Revealing the high-resolution three-dimensional network of chromatin and interchromatin space: a novel electron-microscopic approach to reconstructing nuclear architecture.
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.
Rouquette
Jacques
J
Biocenter, Ludwig Maximilians University (LMU), Grosshaderner Str. 2, D-82152 Martinsried, Germany. Jacques.Rouquette@lrz.uni-muenchen.de
Genoud
Christel
C
Vazquez-Nin
Gerardo H
GH
Kraus
Bernd
B
Cremer
Thomas
T
Fakan
Stanislav
S
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure*; D002843:Chromatin / Q000648:ultrastructure*; D042783:Endothelial Cells / Q000648:ultrastructure*; D022781:Hepatocytes / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission; D051381:Rats
D002843:Chromatin