Nucleolus degradation and growth induced by uv-microbeam irradiation of interphase cells grown in culture

In contrast to total cell irradiation, local UV-microbeam irradiation can stimulate a significant diminution in the irradiated mature nucleoli in interphase mammalian cells in culture. This diminution is accompanied by the concomitant expansion of the unirradiated nucleoli within the same nucleus, and the total nucleolar volume per nucleus does not change appreciably. It is suggested that these nucleolar volume changes are the result of the dispersion, migration, and redistribution of the nucleolar material between competitive nucleolar organizer regions of the interphase nucleus.     

Daily fluctuations of nucleoli in neurosecretory cells of the rat supraoptic nucleus

Summary Daily fluctuations of nucleoli and nucleolar fibrillar centres in neurosecretory cells from the supraoptic nucleus (SON) were investigated in rats artificially synchronized for 3 weeks to a set 12 h light/12 h dark cycle with free access to food and water. Groups of 3 animals were sacrificed by intracardiac perfusion every 4 h for a 24-h period and every 2 h between 22.00 h and 07.00 h. The SON of each animal was removed, and the mean nucleolar volume and the mean volume of the nucleolar fibrillar centres were estimated by a stereological analysis. The quantitative data showed that the fluctuations in the nucleolar volume of SON neurons depend on the time of sacrifice. A peak value was found in animals sacrificed at 03.00 h which was 1.5 times the value found in animals sacrificed at 19.00 h. The volume of fibrillar centres underwent small, but not significant changes over the 24-h period. None of the large fibrillary centres that can be observed in the superior cervical ganglion were found in the SON. Our results demonstrate that in these neurons the size of the nucleolus undergoes daily fluctuations. These results are discussed in the light of previous studies conducted at our laboratory on the circadian rhythm of nucleolar volume and of nucleolar components in neurons of the superior cervical ganglion.     

Relations between nucleolar morphometric parameters and pre-rRNA synthesis in animal and plant cells

In order to study if there are differences between cells of the same tissue with one and two nucleoli, nuclear and nucleolar volume, density of tritiated uridine incorporation, amount of DNA per nucleus and intensity of cytoplasmic basophilia were measured in mononucleolated and binucleolated rat epithelial endometrial cells, in onion root meristematic cells and in chick embryo matrix cells of the central nervous system, neuroblasts and neurons. No significant differences in nuclear volume, density of tritiated uridine incorporation and amount of DNA per nucleus were found between cells of the same type with diverse numbers of nucleoli. Binucleolated endometrial cells, matrix cells, and root meristematic cells have biphasic distributions of nucleolar volumes. One peak of this distribution roughly coincides with the nucleolar volume of mononucleolated cells, the other peak corresponds almost to double the volume. As the density of uridine incorporation is the same irrespective of the nucleolar number and volume, the cells with larger nucleolar volumes have higher pre-rRNA synthesis. These cells also have higher amounts of ribosomes in the cytoplasm, as revealed by the photometric study of basophilia. It is concluded that in this population of cells the ribosomal production is regulated to a higher steady equilibrium than in the general population. This difference is not due to polyploidism or to the increased DNA content of G2 phase cells. Binucleolated neuroblasts and neurons have nucleolar volumes similar to those of mononucleolated ones.     

Evaluation of fixative composition,fixative storage,and fixation duration on the fine structure and volume of root-cell nucleoli

