Summary
This chapter describes the electron microscopic fine structure of early and late autophagic vacuoles in mammalian cells. Detailed instructions are given for the preparation of cells for conventional electron microscopy and for the identification of autophagic vacuoles by morphology. Electron microscopy remains one of the most accurate methods for quantitation of autophagic vacuole accumulation. Therefore, quantitation of autophagic vacuoles by electron microscopy and point counting is also described. Finally, a short description is given for preparation of ultra thin cryosections for immunogold labeling of autophagic vacuoles.
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References
Eskelinen, E. L. (2005) Autophagy in mammalian cells, in Lysosomes (Saftig, P., ed.), Landes Bioscience/Eurekah.com, Georgetown, pp. 166–180
Klionsky, D. J., and Emr, S. D. (2000) Autophagy as a regulated pathway of cellular degradation. Science 290, 1717–1721.
Boya, P., Gonzalez-Polo, R. A., Casares, N., et al. (2005) Inhibition of macroautophagy triggers apoptosis. Mol. Cell Biol. 25, 1025–1040.
Lum, J. J., Bauer, D. E., Kong, M., et al. (2005) Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248.
Kuma, A., Hatano, M., Matsui, M., et al. (2004) The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036.
Eskelinen, E. L., Tanaka, Y., and Saftig, P. (2003) At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13, 137–145.
Tanaka, Y., Guhde, G., Suter, A., et al. (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2 -deficient mice. Nature 406, 902–906.
Liang, X. H., Jackson, S., Seaman, M., et al. (1999) Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672–676.
Qu, X., Yu, J., Bhagat, G., et al. (2003) Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J. Clin. Invest. 112,1809–1820.
Melendez, A., Talloczy, Z., Seaman, M., Eskelinen, E. L., Hall, D. H., and Levine, B. (2003) Autophagy genes are essential for dauer development and lifespan extension in C. elegans. Science 301, 1387–1391.
Ravikumar, B., Vacher, C., Berger, Z., et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585–595.
Hara, T., Nakamura, K., Matsui, M., et al. (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441, 885–889.
Komatsu, M., Waguri, S., Chiba, T., et al. (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441, 880–884.
Talloczy, Z., Jiang, W., Virgin IV, H. W., et al. (2002) Regulation of starvation and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc. Natl. Acad. Sci. USA 99, 190–195.
Nakagawa, I., Amano, A., Mizushima, N., et al. (2004) Autophagy defends cells against invading group A Streptococcus. Science 306, 1037–1040.
Arstila, A. U., and Trump, B. F. (1968) Studies on cellular autophagocytosis. The formation of autophagic vacuoles in the liver after glucagon administration. Am. J. Pathol. 53, 687–733.
Dunn, W. A. (1994) Autophagy and related mechanisms of lysosomal-mediated protein degradation. Trends Cell Biol. 4, 139–143.
Eskelinen, E. L. (2005) Maturation of autophagic vacuoles in mammalian cells. Autophagy 1, 1–10.
Dunn, W. A. (1990) Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J. Cell Biol. 110, 1923–1933.
Dunn, W. A. (1990) Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J. Cell Biol. 110, 1935–1945.
Klionsky, D. J., Cregg, J. M., Dunn, W. A. J., et al. (2003) A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5, 539–545.
Kabeya, Y., Mizushima, N., Ueno, T., et al. (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728.
Jäger, S., Bucci, C., Tanida, I., et al. (2004) Role for Rab7 in maturation of late autophagic vacuoles. J. Cell Sci. 117, 4837–4848.
Griffiths, G. (1993) Fine Structure Immunocytochemistry, Springer-Verlag, Berlin Heidelberg.
Howard, C. V., and Reed, M. G. (1998) Unbiased Stereology. Three-Dimensional Measurement in Microscopy, Springer-Verlag, New York.
Liou, W., Geuze, H. J., and Slot, J. W. (1996) Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106, 41–58.
Acknowledgments
The author would like to thank Pirkko Hirsimäki (Turku, Finland) and Hilkka Reunanen (Jyväskylä, Finland) for the initial introduction to the secrets of electron microscopy and autophagy. John Lucocq (Dundee, UK) is thanked for sharing his knowledge on the quantitative aspects of microscopy, and Paul Saftig (Kiel, Germany) is acknowledged for his generous support during the years we have used to explore the functions of lysosomal membrane proteins. Work described in this chapter was supported by The Royal Society (Dundee), Helsel Stiftung (Kiel), University of Helsinki Research Funds, and Biocentrum Helsinki
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Eskelinen, EL. (2008). Fine Structure of the Autophagosome. In: Deretic, V. (eds) Autophagosome and Phagosome. Methods in Molecular Biology™, vol 445. Humana Press. https://doi.org/10.1007/978-1-59745-157-4_2
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DOI: https://doi.org/10.1007/978-1-59745-157-4_2
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