The nuclear higher-order structure defined by the set of topological relationships between DNA and the nuclear matrix is species-specific in hepatocytes.

During the interphase the nuclear DNA of metazoan cells is organized in supercoiled loops anchored to constituents of a nuclear substructure or compartment known as the nuclear matrix. The stable interactions between DNA and the nuclear matrix (NM) correspond to a set of topological relationships that define a nuclear higher-order structure (NHOS). Current evidence suggests that the NHOS is cell-type-specific. Biophysical evidence and theoretical models suggest that thermodynamic and structural constraints drive the actualization of DNA-NM interactions. However, if the topological relationships between DNA and the NM were the subject of any biological constraint with functional significance then they must be adaptive and thus be positively selected by natural selection and they should be reasonably conserved, at least within closely related species. We carried out a coarse-grained, comparative evaluation of the DNA-NM topological relationships in primary hepatocytes from two closely related mammals: rat and mouse, by determining the relative position to the NM of a limited set of target sequences corresponding to highly-conserved genomic regions that also represent a sample of distinct chromosome territories within the interphase nucleus. Our results indicate that the pattern of topological relationships between DNA and the NM is not conserved between the hepatocytes of the two closely related species, suggesting that the NHOS, like the karyotype, is species-specific.

New ultrastructural observations on the skeletal matrix of Tritrichomonas foetus.

Tritrichomonas foetus, a parasitic protozoon of the urogenital tract in cattle, presents a poorly known cytoskeleton, formed by rootlets and proteinaceous structures, many of which have not yet been characterized. Studies on its skeletal organization sheds light on the evolution of the matrix system, characteristic of higher eukaryotes. The skeletal matrix system of T. foetus in interphasic and dividing cells were studied using whole mount cell procedures observed either in field emission scanning electron microscopy (FESEM) or in transmission electron microscope (TEM) after the cell-sandwich technique, where the plasma membrane was mechanically removed. Three-dimensional-like images of the cell matrix were attained revealing a network of filaments that has not been described previously. Freeze-etching and cytochemistry using acridine orange for TEM, were also used. Membrane-skeleton interactions were examined in the hydrogenosomes, on the nuclear envelope at mitosis and interphase, and in the overall matrix filling of the cytoplasm and nucleoplasm. It was demonstrated that this eukaryote has a complex skeletal matrix other than just the rigid cytoskeletal structures. Our analysis indicated that the nucleus has a defined position, and fibrils perform an anchoring system for the nucleus. The possibility of a mechanism for nuclei fidelity migration during mitosis is discussed.

Birefringence of cells grown in vitro and of mitotic chromosomes after staining with picrosirius red

Fibroblasts and neuroblastoma cells kept in monolayer cultures, as well as surface spreads of mitotic chromosomes, were stained with picrosirius red. Red staining (in normal light) and optical anisotropy of the stained structures (in polarized light) were observed intracellularly and in the chromosomes. The intracellular and extranuclear birefringence induced by staining with sirius red could not be abolished by digestion with collagenase prior to staining, or by treatments used to disrupt microtubules (vinblastine, colcemid) or microfilaments (cytochalasin B). We therefore propose that the parallelly-arranged intermediate filaments are responsible for the optical anisotropy induced by sirius red staining in these cells. In addition, the spatially oriented scaffold of chromosomes can be detected by sirius red-induced birefringence. These data argue against the collagen-specificity of picrosirius red staining and of the birefringence induced by this technique. Our results also suggest that picrosirius red staining combined with polarized light microscopy can be used to study the spatial orientation pattern of the intermediate filaments and chromosome scaffold.

Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

Immunolocalization of tubulin isoforms and post-translational modifications in the protists Tritrichomonas foetus and Trichomonas vaginalis.

In the present report we show the distribution of multiple tubulin isoforms in Trichomonas vaginalis and Tritrichomonas foetus, flagellated parasitic protists of the urogenital tracts of human and cattle, respectively, using immunofluorescence and immunoelectron microscopy. We used several monoclonal and polyclonal anti-tubulin antibodies from different sources and recognizing variant tubulin isoforms. Our results demonstrate that: (1) there is a heterogeneous distribution of the different tubulin isoforms in the main microtubular cell structures, such as axostyle, flagella, basal bodies, and mitotic spindle, (2) the axostyle-pelta junction is a structure with high affinity for glutamylated tubulin antibodies in T. foetus, (3) the spindle labeling is positive to anti-glutamylated tubulin and anti-alpha-tubulin (TAT1 and purchased from Amersham) antibodies in T. vaginalis but it is negative in T. foetus, (4) the nuclear matrix and the cytosol presented positive reaction using glutamylated and TAT1 (anti-alpha-tubulin) antibodies only in T. vaginalis, and (5) the Golgi complex exhibited staining using the glutamylated tubulin antibody. The present data corroborate with the idea of the existence of a heterogeneous population of microtubules in these protists and of a subset of intracytoplasmic microtubules. Microtubule diversity may reflect distinct tubulins, diverse microtubule-associated proteins, or a combination of both.

Intranuclear network of 3-5 nm and 8-10 nm fibers in EL-4 lymphoma cells.

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3-5 nm and 8-10 nm. The 3-5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8-10-nm fibers frequently split into two 3-5-nm filaments. Some 3-5-nm fibers appear to be connected at 90 degrees angles with the 8-10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8-10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.