Summary— The effect of various combinations of three fixative compositions (glutaraldehyde buffered in veronal acetate, cacodylate, and piperazine-N, N'-bis[2-ethanesulfonic acid]—PIPES], two fixative storage times (fresh vs 6 weeks), and two fixation durations (3 h vs 9 days) on nucleolar fine structure and nucleolar volume in three root cell-types of oat seedlings (Avena sativa L, cv Seger) were evaluated. All fixatives show overall good preservation of fine structure. Nucleolar components are distinct and well delineated in cells fixed in solutions buffered with either cacodylate or veronal acetate; the components are more condensed when preserved in fixative buffered with PIPES. Nucleolar volume is greatest in cells fixed in the cacodylate fixative, and smallest in those preserved in the PIPES fixative. Among the treatments tested, the PIPES fixative evidently best maintains nucleolar volume. Distracting particulate deposits are abundant on nuclei and nucleoli in cells preserved in the veronal-acetate fixative. Contrary to common assumptions, aging of buffered fixative at room temperature for 6 weeks seems to affect neither the general quality of cellular preservation nor the pH of the fixatives, although nucleolar volume is reduced by such treatment. Long-period fixation (9 days) results in destruction of membrane integrity (mitochondria, plastids, ER), and shrinkage of organelles from the cytoplasm. Nucleolar volume is reduced with prolonged fixation.     

Quantitative analysis of Ehrlich tumour cell nucleoli during interphase

Nuclear and nucleolar growth throughout interphase has been analysed by stereological methods. The nuclear volume and the volumes of the different nucleolar components have been determined in Ehrlich tumour cells selected at various stages of the cell cycle. These quantitative electron microscopic investigations demonstrate that the nuclear and nucleolar volumes are twice as large in G2 as in G1 cells. In addition, all the nucleolar constituents participate in this latter process.     

Nuclear and Nucleolar Size Changes and Nuclear Pore Frequency in Cultured Explants of Jerusalem Artichoke Tubers (Helianthus tuberosus L.)

Nucleolar and nuclear envelope size changes in cultured explantsof H. tuberosus L. were studied prior to the first mitotic division.Using the technique of nuclear isolation to facilitate measurementsresults were obtained showing an almost immediate increase innuclear envelope surface area, while nucleolar volume showedno appreciable increase until 4 h after excision. The sharpincrease in nucleolar volume shown at this time reaches a maximumat 18 h which is maintained until mitosis occurs. The frequencyof nuclear pores remains constant. These results are discussedin the light of previous work on levels of RNA throughout theactivation process.     

Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein.

DNA topoisomerase (topo) I is an essential nuclear protein and a target for anticancer drug camptothecin derivatives. As a nuclear protein, topo I is concentrated in the nucleolus. However, this nucleolar distribution of topo I is dynamic. It has been shown recently that topo I rapidly moves out of the nucleolus (nucleolar delocalization) in response to topo I inhibitors. In the present study, we demonstrated that nucleolar delocalization of topo I is associated with its conjugation by SUMOs (small ubiquitin-like modifiers) in response to the topo I inhibitor topotecan. Time-course experiments revealed that SUMO-topo I conjugation occurred at as early as 5 min after drug treatment, which was earlier than its observed nucleolar delocalization. Furthermore, heat shock blocked sumoylation of topo I; it also blocked the nucleolar delocalization of topo I fusion proteins. UBC9 is an E2 (ubiquitin carrier protein)-conjugating enzyme essential for sumoylation. Although overexpression of wild-type UBC9 enhanced both sumoylation and nuclear delocalization of topo I, overexpression of a UBC9 dominant negative mutant attenuated topo I sumoylation and its nucleolar delocalization. Taken together, our results suggest that sumoylation of topo I might serve as an addressing tag for its nucleolar delocalization in response to topo I inhibitors.     

Effects of 2-mercapto-1-(beta-4-pyridethyl)-benzimidazole (MPB) on Ehrlich cell nucleoli: stereological analysis

The effects of MPB, a strong inhibitor of RNA synthesis, have been analysed at the ultrastructural level by means of stereological methods. After treatment, an increase in the nucleolar volume is observed. This enlargement is due to vacuolization of the nucleoli. However, the relative volumes of the nucleolar components are modified in various directions: the volume of the granular component decreases whereas the fibrillar centres increase in size. These results are discussed in terms of relations between morphology and function of the nucleolus.     

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.     

