Chromosome Research

, 17:801 | Cite as

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

  • Jacques Rouquette
  • Christel Genoud
  • Gerardo H. Vazquez-Nin
  • Bernd Kraus
  • Thomas Cremer
  • Stanislav Fakan
Article

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.

Keywords

chromatin interchromatin space 3D reconstruction electron microscopy 3View SBFSEM 

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

Supplementary material

10577_2009_9070_MOESM1_ESM.mov (6.5 mb)
Supplementary Movie S1A complete stack of images reconstructed from one hepatocyte nucleus (MOV 6.46 mb)
10577_2009_9070_MOESM2_ESM.mov (6.8 mb)
Supplementary Movie S2A 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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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Jacques Rouquette
    • 1
    • 2
  • Christel Genoud
    • 3
  • Gerardo H. Vazquez-Nin
    • 4
  • Bernd Kraus
    • 3
  • Thomas Cremer
    • 1
    • 2
  • Stanislav Fakan
    • 1
    • 2
  1. 1.BiocenterLudwig Maximilians University (LMU)MartinsriedGermany
  2. 2.Center for Integrated Protein Science Munich (CIPSM), LMU MunichMunichGermany
  3. 3.Gatan GmbHMunichGermany
  4. 4.Laboratory of Electron Microscopy, Department of Cell Biology, Faculty of SciencesNational Autonomous University of Mexico (UNAM)Mexico CityMexico
  5. 5.Friedrich Miescher Institute for Biomedical ResearchBaselSwitzerland

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