Nucleolus: the fascinating nuclear body

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.     

Imaging of Nucleolar Dynamics During the Cell Cycle of Cancer Cells in Live Mice

The synthesis and assembly of ribosomal subunits take place in the nucleolus. The nucleolus forms in the nucleus around the repeated ribosomal gene clusters and undergoes cyclic changes during the cell cycle. Although the nucleolus is easily visualized by light microscopy of cells in vitro, the nucleolus has not been imaged in cells in vivo. We report here development of a mouse model to visualize the nucleolus cycle of cancer cells in live mice. HT-1080 human fibrosarcoma cells were labeled in the nucleus with histone H2B-GFP and with retroviral RFP in the cytoplasm. The nucleolus was visualized by contrast to the fluorescence of GFP expressed in the nucleus. HT-1080 dual-color cells were seeded on the surface of a skin-flap of nude mice. The inside surface of the skin-flap was directly imaged with a laser scanning microscope 24 hours after seeding. The nucleoli of the cancer cells were clearly imaged in real-time. The appearance of the nucleoli changed dramatically during the cell cycle. During mitosis, the nucleolus disappeared. After mitosis, the nucleoli decreased in number and increased in size. The nucleolus appears to have a major role in cell cycle regulation effected at least in part by sequestering proteins which affect cell cycle progression. Nucleolar imaging could be used for more precise determination of cancer-cell position in the cell cycle in vivo.     

Morphology of healthy human parathyroid glands in cytologic smears

OBJECTIVE: To compare the morphology of different parenchymal cell types in healthy human parathyroid glands (HPGs) in cytologic smears with their structure on histologic sections and to establish criteria for their recognition in smears. STUDY DESIGN: The HPGs of 47 subjects (27 females and 20 males) were incidentally removed during surgery on the thyroid gland. The tissue of glands with a normal macroscopic and microscopic appearance was analyzed in cytologic smears and on histologic sections. RESULTS: In cytologic smears (as well on histologic sections), dark and light chief cells predominated. Dark chief cells, which were more numerous than light ones, had a smaller nucleus without a visible nucleolus. The nucleus of light chief cells was larger with 1 or 2 nucleoli visible. The cytoplasm of both types of chief cells was poorly defined, grey-blue and often vacuolated. In the smears, the cytoplasm of oxyphilic cells was dense, gray-rose and well defined. In dark oxyphilic cells, the nuclei were smaller and without a visible nucleolus. Light oxyphilic cells had a larger nucleus and visible nucleolus. CONCLUSION: The identification and distribution of 4 parenchymal cell subtypes in the smears of pathologically altered HPGs may yield insights into the possible role of these cells in a specific disease.     

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain.

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.     

Vesicle trafficking maintains nuclear shape in Saccharomyces cerevisiae during membrane proliferation

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope "flare" that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7Δ cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation.     

The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick

Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation. By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyribonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.     

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.     

Age-related quantitative changes in the organelles of rat neocerebellar Purkinje cells.

A quantitative study regarding the age-related changes occurring in the somatic organelles of the neocerebellar Purkinje cell is carried out, using female rats aged 2 to 24 months. Standard manual morphometric techniques are used to calculate the following parameters: somatic volume, volumetric fractions and absolute volumes of the intracellular structures as well as the membrane profile concentration, the membrane surface concentration and the mean surface of the rough endoplasmic reticulum cisternae per cell (RER-S). From a statistical point of view, all the cell components significantly modify their volumetric fractions (except the multivesicular bodies and nucleolus; the latter in relation to the nucleus) and their absolute volumes (except the mitochondria and the multivesicular bodies); the parameters regarding the reticulum are also modified during ageing. There is a linear trend between the age and either the somatic volume of the RER-S or the absolute volumes of the following structures: mitochondria, dense bodies, ground substance and total cytoplasm. A linear correlation is also observed between the cell volume and either the RER-S or the absolute volume of intracellular structures (the Golgi apparatus, the multivesicular bodies and nucleolus being excluded). Anatomophysiological considerations about the findings are discussed. The role of the ground substance as the major modulator of the volumetric plasticity of the Purkinje cell during ageing, is emphasized as a conclusion.     

