Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Rearrangement of nucleoli in hepatocytes stimulated for proliferation and enhancement of their transcription function

The nucleoli of normally functioning guinea-pig hepatocytes that have a nucleolonemal (strand-like) organization differ from identical nucleoli of other cells. Their nucleolonema consists as a rule of a fibrillar component with 45S RNA and is poor in granulas that contain pre-rNA molecules of an intermediate size and 28S rRNA, a dense fibrillar component with nascent rRNPs in its composition was not revealed. In hepatocytes stimulated by a 2/3 liver resection rearrangements in nucleoli were found. This brought to a conclusion that rRNA metabolism undergoes some changes. In 2.5 and 5 hours after the resection the hepatocytes' nucleoli were characteristic of a greater thickness of strands and a smaller size of vacuoles, appearance of distinct zones of the dense fibrillar component and an increased amount of RNP-granules. All these observations taken together point out at an increased synthesis and processing of rRNA at early stages of the prereplicative period. In 9 hours the character of changes in nucleoli was different: the vacuoles were considerably widened, whereas the thickness of strands that consisted of a well-expressed dense fibrillar, fibrillar and granular components was lesser. Such rearrangement points out at an increased transport of preribosomes from the nucleolus, a high level of synthesis and processing of nascent RNP-product being maintained. The changes of nucleolar RNP-component were followed by appearance of greater blocks of perinucleolar condensed chromatin, which may be connected with "cutting-off" some tissue-specific genes and initiation of functioning of the mitotic operon genes.     

Ultrastructure of the ring-shaped nucleoli in the hepatocytes of chick embryos]

The fine structure of ring-shaped nucleoli in hepatocytes of chick embryos at different terms of development was studied. Structural differences were shown between annular nucleoli and nucleoli with typical nucleolonemal organization. Ring-shaped nucleoli, as a rule, are devoid of nucleolonemal structure and their fibrillar component is reduced. The material which fills the central cavity always appears similar to the nucleoplasm in its structure. On the basis of serial sections, we propose that central cavity is isolated from the nucleoplasm. From the hypotonical treatment and EDTA staining application it is concluded that the central cavity of ring-shaped nucleoli contains DNP associated with the intranucleolar chromatin. The number of these nucleoli increased after the injection of a liver homogenate cytoplasmic fraction extracted from adult hen. The physiological significance of such nucleoli is discussed.     

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7–1.5 µm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5–2.5 µm) have a higher level of activity, contain 4–7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5–3.5 µm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18–22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2–7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.     

Relationship between the Ag-NOR proteins and ribosomal chromatin in situ during drug-induced RNA synthesis inhibition

In human TG tumor cells, the role of silver-NOR proteins was investigated by examining their relationship with the chromatin structure during inhibition of RNA synthesis by actionomycin-D treatment. This induced segregation of the nucleoli into four distinct zones and weakened the silver reaction. The fibrillar components were found to constitute the site of silver-stained proteins segregation. Feulgen-like osmium-ammine staining revealed that the DNA disappeared from the segregated nucleoli except for a network of nonnucleosomal filaments. When Ag-NOR protein detection was combined with chromatin visualization, we found constant overlapping of the silver reaction sites with the nonnucleosomal DNA filaments. Our results indicate that certain Ag-NOR proteins are not directly linked to active rRNA synthesis, but might rather affect the structure of ribosomal genes.     

