DISTINCTIVE CHARACTERISTICS OF NUCLEOLI OF TWO ESTABLISHED CELL LINES

Nucleoli of cultured cells of the established lines KB and L were found to possess a distinctive fine structural organization. The major portion of the nucleolar volume was composed of compact, particulate material. Spheroidal fibrillar zones about 0.4 µ in diameter occurred within the particulate mass. These fibrillar zones had a central light area and a denser rim. Toyocamycin treatment, which sharply inhibited the appearance of newly synthesized RNA in the cytoplasm, caused the gradual disappearance of the fibrillar material from nucleoli. Actinomycin D treatment, which inhibited virtually all RNA synthesis, caused varying types of segregation of nucleolar components. The morphology of nucleoli of KB and L cells and the reorganization of these nucleoli in response to drugs appear to be different from those of nucleoli of freshly initiated Chinese hamster and mouse cell lines.     

A comparative study of the main components of the nucleolus in different populations of rat trophoblast cells during differentiation. II. The nucleoli of the trophospongium cells, the glycogen cells and the secondary giant cells

A comparative study was performed of the arrangement of different nucleolar components during differentiation of trophoblast cell populations in the junctional zone of placenta (glycogen cells and trophospongium) and in the secondary giant cells. Each cell type is characterized by specific interrelation of nucleolar components. Some glycogen cells show signs of segregation of nucleolar components: strands of nucleolar components with fibrillar centers (FCs) are displaced to the periphery of the nucleolus and contact with the perinucleolar chromatin. Large reticular nucleoli in trophospongium cells contain many FCs which are gathered into several "chains" by strands of dense fibrillar component. Such a "chain" has also been found in nucleoli of secondary giant cells, with greater number of FCs in each "chain". Relationship between the arrangement of nucleolar components and the level of cell differentiation is discussed.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

Structure of the nucleoli of developing microsporangiate strobili and root tips of scotch pine

Summary The characteristics of the nucleoli of the microsporangiate strobili and the root tips of Scotch pine (Pinus sylvestris L.) vary both during the course of the cellular cycle and, with regard to the pattern and stage of organ and tissue differentiation. Nucleologenesis takes place in interphase and the nucleoli last until prophase. Several types of nucleoli occur during the nucleolar cycle, the pattern and age of tissues determining which type or types dominate. In the strobilus primordia collected at the end of July and in August, the mitotic frequency is high. Nucleoli remain small throughout the nucleolar cycle, and at the electron microscopic level, they display intermingled fibrillar and fibrillogranular components. Strobilus primordia collected in September contain larger nucleoli in the sporogenous nuclei than in the nuclei of the tapetum or of the wall cells. Amongst the nucleoli with completely intermingled fibrous and granular material, nucleoli with nucleolonema or with vacuoles occur frequently. Small balls of fibrous material are seen on the nucleolar surface and in the nucleoplasm. In October, the mitotic frequency of strobilal cells is low. Nucleoli with completely intermingled fibrillar and granular components have vanished whereas a new, compact type of nucleolus with a dense fibrillogranular main portion and with nucleolonema, has developed. The nucleoli of the sporogenous cells have enlarged continuously whereas those of the wall cells are small. The nucleoli of the root tip cell resemble, to a certain extent, those of the strobilus primordia collected in September. In squashed preparations, the nucleoli of the strobilal cells bind the common nucleolar stains poorly whereas the nucleoli of the root cells can be stained with all the methods used. In certain cases, DNase treatment improves the stainability of the strobilal nucleoli. AgNO₃-staining is successful after acetic acid: alcohol fixation but not after formalin: hydrochinone fixation.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

