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Three-Dimensional Reconstruction of Nucleolar Components by Electron Microscope Tomography

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The Nucleus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 463))

Abstract

The nucleus is a complex volume constituted of numerous subcompartments in which specific functions take place due to a specific spatial organization of their molecular components. To understand how these molecules are spatially organized within these machineries, it is necessary to investigate their three-dimensional organization at high resolution. To reach this goal, electron tomography appears to be a method of choice; it can generate tomograms with a resolution of a few nanometers by using multiple projections of a tilted section several hundred to several thousand nanometers in thickness imaged by transmission electron microscopy (TEM).

Specific identification of molecules of interest contained within such thick sections requires their specific immunocytochemical labelling using electron-dense markers. We recently demonstrated that electron tomography of proteins immunostained with nanogold particles before embedding, and subsequently amplified with silver, was very fruitful due to the inherently high spatial resolution of the mediumvoltage scanning and transmission electron microscope (STEM). Here we describe this approach, which is very efficient for tracing the 3D organization of proteins within complex machineries by using antibodies raised against one of the proteins, or against GFP to analyse GFP-tagged proteins.

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Acknowledgments

The financial support for this work was received from the Association pour la Recherche sur le Cancer (contract N° 4497) and the Ligue contre le Cancer (Departements de l’Aube, de la Marne, et des Ardennes). A long-term grant for invited scientists was obtained from the Région Champagne-Ardenne for Prof. P. Tchelidze.

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Authors and Affiliations

  1. Department of Morphology, Institute of Biology, Tbilisi State University, Tbilisi, Georgia

    Pavel Tchelidze

  2. Unité MEDyC, CNRS UMR 6237, IFR 53, Université de Reims-Champagne Ardenne, Reims, France

    Pavel Tchelidze, Hélène Bobichon, Nathalie Lalun & Dominique Ploton

  3. IFR53, Reims, France

    Hervé Kaplan

  4. DTI, UMR 6107, UFR de Sciences, Reims, France

    Adrien Beorchia

  5. UMR CNRS 5099, IFR 109, Université Paul Sabatier Toulouse III, F 31062, Toulouse, France

    Marie-Françoise O’Donohue

  6. Laboratoire de Microscopie Électronique, INSERM ERM 0203, Reims, France

    Laurence Wortham

Authors
  1. Pavel Tchelidze
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  2. Hervé Kaplan
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  3. Adrien Beorchia
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  4. Marie-Françoise O’Donohue
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  5. Hélène Bobichon
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  6. Nathalie Lalun
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  7. Laurence Wortham
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  8. Dominique Ploton
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Editor information

Editors and Affiliations

  1. Hôtel-Dieu Hospital, Laval University Cancer Research Centre, Québec, Canada

    Ronald Hancock

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© 2008 Humana Press

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Tchelidze, P. et al. (2008). Three-Dimensional Reconstruction of Nucleolar Components by Electron Microscope Tomography. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 463. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-406-3_10

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  • DOI: https://doi.org/10.1007/978-1-59745-406-3_10

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-977-2

  • Online ISBN: 978-1-59745-406-3

  • eBook Packages: Springer Protocols

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