Behaviour of nucleolus during mitosis

The aim of the present work was to study the distribution and the behaviour of the silver-staining nucleolar organizer region (Ag-NOR) proteins at the ultrastructural level during interphase and mitosis in five human and murine cancerous cell lines each characterized by a typical nucleolar morphology. During interphase the Ag-NOR proteins are restricted to the fibrillar centres (F.C.) and/or to the dense fibrillar component (D.F.C.). During prophase the silver-staining components come into close contact with some chromosomes and are arranged with a typical polarity: chromosome, F.C. and D.F.C. Then F.C. and D.F.C. together form roundish silver-stained structures and integrate in part within indentations at the periphery of the metaphase chromosomes. During anaphase and telophase large and small spherical silver-staining structures may be seen. They correspond respectively to the metaphase NORs and to numerous structures which appear de novo within ribonucleoprotein (RNP) material localized between the chromosomes. During late telophase the number of the small silver-staining structures decreases whereas the size of the larger ones increases. Then the interphase nucleoli recover their typical shape. These results suggest that when rRNA synthesis is impaired during mitosis the inactive NORs assume a structure and a localization which are not typical of the cell line. In contrast the F.C. and D.F.C. are probably two aspects of the NORs whose typical distribution, relative to the other nucleolar components, gives the interphasic nucleolus its characteristic morphology.     

Structural organization of chromatin in nucleolar organizer regions of nucleoli with a nucleolonema-like and compact ribonucleoprotein distribution

We have studied the relationship between the structural organization of intranucleolar chromatin and fibrillar nucleolar structures, fibrillar centers, and RNP fibrillar component, which are the interphase counterpart of metaphase nucleolar organizer regions (NORs), in regenerating rat hepatocytes and in a human tumor cell line (TG cells). These two cell types were characterized by a nucleolonema-like and compact nucleolar RNP distribution, respectively. We found that, in sections selectively stained for DNA, the intranucleolar chromatin composed of extended, nonnucleosomal DNA filaments formed roundish agglomerates with a spatial distribution which was superimposable on that of the fibrillar centers and the RNP fibrillar component around them and on sites of the silver reaction in samples selectively stained for Ag-NOR proteins. The agglomerates of extended nonnucleosomal DNA filaments were small and numerous in regenerating hepatocyte nucleoli, in which the RNP components had a nucleolonema-like distribution, whereas they were large and few in TG cell nucleoli, in which the RNP components showed a compact organization. Since the pattern of ribosomal RNA synthesis and processing was similar in the two cell types, a model was proposed in which the difference in size and shape of the agglomerates of extended DNA might be responsible for the different structural organization of the RNP components.     

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei

Summary Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver.In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes.In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules.In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes.In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes.In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components.The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.     

RNA synthesis in the circular nucleoli of hepatocytes]

By analysis of serial sections it has been revealed that the so-called ring-like nucleoli of hepatocytes consist of a cavity with an amorphous contents surrounded by fibrillar and granular material. Such nucleoli are sometimes encountered in normal animals; the number of ring-like nucleoli increases considerably in chronic pathological process caused by repeated CCl4 injections. The capacity of RNA synthesis in the ring-like nucleoli was revealed by means of electron-microscopic autoradiography.     

Three-dimensional distribution of Ag-NOR proteins in rat superior cervical ganglia nucleoli according to circadian rhythm

A three-dimensional reconstruction of the distribution of Ag-NOR proteins in nucleoli of sympathetic neurons of a rat killed during the dark period of its light-dark cycle was compared with previously reported analyses on the three-dimensional distribution of fibrillar centers, the high-resolution localization of these proteins, and the morphometric results. The domain occupied by these proteins appeared to far exceed that of the fibrillar centers and included the dense fibrillar RNP component. In the present material this component in turn provided partial bridging between the units consisting of the fibrillar centers plus their surrounding dense fibrillar component.     

A comparative study of the main components of the nucleolus in different populations of rat trophoblast cells during differentiation. I. The nucleoli of the trophoblast cells in the labyrinth section of the placenta

The nucleolus undergoes some steps of structural transformation during differentiation of the labyrinth trophoblast cells. Primarily (on day 13 of gestation) the nucleolar components become rather disjoined. The nucleolus is composed of a loose net of strands of granulofibrillar and dense fibrillar components bearing fibrillar centers (FCs). Strands are separated by large lacunae. This rare-occurring type of nucleoli is replaced on the next (14th) day by the nucleolonemal type and later--by the compact nucleolar type. FCs with dense fibrillar component strands become extended into the masses of granulofibrillar component. Such transformations of nucleolar structure seem to be an expression of a fast-proceeding differentiation of the labyrinth trophoblast cells.     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.     

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.     

Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.