The nucleolus: a model for the organization of nuclear functions

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.Robert Feulgen Lecture presented at the 48th Symposium of the Society for Histochemistry in Stresa, Lake Maggiore, Italy, 7–10 September 2006.     

Study of the positional relationship of nucleolar chromatin and nucleolar compartments in somatic nuclei OPF the ciliate Didinium nasutum

We showed earlier that nucleoli in interphase ciliates Didinium nasutum, appearing on single ultrathin sections as individual structures, actually are parts of more complex network-like structures in which fibrillar component is located on periphery, and granular--in the central part of a nucleolus. It is known, that nucleolar organizers in D. nasutum are represented by chromatin bodies connected with nucleoli. In this work we used 3D reconstruction on the basis of serial ultrathin sections to study localization of chromatin bodies which by morphological criteria might correspond to nucleolar organizers. Our data showed, that all such chromatin bodies settled down outside of nucleoli, near the periphery of fibrillar component. Even those chromatin bodies which on single sections looked completely surrounded by fibrillar nucleolar component, actually settled down in fibrillar component cavities open to nucleoplasm. Analysis of distribution of nucleolar chromatin bodies allowed us to conclude that activity in different parts of interphase complex network-like nucleoli of D. nasutum is approximately the same.     

Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.     

Electron Microscopic Studies on the Silver-stained Nucleolar Cycle of Physarum polycephalum

The dynamic changes of nucleolar ultrastructure in the cell cycle of Physarum polycephalum Schw. were studied by an en bloc silver-staining method. The results showed that the nucleolus was large in size and situated in the center of the nucleus in late G2-phase, and the fibrillar centers, dense fibrillar components and granular components could be observed in the nucleolus. During prophase, the nucleolus moved towards the periphery of the nucleus and in late prophase disintegrated near the nuclear envelope. In metaphase, the disintegrated nucleolar components were dispersed in masses and located at the periphery of the chromosomal region of the nucleus. No specifically silver-stained area and argentophilic protein sheath were observed on the chromosomes, but there were some big dispersed silver particles within the chromosomes. During telophase the nucleolar components moved towards the two poles along with the chromosomes and co-existed with the decondensing chromatin in daughter nuclei. The nucleolar components then gradually converged with one another and separated from the chromatin. A big nucleolus was formed in the nucleus about 120 min after the completion of mitosis.     

多头绒泡菌银染核仁周期的电镜研究

以同步化培养的多头绒泡菌（Physarum poldycephalum Schw.)原生质团为材料,应用整体银染技术,电镜下研究了核仁在细胞周期中的超微结构变化。结果变化：核仁成熟时比较大,位于细胞核中央,核仁内可区分出纤维中心、密集纤维成分和颗粒成分等。前期时,核仁向边缘移动,前期末在近核膜处解体,解体的核仁物质主要呈团块状散开。中期时,解体的核仁物质位于细胞核中央染色体区域的周围,染色体上没有特异的银染区域,染色体周边也看不到银染的“鞘”状结构,但在染色体中可见一些散在的银染大颗粒。末期时,核仁物质与染色体一起到达两极,在子细胞核中与正在解集缩的染色质共存一起,以后核仁物质逐渐汇合并与染色质分开。大约在有丝分裂结束120min后,在细胞核中形成一候 中央位置的大核仁,结果提示,低等真核生物的核仁结构和周期变化与高等真核生物的不完全相同。     

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.     

Localization of Nopp140 within mammalian cells during interphase and mitosis

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.     

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.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.     

Ultrastructural Changes of the Nucleolus During Cell Cycle in Triticum aestivum

The ultrastructural changes of the nticleolus during cell cycle in common wheat (Triticum aestivum L. ) were studied by an "en bloc" silver-staining method. It was observed that in interphase, the nucleolus was heavily stained, within which fibrillar centres, dense fibrillar component, granular component and nucleolar vacuoles could be identified. A large quantity of argentine fine granules were distributed in the condensed chromatin. Dur-ing prophase, along with the disintegration of the nucleolus and condensation of the chromatin, the larger heavily-stained granules gradually appeared at the periphery of the chromatin. At late prophase, the materials derived from the nucleolus were spread and deposited on the surface of the chromosomes. The silver-stained, larger granules, deriving from the disintegrated nucleolus, accumulated at the periphery of the metaphase chromosomes and formed an uneven and discontinuous "sheath"-like structure. This "sheath"-like structure was also observed at anaphase. In telophase, the silver-stained nucleolar materials were progressively separated from the "sheath' and fused with each other to form prenucleolar bodies, and at last, participating in the formation of new nucleoli. The results showed that the nucleolar materials were transferred directly to the surface of the chromosomes and formed a discontinuous coat, but not incorporated into the interior of the chromosomes. The silverstained granules inside the chromosomes were neither related to the nucleolus nor to the materials from the disintegrated nucleolus.