Cadmium can induce alterations in the cellular localization and expression of three major nucleolar proteins in root tip cells of Vicia faba L

Aims

The findings of our earlier investigations indicated that cadmium (Cd) could induce some nucleolar materials containing the argyrophilic proteins scattered in the nuclei and located in cytoplasm in the root tip cells of Allium sative and Vicia faba. However, what kinds of nucleolar proteins are affected has not been reported up to now. In order to further confirm the cytological effects of Cd on nucleolus and nucleolar proteins, alterations in the cellular localization and expression of three major nucleolar proteins: nucleophosmin, fibrillarin and nucleolin were investigated under the treatment with Cd in the root tip cells of V. faba in this study.

Methods

Silver methods, Indirect immunofluorescent microscopy; Western blotting.

Results

The obvious toxic effects on nucleoli in the cells were observed after Cd treatment. Nucleolar proteins, nucleophosmin, fibrillarin, and nucleolin in root tip cells exposed to Cd (50 μM) for 48 h were translocated from nucleolus to nucleoplasm and cytoplasm and expression of the three major nucleolar proteins was relatively higher when compared with control.     

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.     

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.     

Rbm19 is a nucleolar protein expressed in crypt/progenitor cells of the intestinal epithelium

Intestinal development and homeostasis rely on the coordination of proliferation and differentiation of the epithelium. To better understand this process, we are studying Rbm19, a gene expressed in the gut epithelium that is essential for intestinal morphogenesis and differentiation in the zebrafish (Development 130, 3917). Here we analyzed the expression of Rbm19 in several biological contexts that feature proliferation/differentiation cell fate decisions. In the undifferentiated embryonic gut tube, Rbm19 is expressed throughout the epithelium, but then becomes localized to the crypts of Lieberkühn of the adult intestine. Consistent with its expression in adult crypt/progenitor cells, expression is widespread in human colorectal carcinomas and dividing Caco-2 cells. Its expression in Caco-2 cells recapitulates the in vivo pattern, declining when the cells undergo confluence-induced arrest and differentiation. Rbm19 protein localizes to the nucleolus during interphase and to the perichromosomal sheath during mitosis, in accordance with the pattern described for other nucleolar proteins implicated in ribosome biogenesis. Interestingly, the loss of nucleolar rbm19, nucleolin/C23, and nucleophosmin/B23 in confluent Caco-2 cells did not signify loss of nucleoli as detected by electron microscopy. Taken together, these data point to the nucleolus as a possible locus for regulating the proliferation/differentiation cell fate decision in the intestinal epithelium.