Association between the nucleolus and the coiled body

By means of light and electron microscopic immunocytochemistry, we have localized p80-coilin, a specific protein marker for coiled bodies, in mammalian cell lines as well as in primary rat neuron cultures. p80-coilin-stained nuclear bodies, which also contained fibrillarin, could be subsequently silver stained by a method specific for the visualization of nucleolar organizer regions. In cycling cells, most coiled bodies were not associated with nucleoli, whereas in rat neurons such as association was frequent. The treatment of cycling cells with actinomycin D or 5,6-dichloro-1-beta-D-ribo furanosyl-benzimidazole led to nucleolar segregation and/or disintegration, and to an association of p80-coilin staining structures with nucleoli. p80-coilin-positive structures contained fibrillarin in both untreated and treated cells. These results support the opinion that there might be a special association between coiled bodies and nucleoli, particularly in neuronal cells.     

Cytochemistry of the microtrabecular network in compact nucleoli of hepatocytes treated with cycloheximide

Summary Compact nucleoli without the segregation of nucleolar components were produced in hepatocytes by the treatment of experimental rats with cycloheximide to facilitate a cytochemical study on the organization of nucleolar components in such nucleoli. The extraction of pepsin pretreated specimens with nucleases (deoxyribonuclease and ribonuclease) demonstrated that compact nucleoli are characterized by a relatively uniform distribution of RNP components which mask a microtrabecular intranucleolar network. This network apparently consists of proteins and contains fine DNA filaments.     

The effect of the Bacillus thuringiensis exotoxin on the fine nucleolar morphology and ultrastructure

Depending on the dose administred to the experimental mice, the Bacillus thuringiensis exotoxin produces striking changes in the nucleolar morphology of hepatocytes such as the formation of ring-shaped nucleoli, micronucleoli and the segregation of nucleolar components. Such changes are apparently related to the decrease and inhibition of nucleolar biosynthetic activities in the production of the nucleolar RNA. In addition, the Bacillus thuringiensis exotoxin causes the formation of nucleolar peripheral dense plaques and, at higher concentrations, the segregation of two distinctly separated granular areas in the nucleolus. Both these light and dense granular areas showed positive staining with Bernhard's EDTA procedure for the preferential demonstration of RNA-containing structures. In some segregated nucleoli the granular components of light granular areas seemed to leave the nucleolus. The presence of discontinuous filamentous shell around micronucleoli produced by the high dose of exotoxin suggests the nucleolar origin of nuclear granular bodies which are surrounded by similar but continuous filamentous shell characteristic of these structures.     

Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry.

Human erythroblasts which are prereticulocyte maturation stages of red blood cells were studied by light microscopic cytochemistry and electron microscopy to provide more information on the ultrastructure of the micronucleoli which are terminal stages of nucleolar changes found during maturation of these cells. As indicated by light microscopy of smeared cells, micronucleoli were virtually the only types of nucleoli present in the last stages of maturing erythroblasts, i.e., polychromatic and orthochromatic (late polychromatic) erythroblasts. Accordingly, they were not portions of the periphery of other nucleoli. Inasmuch as most of the micronucleoli exhibited characteristic segregation of nucleolar fibrillar and granular components they presumably are producing little if any preribosomal RNA, since such segregation generally reflects inhibition of nucleolar RNA synthesis.     

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Summary Brief fixation in a mixture of glutaraldehyde and OsO₄ caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO₄ prevented this fixation artifact. In a fixative mixture, OsO₄ apparently reacted with cell contents before glutaraldehyde.     

Nucleolus-like bodies in micronuclei of cultured Xenopus cells

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [³H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.     

THE FINE STRUCTURE OF THE TRIGGER HAIR IN VENUS'S FLYTRAP

Trigger hairs of Dionaea muscipula fixed in glutaraldehyde and OsO₄ were prepared for study in the electron microscope. Electron micrographs of the active zone of the trigger hair reveal three regions in which the cells differ in size, shape, and cytoplasmic content. Each region contains large numbers of protein bodies and mitochondria with densely packed tubular cristae. Vacuole-like structures containing protein bodies or an anastomosing system of cisternae, or occasionally both, are also present. Found only in the indentation cells is a complex, whorled endoplasmic reticulum. A concentric lamellar arrangement of the endoplasmic reticulum around the vacuolar structures is often observed. The lateral walls of the indentation cells are disproportionately thick while end walls are thin. The basal walls of these cells contain many plasmodesmata. Plasmodesmata in the anticlinal and podium cells pass through constricted zones in the cell wall and are particularly numerous in the peripheral podium cells. The possible functional significance of these structures is discussed.     