Development of the accessory glands in the genital tract of female Teleogryllus commodus WALKER (Insecta, Orthoptera)

The development of the female accessory glands in Teleogryllus commodus was studied by detailed morphometric and stereological analyses. In addition, a microsurgical method was developed to quantify the gland secretion. The morphometric results yield evidence that the glands are subject to a significant growth during peak differentiation, starting immediately after the adult moult. The gland growth is exclusively caused by a hypertrophy of single gland cells with respective volume gains between 400 and 700%. According to the stereological results, the volume of mitochondria per cell is marked by an up to fourfold increase during peak differentiation. Other cell structures (rER, sER) are characterized by a similar propagation behavior. The nucleus and nucleolus grow simultaneously with the cell, indicating high production of site-specific macromolecules. Infolds of the basal cell membrane cause a progressive enlargement of the basal cell surface ensuring an increased uptake of secretory precursors from the hemolymph. Quantitative studies show that the total production of secretion increases with proceeding age and can be correlated with a rising egg-laying activity, starting on the eighth day of adult's life. This underlines the main function of the secretion as a lubricant for a facilitated transport of the eggs through the ovipositor.     

Changes in the thyrocytes to the administration of intermedin under conditions of the aminazine-altered functional state of the nervous system]

Histological and electron microscopial investigations of the thyroid gland were performed in white mice after exogeneous administration of intermedin (MSH) and intermedin against the background of aminasin. It may be concluded that while a prolonged injection of MSH results in the stimulation of the thyroid gland, the administration of the hormone in question against the background of aminasin results in the same ultramorphological structural changes of the gland which are observed after injection of aminasin alone, causing a decreased functional activity of the thyroid gland. The effect of intermedin upon the thyroid is supposed to be realized through those hypothalamus structures which are responsible for the regulation of the thyrotropic function of the hypophysis.     

Elimination of the nucleolus from the nucleus of a living cell by centrifugation (a literature review)

The following effects involving the nucleolus take place during centrifugation of living cells at centrifugal forces of several thousand g to several hundred thousand g: settling of the nucleolus in centrifugal direction on the nuclear envelope; pulling the latter as a long stalk with the nucleolus at its end (or alternatively an easy perforation of the nuclear envelope by the nucleolus); release of the nucleolus into the cytoplasm or its expulsion out of the cell; occasional stratification of the nucleolus in the nucleus; fusion of many nucleoli together under centrifugal pressure. The asymmetric topography of the nuclear envelope is considered to be one of the causes of its different resistance to the penetration of the nucleolus. Elimination of the nucleolus from cancer cell nuclei to test the nucleolar contribution to cell malignancy is suggested as one conceivable application of the centrifugal technique of cell enucleolation.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The pars intermedia of Sarotherodon mossambicus (Tilapia mossambica) contains two cell types which can be differentiated at both the light and electron microscopic level. The predominant cell type is lead haematoxyline positive, and has been shown to be the MSH producing cell type by means of immunocytochemical staining at the ultrastructural level. The changes in cellular and nuclear volume, as well as the results of stereological measurements on the cytoplasmic organelles, show that the activity of MSH cells is high on a black background and low on a white background or in total darkness. In blinded fish under a normal day-night regime the activity of the MSH cell is as high as that in black adapted fish, whereas the activity is low when the blinded fish are kept in total darkness. From the observed differences in activity of the MSH cells between the experimental groups, it is concluded that the MSH cells are not activated by the absence of reflected light, but by a high ratio between direct and reflected light. A second light-sensitive organ, supposedly the pineal gland, is also involved in the background response of the MSH producing cells.     

