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.     

Three-dimensional structure of the ciliate Didinium nasutum nucleoli

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.     

Further studies of the nucleolar material in salivary gland nuclei of Sciara coprophila

The nucleolar-like bodies or micronucleoli of Sciara coprophila salivary gland nuclei have been studied with phase contrast, the Nomarski optics, Azure B staining, and electron microscopy. In the late fourth instar the main nucleolus formed at the X-chromosome may become extensively fragmented and may appear as a large aggregate of micronucleoli. At about the same time large numbers of micronucleoli in a more peripheral location are also found. Studies in partially squashed and stained nuclei, as well as in unfixed glands have shown that, at a time when the nucleolar material is abundant, the X-NOR is highly ramified with its branches permeating much of the nuclear space. These observations make it appear probable that most or all of the nucleolar material, even the more peripherally located, is actually in contact with the main nucleolar organizer or its branches. On the other hand, many chromosomal bands are also in close association with micronucleoli. At the level of electron microscopy some of the associations between chromo somal bands and micronucleoli are very intimate with the nucleolar material often found deep within the band. In other instances there seems to be physical continuity between extensions of band chromatin and certain areas of the fibrillar component. The bands in question could be the sites of secondary nucleolar organizers. In the electron microscope a large aggregate of micronucleoli, interspersed with portions of chromatin can often be seen in an approximately central location. This is interpreted as the main nucleolus with portions of its NOR. Both the main nucleolus and the more peripheral micronucleoli are indistinguishable in their fine structure and show the components typically found in nucleoli, i.e., fibrils and granules. On the other hand the fine structure of both RNA and DNA puffs is strikingly different.Supported by funds from Public Service Grants GM 12191 from the National Institute of General Medical Sciences and 5 RO 1 AM 10016-06 from the National Institute of Arthritis and Metabolic Diseases (to Dr. A. M. Garcia).     

Differences in the fine structure and chemical constitution of nucleolar bodies and the true nucleolus in Xenopus laevis embryos

Numerous bodies resembling nucleoli, named “prenucleolar bodies”, were seen in the interphase nucleus of Xenopus laevis embryos between stages 7 and 11 of Nieuwkoop and Faber (1956) but not at stage 12. These bodies are composed of thick strands, 200 A in diameter, and apparently differ from the fibrillar component of the true nucleolus which consists of thin fibrils, 50 A in diameter. The granular component of the true nucleolus consists of fibers and granules which are both also 150–200 A in diameter, but which differ in chemical nature from the prenucleolar bodies. The granular component and fibrillar component are readily digested by RNase with or without pretreatment with trypsin, while the prenucleolar body is only digested with RNase after pretreatment with trypsin. This suggests that the prenucleolar body consists of strands of RNA coated with protein. At stage 9, another type of nucleolus-like body is formed, which is larger (2–2.6 μ in diameter) than the prenucleolar body (0.2–1 μ) and consists of thin fibrils of 50 A. This body resembles the fibrillar component of the true nucleolus in the size of the elemental fibrils as well as in its susceptibility to actinomycin D, RNase and trypsin. It seems to be a precursor of the true nucleolus and for this reason was named the “primary nucleolus.” From stage 9 to stage 10, each nucleus in the presumptive ectodermal and mesodermal areas contains 2 primary nucleoli together with multiple prenucleolar bodies. At stage 12, the prenucleolar body is not seen at all, but a new type of nucleolus-like body appears. There are usually 2 of these bodies in each nucleus, and they consist of 2 components: a network of 50 A fibrils, and a group of strands, 150–200 A in diameter, containing some granule-like elements. The former has the same susceptibility to actinomycin D, RNase and/or trypsin as the fibrillar component of the definitive nucleolus and the primary nucleolus, while the latter has the same susceptibility as the granular component of the definitive nucleolus. Thus, this body may     

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.     

Ultrastructural Distribution of DNA within Plant Meristematic Cell Nucleoli during Activation and the Subsequent Inactivation by a Cold Stress

We have investigated the precise location of DNA within the meristematic cell nucleolus ofZea maysroot cells andPisum sativumcotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with thein situterminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.     

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

Summary The silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.The work reported in the paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.     

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.     

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     

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.     

Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.     

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

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.