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.     

NUCLEOLAR VARIATION DURING DIFFERENTIATION OF PHLEUM ROOT EPIDERMIS

Striking differences in nucleolar volume were found between trichoblasts and hairless initials all along the differentiation gradient of the tissue. The larger nucleoli of the trichoblasts were evident from the onset of their differentiation in the meristem and remained so throughout the growing 1000μ of epidermis. At the same time, nucleoli of the alternating, hairless initials rapidly became reduced in volume and virtually disappeared in maturing cells. Nuclear volume was relatively constant throughout the epidermis, so that nuclear:nucleolar volume ratios clearly indicated the nucleolar differences between the shorter trichoblasts and longer hairless initials. Along with the first signs of nucleolar volume difference in the meristem, there were higher concentrations of RNA and ribonucleoprotein in nucleoli and cytoplasm of trichoblasts compared with hairless initials. Although these chemical differences occurred principally in meristem cells, protein content was much higher in trichoblasts than in the alternating hairless initials of the enlargement zone, 300–500μ from the root tip apex. There was no essential difference in protein content between the 2 cell types in the meristem or in older enlarging cells. The data suggested that the initial increase in nucleolar volume and content of the meristem trichoblasts led to their increased protein content and enzyme activities during the enlargement phase of their differentiation. The sharp reduction in nucleoli of the hairless initials, at the same time, led to their generally lower metabolism during tissue differentiation.     

Predictive value of discriminant analysis of monocyte ultrastructure in malignant lymphoma

Discriminant analysis was applied to morphometric data obtained from ultrastructural studies of blood monocytes from 20 normal subjects, 23 patients with Hodgkin's disease and 12 patients with non-Hodgkin's lymphoma. The aim was to assess the efficiency of predicting subject groups from such data. The analysis, performed on a microcomputer using a standard statistical package, considered nuclear volume, nuclear surface area, nucleolar volume, nucleolar surface area, nucleolar volume fraction, number of nucleoli per section, cell surface area, mitochondrial surface area and subject age. The overall agreement between predicted and actual subject groups was 64%; considering only normality and disease, the agreement was 80%. While the predictive value of such data from circulating monocytes would appear insufficient for diagnostic purposes, discriminant analysis as used here might be of value in indicating the state of host defense in malignancy.     

Changes in nucleolar morphology during human macrophage development: a morphometric study

Morphometric methods were used to study the nucleolar ultrastructure during the development of human blood monocytes into macrophages in suspension culture. Nucleolar volume (Vn), surface area (Sn), volume fraction within the nucleus (VVn), surface-to-volume ratio [(S/V)n] and number of nucleolar profiles per section were measured in 20 healthy adults over a 6-day period, and the results examined using multivariate and univariate analyses of variance. Highly significant increases in Vn, Sn and VVn occurred, with no significant change in the number of nucleolar profiles per section; (S/V)n decreased during culture; no significant differences were found between male and female subjects. These nucleolar changes would be consistent with an increased protein synthesis during macrophage development. The results provide quantitative data against which changes in nucleolar morphology during macrophage development in disease states may be assessed.     

Fluctuations in ribosomal RNA gene content and nucleolar activity in the cambial region of Abies balsamea (Pinaceae) shoots during reactivation

Tissue was collected from the vascular cambial region of 1-year-old balsam fir shoots over an 11-week period during which cambial reactivation occurred. The amount of rDNA (ribosomal RNA genes) relative to total genomic DNA was determined by quantitative slot blots for three trees, one of which showed a 3-week delay in reactivation. In addition, nucleolar activity was estimated by measuring nucleolar volume, number, and staining intensity. Relative rRNA gene content increased transiently prior to the onset of cambial cell periclinal division. Nucleolar volume also increased transiently, but 1–2 weeks prior to the maximal relative rDNA value. The increases in relative rDNA and nucleolar activity were delayed in the tree in which reactivation was late. We interpret these changes as reflecting the amplification and loss of genes encoding rRNA to facilitate cambial cell reactivation.     

