Cementum protein 1 transfection does not lead to ultrastructural changes in nucleolar organization of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Transfection of cementum protein 1 (CEMP1) into human gingival fibroblasts (HGFs) notably increases cell metabolism and results in overexpression of molecules related to biomineralization at transcriptional and protein levels. Therefore, HGF-CEMP1 cells are considered as putative cementoblasts. This represents a significant advance in periodontal research because cementum neoformation is a key event in periodontal regeneration. In addition, it is well known that important changes in cell metabolism and protein expression are related to nucleolar structure and the function of this organelle, which is implicated in ribosome biogenesis. The aim of this study was to determine the effect of transfecting CEMP1 gene in human HGF on the ultrastructure of the nucleolus. MATERIAL AND METHODS: Cells were processed using the conventional technique for transmission electron microscopy, fixed with glutaraldehyde, postfixed with osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections were stained with Toluidine blue and observed by light microscopy. Thin sections were stained with uranyl acetate and lead citrate. For ribonucleoprotein detection, the staining method based on the regressive effect of EDTA was used. In addition, the osmium ammine technique was used for specific staining of DNA. RESULTS: The results obtained in this study suggest that transfection of CEMP1 into HGFs does not produce changes in the general nucleolar ultrastructure because the different components of the organelle are present as fibrillary centers, and dense fibrillar and granular components compared with the control. CONCLUSION: The transfection of CEMP1 into HGFs allows these cells to perform cementoblast-like functions without alteration of the ultrastructure of the nucleolus, evaluated by the presence of the different compartments of this organelle involved in ribosomal biogenesis.

Cellular organization of pre-mRNA splicing factors in several tissues. Changes in the uterus by hormone action.

In the mammalian cell nucleus, splicing factors are distributed in nuclear domains known as speckles or splicing factor compartments (SFCs). In cultured cells, these domains are dynamic and reflect transcriptional and splicing activities. We used immunofluorescence and confocal microscopy to monitor whether splicing factors in differentiated cells display similar features. Speckled patterns are observed in rat hepatocytes, beta-cells, bronchial and intestine epithelia and also in three cell types of the uterus. Moreover, the number, distribution and sizes of the speckles vary among them. In addition, we studied variations in the circular form (shape) of speckles in uterine cells that are transcriptionally modified by a hormone action. During proestrus of the estral cycle, speckles are irregular in shape while in diestrus I they are circular. Experimentally, in castrated rats luminal epithelial cells show a pattern where speckles are dramatically rounded, but they recover their irregular shape rapidly after an injection of estradiol. The same results were observed in muscle and gland epithelial cells of the uterus. We concluded that different speckled patterns are present in various cells types in differentiated tissues and that these patterns change in the uterus depending upon the presence or absence of hormones such as estradiol.

Localization of intranuclear RNA by electron microscopy in situ hybridization using a genomic DNA probe.

BACKGROUND: The presence of RNA in the cell nucleus is well known. However, a high resolution in situ hybridization evidence for the presence of RNA in some nuclear particles is still lacking. The aim of this work is to localize RNA in subnuclear particles using a novel ultrastructural in situ hybridization procedure. In this study, biotinylated genomic mouse DNA as a probe to localize total RNA in the nuclei of mouse hepatocytes was used. METHODS: The procedure is based on paraformaldehyde fixation and embedding in lowicryl resin. Thin sections are mounted in formvar-coated gold grids. Hybridization is performed on non-denatured thin sections. DNA-RNA hybrids are detected with streptavidin-10 nm gold particles complex. By controlling the time of nick-translation during incorporation of biotin into the probe, labeling in the fibrillar portions of the nucleoplasm is obtained. More digested probes generate more labeling in the granular components. Nucleoli were similarly labeled. RESULTS: As expected, no label was observed in the compact chromatin clumps. These results indicate that granular components as perichromatin granules in the nucleus contain more processed RNA than fibrillar portions. As a comparison, viral DNA sequences on denatured RNase-treated thin sections of adenovirus-2 (Ad-2)-infected human cells were detected. As previously reported, at late stages DNA was observed in the viral particles and surrounding nucleoplasm, where Ad-2 DNA is synthesized. CONCLUSIONS: The present procedure allows the study of intranuclear RNA distribution and will be useful for the analysis of RNA processing in several types of cells.

Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis.

We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.

Electron microscopic localization of ribosomal DNA in rat liver nucleoli by nonisotopic in situ hybridization.

We have used postembedding nonisotopic in situ hybridization, with biotinylated rat ribosomal DNA (rDNA) as a probe and streptavidin coupled to 10-nm colloidal gold particles as the detection system, to localize rDNA sequences in rat liver nucleoli at the electron microscopic level. For comparison purposes, immunoelectron microscopy was performed for the detection of DNA. Our results indicate that ribosomal DNA sequences are enriched in the dense fibrillar component of the rat liver nucleolus. These data are discussed in relation to the putative site(s) for transcription of ribosomal genes.