Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes

Abstract

The arrangement of compact chromatin of G0 lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial software package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G0 lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.

Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks

“…EM investigations have provided evidence that the IC comprises about half of the nuclear space and that its interior is mostly DNA-free (Lopez-Velazquez et al 1996, Esquivel et al 1989. A border zone of decondensed chromatin, called the perichromatin region, separates the interior of IC lacunas and channels from the compact interior of higher-order chromatin domains (for review see Cremer et al 2004, Fakan 2004a.…”

The functional architecture of the nucleus as analysed by ultrastructural cytochemistry

“…This approach, extensively applied on different cell models (Biggiogera et al 1996), can be combined with other methods such as immunocytochemistry and electron spectroscopic imaging (Biggiogera et al 2001). Analysis of reconstructions from serial ultrathin sections of interphase nuclei revealed that about 50-60% of the nuclear space is filled by chromatin (Esquivel et al 1989;López-Velµzquez et al 1996), while the remaining volume is occupied by the interchromatin space with its structural interchromatin domains. The interchromatin space is largely devoid of DNA (Fig.…”

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

“…3D electron-microscopic studies carried out so far have been based on the reconstruction of transmission electron microscopy (TEM) images obtained from serial ultrathin sections either contrasted after ultramicrotomy or prepared from specimens contrasted en bloc during the preparation of the sample before resin embedding. In both cases, the main goal has been to explore the spatial relationships between structural constituents of the nuclear architecture (Dupuy-Coin et al 1986;Esquivel et al 1987;Esquivel et al 1989;Hozak et al 1989;Lopez-Velazquez et al 1996). Cytochemical techniques such as immunocytochemistry using specific antibodies against nuclear components or in-situ hybridization with molecular probes recognizing DNA or RNA sequences in the specimen are routinely used at both the light/fluorescence and the electron-microscopic levels of visualization.…”