Prestress mediates force propagation into the nucleus

Several reports show that the nucleus is 10 times stiffer than the cytoplasm. Hence, it is not clear if intra-nuclear structures can be directly deformed by a load of physiologic magnitudes. If a physiologic load could not directly deform intra-nuclear structures, then signaling inside the nucleus would occur only via the mechanisms of diffusion or translocation. Using a synchronous detection approach, we quantified displacements of nucleolar structures in cultured airway smooth muscle cells in response to a localized physiologic load ( approximately 0.4 microm surface deformation) via integrin receptors. The nucleolus exhibited significant displacements. Nucleolar structures also exhibited significant deformation, with the dominant strain being the bulk strain. Increasing the pre-existing tensile stress (prestress) in the cytoskeleton significantly increased the stress propagation efficiency to the nucleolus (defined as nucleolus displacement per surface deformation) whereas decreasing the prestress significantly lowered the stress propagation efficiency to the nucleolus. Abolishing the stress fibers/actin bundles by plating the cells on poly-L-lysine-coated dishes dramatically inhibited stress propagation to the nucleolus. These results demonstrate that the prestress in the cytoskeleton is crucial in mediating stress propagation to the nucleolus, with implications for direct mechanical regulation of nuclear activities and functions.     

Nucleolar topography of nuclei in histologic sections. Application of a nonparametric approach to the study of breast cancer and non-Hodgkin's lymphoma

The position of the nucleolus within a near-convex nuclear profile was defined, without taking stereology into account, in terms of two distances, P and G, where P = pi p2/SN, G = pi 2/SN, p and g being, respectively, the smallest and greatest distances between the center of the nucleolus (considered as a disk) and the superficial nuclear membrane, and SN being the surface area of the nucleus. Nuclear elongation (ND) was defined by the ratio ND = SN/A, where A is the area of the largest inscribed disk. The nucleolus-nucleus ratio RS = Sn/SN (i.e., the surface of the nucleolus over that of the nucleus) describes the size of the nucleolus. A four-class classification of nucleolar topography was then developed from the model of an ellipsoid nuclear profile (1 less than ND less than 2.5), for which the probability that a randomly located nucleolus (with RS less than 0.23) will be classified as central (G less than 2.5 and 0.40 less than or equal to P less than 1) is less than 0.14, as paracentral (G less than 2.5 and 0.23 less than or equal to P less than 0.40) is less than 0.13, as "transversely eccentric" (G less than 2.5 and P less than 0.23) is less than 0.10 and as "longitudinally eccentric" (G greater than or equal to 2.5) is greater than 0.66. This theoretical distribution into four classes was compared to that of nuclear profiles in four cases of breast cancer and in typical cases of immunoblastic, centroblastic and Burkitt's non-Hodgkin's lymphomas. The findings illustrate the ability of this nonparametric method to indicate characteristic nucleolar locations in relation to the number of nucleoli, their size and their nuclear profile elongation.     

THE ROLE OF THE NUCLEOLUS : I. Tritiated Cytidine Activity in Liver Parenchymal Cells of Thioacetamide-Treated Rats