Bismuth staining of a nucleolar protein

A major nucleolar protein in Chinese hamster ovary cells with a molecular weight (MW) of 100 kD has been found to stain selectively with the bismuth tartrate technique of Locke & Huie [19]. After glutaraldehyde fixation and bismuth staining of electrophoretic transfers of total nucleolar proteins separated by SDS-PAGE, a single band corresponding to the 100 kD protein is revealed. When the technique is applied to whole cells, small punctate regions of the nucleoli are strongly stained. At the ultrastructural level, bismuth selectively contrasts the fibrillar centers and the adjoining cords of the dense fibrillar component. The remainder of the dense fibrillar component is not stained. It is proposed that the high phosphorylation level of the 100 kD protein is responsible for its glutaraldehyde-insensitive bismuth staining. The concentration of this protein in certain localized regions of the nucleolus suggests that it plays a metabolic rather than a structural role.     

A STUDY OF NUCLEOLAR VACUOLES IN CULTURED TOBACCO CELLS USING RADIOAUTOGRAPHY, ACTINOMYCIN D, AND ELECTRON MICROSCOPY

Previously it has been found that in tobacco callus cells nucleolar vacuoles repeatedly form and contract. In this study, nucleolar vacuoles were investigated by using radioautography, actinomycin D, and electron microscopy. It was found, from grain counts of nucleoli labeled with uridine-³H, that nucleoli containing vacuoles had more than three times as many grains/µ² of nucleolar substance as did nucleolei without vacuoles. Treatment of tobacco callus cells with various concentrations of actinomycin D caused the percentage of cells containing nucleolar vacuoles to decrease; with the highest concentration the percentage of these cells dropped from the normal level of about 70% to less than 10%. However, after removal of actinomycin D the cells regained nucleolar vacuoles up to the control level. When radioautography was used with actinomycin D, it was found that the actinomycin D inhibited the uptake of uridine-³H, i.e. inhibited RNA synthesis, in those nucleoli which lost their nucleolar vacuoles. In addition, after removal of the cells from actinomycin D, it was found that as the cells regained nucleolar vacuoles the nucleoli also began to incorporate uridine-³H. Electron micrographs showed the nucleoli to be composed of a compact, finely fibrous central portion surrounded by a layer of dense particles 100–150 A in diameter. Nucleolar vacuoles occurred in the fibrous central portion. Dense particles similar to those in the outer layer of the nucleoli were found scattered throughout the vacuoles and in a dense layer at their outer edge. These data suggest that in cultured tobacco callus cells the formation and contraction of nucleolar vacuoles is closely related to RNA synthesis in the nucleolus.     

Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures

EM investigation of Ag-AS-NOR staining after short glutaraldehyde prefixation followed by Carnoy fixation maintained good ultrastructural preservation and reactive selectivity. This enables exact localization of silver deposits both in the fibrillar centers of typical or segregated nucleoli during interphase, and in chromosome NORs during mitosis. These results argue in favour of the possibility that fibrillar centers are the interphasic counterpart of chromosome NORs. Special structures such as nucleolar blobs and remnants usually considered to be of nucleolar origin, were also stained. — These findings seem to indicate a relationship between the distribution of the silver-stained proteins, the arrangement of the nucleolar structures and the degree of nucleolar activity resulting from the experimental conditions. These results are of interest at the time when the concept of the nucleolar matrix is gradually emerging.