Control of nucleolar growth during interphase in higher plant meristem cells

Summary The evolution of nuclear and nucleolar sizes throughout interphase have been studied in synchronous caffeine-labeled binucleate cells of onion root meristems by using silver impregnation and stereological methods over semithin sections. Nucleus and nucleolus grow independently, since nucleolus enlarges at its fastest rate in G 1, while nucleus grows mostly in two periods: onset of replication and G 2. Nucleolar size in the cycle seems to be a genecontrolled function, hardly affected by protein synthesis inhibition. Hence, there is a biphasic response to cycloheximide (CHM) in the fast growing nucleoli of both early and late G 1 with an initial stimulation later counterbalanced by a depressed rate, so that nucleolar size in S was similar to control shortly afterwards the start of the CHM treatment. The initial enlargement under CHM was due to an increase of all nucleolar structural components, i.e., fibrillar, granular, vacuolar, and lacunar regions. No cycloheximide effect whatsoever was detected in S and G 2 nucleoli.Abbreviations CHM cycloheximide - F fibrillar component - G granular component - L lacunae - V vacuoles - VN nuclear volume - VNu nucleolar volume - VvNu volume density of the nucleoli     

Nucleolar variation in response to nutritional condition in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

The variations of the nucleolar characteristics size and number were studied in the gill and liver of Odontesthes bonariensis under different feeding regimes in order to test the hypothesis that functional genomes at the nucleolus, which determine variation of size (heterogeneity) in juvenile fishes, are modulated by environmental factors such as food availability. The programme that controls genetic changes of the nucleolar characteristics of the diploid cells of O. bonariensis was different from that described in the diploid cells of cyprinid fishes. No variations of the mean nucleolar number per nucleus were observed in liver or gill cells in relation to the feeding regime. Conversely, significant reduction of the mean nucleolar volume per nucleus ( V _MN) and change in the shape of the frequency distribution of the total nucleolar volume per cell was evident in both cell types in response to starvation or food restriction. Tissue specificity was observed in relation to the absolute values of the nucleolar characteristics; however, the relative behaviour was comparable in digestive and non-digestive tissues. The time-course reduction of V _MN in gill and liver cells of restrictively fed fish followed a negative exponential. A good correlation between V _MN and the condition index of the fish ( K ) was observed after long-term food restriction; however, a faster response of V _MN was observed after short-term fasting events. Results supported the stated hypothesis and showed nucleolar activity as a direct, rapid and sensitive indicator of the nutritional status of the fish.     

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis (RB), and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting RB. However, there is a need for a more direct high-throughput assay for a nucleolar function to further evaluate hits. Previous reports have monitored nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay that enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay uses two siRNA controls: a negative non-targeting siRNA control and a positive siRNA control targeting RNA Polymerase 1 (RNAP1; POLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small-molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dispersion. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease.     

The pinealocyte nucleolus

Summary An ultrastructural and morphometric analysis was made of the nucleolar components in pinealocytes of 40 male Fischer rats sampled at eight times in an LD 1212 photoperiod cycle. Comparisons of results from the eight times showed variation in estimated mean volume of the granular component of ±29%, and of the fibrillar component ±11%, in relation to daily means. Peaks in mean volume of total nucleolus and its granular component occurred at 1 h of light. Near maximal and minimal mean volumes of the fibrillar component both occurred during both light and dark. Fibrillar centers (nucleolar organizer regions) of different sizes were found at all sampling times. It is concluded that temporal patterns in 24-h changes in the nucleolar components are most prominent in the granular component, and are more complex than suggested by changes in total nucleolar size or mean dimensions, and than represented by a simple biphasic circadian rhythm. Examples of different stages in the migration of the granular component, and of possible sites of nucleo-cytoplasmic transfer of nucleolar material, are described.