Male rats of the Sherman strain were fed for 2 weeks a diet of ground purina rat chow containing 0.04 per cent thioacetamide. Animals were injected intraperitoneally with tritiated cytidine, 200µc/100 gm body weight, and sacrificed in pairs, a control and a thioacetamide-treated rat, at prescribed intervals. Liver tissues were preserved with the freeze-substitution method and postfixed in anhydrous OsO₄. Other samples were fixed directly with an acetic acid-ethanol mixture (1:3). AR-10 stripping film was applied to 2- and 4-µ sections and exposed for appropriate lengths of time. Nuclear and nucleolar volumes were obtained by direct measurement. Cytoplasmic volumes were obtained with the aid of Chalkley ratios. Nucleolar and cytoplasmic RNA concentrations were calculated from cytophotometric extinction (E_{540 mµ}) measurements. Data were expressed as grains/unit area, grains/unit area/concentration (or specific activity) and grains/total structure. In the liver parenchymal cells of thioacetamide-treated rats, the nucleolus shows vast increases in volume, RNA content, and grain count/total structure, 14-fold, 25-fold, and over 30-fold, respectively. The nucleus increases 2-fold in volume and about 3-fold in total grain count. Cytoplasmic volume increases only 20 per cent and displays a total grain count about equal to that in the control. The time course of incorporation curves for nucleolus and non-nucleolar nucleus (NNN) contain 2 distinct turnover fractions, rapid and slow. Both fractions were increased after thioacetamide treatment but remained proportional to those of controls. The unique stimulated RNA turnover in the nucleus and nucleolus, coupled to a "normal" turnover in the cytoplasm, suggests that this nuclear-nucleolar loss of label does not represent an exclusive passage of formed nuclear RNA to the cytoplasm.     

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes.

BACKGROUND: Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. MATERIALS AND METHODS: Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G(0) to G(1) transition and the cell cycle progression. RESULTS: During the first 24 h of stimulation, when the cells underwent G(0) to G(1) transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24-48 h of stimulation, when cells were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G(0) cells. CONCLUSIONS: While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G(0) to G(1) transition, the cells progressing through the remainder of the cycle are better distinguished from G(0) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments.     

Effect of carbon tetrachloride on the hepatic alkaline and acid phosphatases in a teleost fish, Heteropneustes fossils.

The effect of carbon tetrachloride on the liver of Heteropneustes fossilis was investigated in relation to the activity of phosphatases. 3 h after intraperitoneal injection, the liver showed binucleate cells, balloon cells and necrosis. The cell membrane of the hepatocytes around the biliary canaliculi is ruptured at a few places and the cellular exudates are accumulated in the canaliculi. The nucleus is enlarged and becomes pycnotic. Intense alkaline and acid phosphatase activity is observed along the cell membrane, around the nucleus and nucleolus. 5 h after injection, the liver cell membrane is ruptured and cirrhosis results. The cell volume is decreased and the contents of the cell are accumulated at a few places. The nucleus is fragmented. Strong phosphatase activity is seen throughout the cells, especially around the nucleus and cell membrane.     

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.     

Fasciola hepatica: Stereological analysis of vitelline cell development

Cytomorphosis of vitelline cells in Fasciola hepatica has been studied quantitatively by means of a Kontron Videoplan computerized image analyser. The process of vitelline cell development was subdivided into four characteristic phases: the stem cell, the intermediate type 1 cell, the intermediate type 2 cell, and the mature cell. The whole cell and the following constituent organelles, the nucleus, nucleolus, mitochondria, granular endoplasmic reticulum (GER), secretory granules, glycogen, heterophagosomes, and lipid, were analysed at each phase. Results indicated that there was a significant increase in nuclear size between the two intermediate cell stages, and a significant increase in nucleolar size between the stem cell and intermediate type 1 cell; the changes were related to possible gene activation, ribosome production, and cell synthesis required for both cell growth and secretory production. Mitochondria increased in number and showed changes in volume and surface area of whole organelles and their cristae such that these correlated with the energy demands of growth and synthesis. The GER increased its average total volume some 16 times and its average total surface area some 25 times between the stem cell and mature cell stages. Even this increase masked the true extent of membrane production by this system, since membrane was transferred to secretory granules; in reality the GER or GER-derived membranes increased more than 42 times between the stem cell and mature cell stages. Shell globules and glycogen eventually contributed 21 and 20%, respectively, to cytoplasmic volume, while heterophagosomes contributed 14% and lipid only 3%. Residual cytoplasm was shown to be an important component in cytomorphosis, comprising never less than 40% of the total cytoplasmic volume and it was shown to increase prior to or parallel with the development and expansion of other systems.