mammals. Science 258, 948–955.
Rodriguez-Boulan, E. and Nelson, W. J. (1989) Morphogenesis of the polarized epithelial cell phenotype. Science 245, 718–725.
Claude, A. and Fullam, E. F. (1945) An electron microscope study of isolated mitochondria, method and preliminary results. J. Exp. Med. 81, 51–62.
Palade, G. E. (1952) The fine structure of mitochondria. Anat. Rec. 114, 427–451.
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.
Bleazard, W., McCaffery, J. M., King, E. J., et al. (1999) The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1, 298–304.
Mozdy, A. D., McCaffery, J. M., and Shaw, J. M. (2000) Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p. J. Cell Biol. 151, 367–380.
Meeusen, S., McCaffery, J. M., and Nunnari, J. (2004) Mitochondrial fusion intermediates revealed in vitro. Science 305, 1747–1752.
Palade, G. E. (1952) A study of fixation for electron microscopy. J. Exp. Med. 95, 285–298.
McLean, W. and Nakane, P. F. (1974) Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083.
Luft, J. H. (1956) Permanganate; a new fixative for electron microscopy. J. Biophys. Biochem. Cytol. 2, 799–802.
Willingham, M. C. and Rutherford, A. V. (1984) The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. J. Histochem. Cytochem. 32, 455–460.
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Perkins, E.M., McCaffery, J.M. (2007). Conventional and Immunoelectron Microscopy of Mitochondria. 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_33
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DOI: https://doi.org/10.1007/978-1-59745-365-3_33
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