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Revealing the high-resolution three-dimensional network of chromatin and interchromatin space: A novel electron-microscopic approach to reconstructing nuclear architecture

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Abstract

The nuclear architecture is considered an important contributor to genome function. Although the fine structural features of the cell nucleus have been investigated extensively by means of ultrastructural cytochemistry, mainly on ultrathin sections in two dimensions (2D), there was a of lack routine methods for a rapid reconstruction of three-dimensional (3D) distribution of different structural constituents throughout the nuclear volume. We have now filled this gap by the application of a novel approach associating a pre-embedding selective visualization of nuclear components with a method making use of ultramicrotomy combined with scanning electron microscopy (microtome serial block face scanning electron microscopy—‘3View’). We have been able to apply this method to the study of DNA distribution within the nuclear volume and reconstruction of 3D chromatin arrangement in nuclei of rat hepatocytes and endothelial cells. Our observations demonstrate that while chromatin appears to occupy the interior of nuclei rather sparsely on 2D images, once reconstructed in 3D from a series of sequential 2D images it gives the impression of considerably filling the nuclear volume. However, quantitative evaluation of the nuclear volume occupied by DNA in the above two types of nuclei leaves a significant part to the interchromatin space (66.2% for hepatic cells and 41.7% for endothelial cells, including nuclear space occupied by nucleoli). Detailed analysis of the reconstructed nuclei reveals a high degree of superposition of chromatin domains, giving rise to a false impression that they fill a much larger part of the nuclear volume than they really do. Our results show the importance of the contribution of such reconstruction techniques to our understanding of the nuclear architecture.

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Abbreviations

2D:

two-dimensional

3D:

three-dimensional

3D EM:

three-dimensional electron microscopy

CT:

chromosome territory

DNA-FISH:

DNA-fluorescent in-situ hybridization

IC:

interchromatin space/compartment

PAS:

periodic acid-Schiff

SBFSEM:

serial block face scanning electron microscopy

SEM:

scanning electron microscopy

TEM:

transmission electron microscopy

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Acknowledgements

We thank Ernestina Ubaldo for her remarkable technical skill, Jitka Fakan for expert technical assistance with the initial experiments, and Patrick Schwarb for his help with image analysis. We are indebted to Drs Boris Joffe and Irina Solovei for critical reading of the manuscript and helpful suggestions and to Professor Dr Gerhard Wanner for stimulating discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Munich Center of Integrated Protein Science (CIPSM), the LMU Bioimaging Network (BIN), and PAPIIT IN203308 and Conacyt 81213 (Mexico). Transmission electron microscopy at the LMU was supported by SFB 646 (Roland Beckmann).

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Author notes

  1. Christel Genoud

    Present address: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

Authors and Affiliations

  1. Biocenter, Ludwig Maximilians University (LMU), Grosshaderner Str. 2, D-82152, Martinsried, Germany

    Jacques Rouquette, Thomas Cremer & Stanislav Fakan

  2. Center for Integrated Protein Science Munich (CIPSM), LMU Munich, Munich, Germany

    Jacques Rouquette, Thomas Cremer & Stanislav Fakan

  3. Gatan GmbH, Munich, Germany

    Christel Genoud & Bernd Kraus

  4. Laboratory of Electron Microscopy, Department of Cell Biology, Faculty of Sciences, National Autonomous University of Mexico (UNAM), A.P. 70-438, Mexico City, Mexico

    Gerardo H. Vazquez-Nin

Authors
  1. Jacques Rouquette
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  2. Christel Genoud
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  3. Gerardo H. Vazquez-Nin
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  4. Bernd Kraus
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  5. Thomas Cremer
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  6. Stanislav Fakan
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Corresponding author

Correspondence to Jacques Rouquette.

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Responsible Editor: Hans-Joachim Lipps.

Electronic Supplementary Material

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Supplementary Movie S1

A complete stack of images reconstructed from one hepatocyte nucleus (MOV 6.46 mb)

Supplementary Movie S2

A model reconstruction of the median portion (2.8 × 9.2 × 8.2 μm) of an image stack of a hepatocyte nucleus. The observation from different angles gives the possibility to better appreciate the real extent of the interchromatin space occurring between chromatin domains (green). (MOV 6.78 mb)

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Rouquette, J., Genoud, C., Vazquez-Nin, G.H. et al. Revealing the high-resolution three-dimensional network of chromatin and interchromatin space: A novel electron-microscopic approach to reconstructing nuclear architecture. Chromosome Res 17, 801–810 (2009). https://doi.org/10.1007/s10577-009-9070-x

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  • DOI: https://doi.org/10.1007/s10577-009-9070-x

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