Electron microscopic study of Ag-protein localization in the nucleoli of human lymphocytes

The present study was undertaken to provide more information on the ultrastructural localization of a silver reaction in normal resting and stimulated lymphocytes as well as leukaemic resting lymphocytes. The results obtained indicated that in the ring-shaped nucleoli of normal mature lymphocytes silver stained proteins (SSPs) were present mostly within single fibrillar centers. In the nucleoli of lymphocyte cultures, being in the presence of phytohemagglutinin (PHA) for 6--72 hours, SSPs formed finger or loop-like protrusions from fibrillar centers towards the adjacent areas of the nucleoli. In the ring-shaped nucleoli of mature leukaemic lymphocytes SSPs are present not only within fibrillar centers, but also in protrusions diverging from fibrillar centers into the surrounding peripheral nucleolar ring. In this respect the nucleoli of leukaemic mature lymphocytes were similar to normal lymphocytes shortly after mitogen stimulation.     

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.     

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.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

Studies on satellite nucleoli in human blastic cells of acute leukaemias.

Blastic cells of acute lymphoid, acute myeloid and acute myelomonocytic leukaemias were studied by means of indirect immunofluorencence to provide more information on the presence of satellite nucleoli in blood cells. According to results, satellite nucleoli were found in a small but constant number of blastic cells disregarding their type and type of acute leukaemia. Satellite nucleoli exhibited a positive immunoreaction for fibrillarin and protein B23 which are characteristic for main nucleolar components. These findings suggest that satellite nucleoli contain fibrillar centers as well as dense fibrillar and granular components or at least proteins characteristic for these nucleolar components. Similarly as in normal and pathological cells of completely different origin, in blastic cells of acute leukaemias the number of satellite nucleoli per cells ranged between 1 and 2.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

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.     

Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes

Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.     

3-dimensional organization of the nucleolus and the nucleolus-organizing regions in differentiated cells. I. Ring-shaped nucleoli in the endotheliocytes, smooth-muscle cells and fibroblasts in the mouse

The method of ultra-thin serial sections was used to study the three-dimensional structure and to perform the quantitative analysis of ring-shaped nucleoli of kidney and liver endotheliocytes, smooth muscle cells of kidney arterioles and fibroblasts of mice. Spatial models of ring-shaped nucleoli with one fibrillar centre are given. For the quantitative analysis the following parameters were measured: the number and volumes of nucleoli, fibrillar centers, RNP-containing structures, the vacuolar system and the RNP-index (the latter is a ratio of RNP-part and fibrillar center volumes). Nucleoli of the same type of cells, occasionally in the same nucleus, were found to differ sharply in their fibrillar center shape. Differences in the mean volume values of nucleoli, fibrillar centers and the RNP-part between some cell populations are sufficiently well pronounced. Within the same population ring-shaped nucleoli have, as a rule, specific volume values of nucleoli, RNP and fibrillar centers. The comparison of quantitative data obtained on different cell types showed that the mean RNP-index values were the most stable parameter. The structural relation between fibrillar centers, intra- and perinucleolar chromatin and lacunar region is shown. The structural organization of intranucleolar chromatin and rRNA in the nucleolar body and in fibrillar centers is discussed.     

Activation of transcription function in lymphocytes after irradiation with He-Ne-laser

The influence of He-Ne-laser irradiation (lambda = 632.8 nm) in dose 56 J/m2 on the ultrastructure of the nucleolus from human peripheral lymphocytes was studied electronmicroscopically. After 1 h irradiation a well-expressed reaction of the nucleolus was observed in 70% of the lymphocytes under examination. Changes consist in the appearance of a wrong-shaped fibrillar center or in its fragmentation, the increase of RNP-containing fibrillar and granular components, and also in expansion of vacuoli. In a number of irradiated lymphocytes nucleoli with several fibrillar centres and with a strand-like organization of RNP part were observed. The size of these nucleoli increases. Following the accepted functional interpretations the observed changes can be connected with the intensification of RNA metabolism including the synthesis, processing of pre-rRNA and preribosome transport from the nucleolus. Similar rearrangements of the nucleoli were revealed in parallel experiments with phytohemagglutinin-treated lymphocytes. They were observed 1 h after the stimulation of lymphocytes. Taking into account the absence of mitogenic action of He-Ne-laser irradiation on lymphocytes, the ultrastructural changes of nucleoli under the action of irradiation are considered as functional activation of rRNA synthesis in the Go-period.