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Birefringence and DNA condensation of liquid crystalline chromosomes - PubMed

Birefringence and DNA condensation of liquid crystalline chromosomes

Man H Chow et al. Eukaryot Cell. 2010 Oct.

Abstract

DNA can self-assemble in vitro into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes-up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.

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Figures

Fig. 1.
Fig. 1.

Polarizing, fluorescence, and bright-field photomicrographs of dinoflagellates. The polarizing and bright-field images in each panel represent the same cell. Each fluorescence image was taken of a different DAPI-stained cell from the same dinoflagellate species. Six different dinoflagellate species (H. triquetra [A], A. catenella [B], K. brevis [C], P. micans [D], A. carterae [E], and C. cohnii [F]) were investigated. Four species (A. tamarense, K. brevis, H. triquetra, and P. micans) showed distinctive birefringence in their chromosomes under the polarizing microscope (A to D, white arrows). These kinds of birefringent chromosomes, however, were absent in both A. carterae and C. cohnii (E and F). Scale bars, 10 μm.

Fig. 2.
Fig. 2.

Metripol photomicrographs of dinoflagellates. Different dinoflagellate species (H. triquetra [A], A. catenella [B], K. brevis [C], P. micans [D], A. carterae [E], and C. cohnii [F]) were studied. Signals of retardance were detected in the birefringent LCCs in A. tamarense, K. brevis, H. triquetra, and P. micans (A to D, white arrows). The relative birefringence varied among the species, as denoted by the intensity of the color in the Metripol |Sinδ| photomicrographs. No obvious retardation signals were detected in the chromosomes of A. carterae and C. cohnii (E and F). Notably, some cellular contents, e.g., starch granules and thecal plates, are birefringent and give strong signals in the Metripol photomicrographs.

Fig. 3.
Fig. 3.

TEM photomicrographs of the chromosomes of the six investigated dinoflagellate species, A. carterae (A), C. cohnii (B), A. tamarense (C), H. triquetra (D), K. brevis (E), and P. micans (F). (A to F) Series of arches were observed in the chromosomes, which resembled the plectonemic structure of the cholesteric liquid crystalline DNA. The similarity in the architectures of the chromosomes among different dinoflagellate species suggested that the structure of the chromosomes is not sufficient to cause birefringence. Magnifications are indicated by the scale bars in the photomicrographs.

Fig. 4.
Fig. 4.

Birefringence was detected in expelled nuclei. (A) (Left to right) Birefringent chromosomes could be observed when the nucleus of C. cohnii, a species that does not have obvious birefringent chromosomes in its intact cell, was expelled by osmotic pressure. White arrows point to the expelled nucleus. (B) (Left to right) Cells of H. triquetra, a species that has birefringent chromosomes, were subjected to a similar study. Birefringence of the chromosomes was retained even in the expelled nucleus.

Fig. 5.
Fig. 5.

Chromosomal birefringence and dinoflagellate genomes. (A) Relationship between DNA content and retardance in dinoflagellates. The DNA content of each dinoflagellate species was defined as the amount of DNA per haploid cell (n) and was obtained from reference (Table 1), while the values of retardance correspond to the average value of |Sinδ| for 100 chromosomes obtained through the Metripol system, as described in Materials and Methods. (B) Relationship between DNA density and retardance in dinoflagellates. The DNA density was defined as the average mass (pg) of the DNA in each chromosome per unit chromosomal volume (μm3). The number of chromosomes of each dinoflagellate species was obtained from the work of Spector (43) and Dodge (9a) and the experiment with induced expulsion of nuclei (data not shown). The chromosomal volume, on the other hand, was calculated under the assumption that the chromosome had a perfect rod shape (see Materials and Methods). A positive relationship can be observed between the DNA density and retardance in the birefringent species.

Fig. 6.
Fig. 6.

Proposed model of dinoflagellate chromosomes. (A) The structure of the dinoflagellate chromosome was proposed by Livoland and Bouligand (31). The dinoflagellate chromosome may be composed of multiple stacks of DNA discs that are twisted very tightly together. Half the helical pitch (P/2) is defined as the distance between two DNA discs (layers) that have 180° of rotation. (B) The corresponding half of the helical pitch seen in the electron micrograph when the fracture plane is along the longitudinal (cholesteric) axis of the chromosome.

Fig. 7.
Fig. 7.

Contribution of half the helical pitch to birefringence. (A) Relationship between the value of half the helical pitch (P/2) and retardance. The value of P/2 for each dinoflagellate species was calculated from transmission electron micrographs, as described in Materials and Methods. As the helical pitch is a direct reflection of the chromosome structure, the positive relationship observed among the dinoflagellates implies a contribution of the chromosome architecture to the level of birefringence. (B) Relationship between the value of half the helical pitch (P/2) and DNA density in dinoflagellates. The negative correlation between the value of P/2 and the DNA density is in agreement with the twisting nature of the dinoflagellate chromosomes.

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