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.     

Sorting of micronuclei from PtK1 cells

We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.     

Ultrastructure of the nuclear apparatus of the infusorian Loxodes magnus. I. The macronuclei, macronuclear anlagen and the interphasic micronuclei]

The nuclear apparatus of Loxodes magnus Stokes (Holotricha) consists of numerous macronuclei which belong to the diploid type and never divide, and of numerous micronuclei. No nuclear groups exist; individual nuclei often lie in cytoplasmic islets surrounded by large lacunae of the smooth endoplasmic reticulum. Interphasic micronuclei have two-membraned envelopes with numerous pores, usually lined at the cytoplasmic side with a layer of vacuoles, channels, or flattened vesicles of the smooth endoplasmic reticulum. The chromatin of the micronuclei consists of anastomosing threads, 0.1--0.2 mum wide, between which several nucleolus-like bodies of microfibrillar structure occur. Adult macronuclei have a similar nuclear envelope and a similar system of vacuoles, channels, and flattened agranular cisternae outside it. The macronucleus contains a single large composite nucleolus with 3 or 4 fibrillar cores inside the common granular cortex. The fibrillar cores are pierced by channels containing nucleolar organizers in the form of strands of condensed chromatin. The peripheral zone of the macronucleus is filled with decondensed chromatin fibrils and contains a number of small chromocenters and several aggregates of RNP granules. No protein inclusions (spheres) have been observed in Loxodes macronuclei. The macronuclear anlagen, developing in the cycle of every cell division, show progressive decondensation of the chromosomes and formation of several nucleoli, each with its own organizer. Later on, the nucleoli fuse into a single nucleolus. The small chromocentres are the last to form.     

Nucleolar apparatus in the macronucleus of Didinium nasutum (Ciliata): EM and 3D reconstruction

The three-dimensional (3D) organization of nucleoli in the somatic nuclei (macronuclei) of recently fed and starved Didinium nasutum was reconstructed on the basis of serial ultra-thin sections. It was shown that nucleoli, looking on the single sections like individual separate structures, appeared to be parts of the large complicated branchy nucleolar networks. A 30 h starvation did not lead to disintegration of this network, but stimulated formation of numerous vacuoles in the granular component of nucleoli, which becomes more condensed. Unlike starved D. nasutum, in fed ciliates numerous holes appeared in the fibrillar component located at the periphery of nucleoli. These holes may presumably serve as channels for transporting newly synthesized rRNA. To our knowledge, this is the first report of a 3D reconstruction of the nucleolar apparatus in ciliates.     

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.Abbreviations NAC nucleolus associated chromatin - CES centromeric structures - NOR nucleolar organizing region - NAB nucleolus associated body - IG interchromatin granules - RNP ribonucleoprotein - Mab monoclonal antibody by M.F. Trendelenburg     

Three-dimensional organization of nucleoli of Didinium nasutum (Ciliata)

A comparative study of nucleolar organization in the somatic nuclei of the ciliate Didinium nasutum was carried out using 3D reconstruction on the basis of serial ultrathin sections. Recently fed interphase ciliates, starved interphase ciliates and cysts were studied. The nucleoli at the interphase stage were shown to have a complex architecture: the fibrillar component forms a complicated network, the granular component is located inside of it. It was shown that nucleoli, which look like individual structures in single sections, are in fact parts of branched nucleolar networks. A 30-h starvation doesn't lead to disintegration of these networks. However in the starved cells the granular component becomes more dense and vacuolized. In the fed ciliates there are many holes in the fibrillar component, whereas in starved ones the fibrillar component is virtually devoid of them. These holes can be proposed to ensure the transport of newly synthesized rRNP. The nucleolar networks didn't occur in D. nasutum cysts. Nucleoli in the cysts look like small individual structures, mainly consisting of fibrogranular component.     

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.     

Number of nucleoli in various cell types of the mouse

The nucleoli of cells of the adult mouse were examined by staining with toluidine blue after removal of deoxyribonucleic acid from tissue sections by deoxyribonuclease treatment. The nuclei of each cell type examined contained one or more nucleoli. This was observed even in lymphocytes and neuroglia, although these cells have occasionally been described as anucleolated. In mature spermatids and spermatozoa, however, it was not possible to detect a nucleolus. The distribution of the number of nucleoli in many diploid cells exhibited a mode of two or three nucleoli per nucleus, and a range from 1 to 6 nucleoli. In presumedly diploid hepatic nuclei, the maximum number of nucleoli was six; but in presumedly tetraploid hepatic nuclei, it was 11. Thus, nearly twice as many nucleoli are present when the chromosome number is doubled. In view of this observation, it is suggested that six nucleolar organizers are present in the diploid chromosomal complement of the mouse. However, through failure of some nucleolar organizers or more probably through fusion of nucleoli, the number of these organelles in most nuclei is less than six.     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.     

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.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

Nucleocytoplasmic sorting of macromolecules following mitosis: fate of nuclear constituents after inhibition of pore complex function

PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68,000 pore complex glycoprotein) or of wheat germ agglutinin (WGA) complete cytokinesis. However, their nuclei remain stably arrested in a telophase-like organization characterized by highly condensed chromatin and the absence of nucleoli, indicating a requirement for pore-mediated transport for the reassembly of interphase nuclei. We have now examined this requirement more closely by monitoring the behavior of individual nuclear macromolecules in microinjected cells using immunofluorescence microscopy and have investigated the effect of microinjecting the antibody or WGA on cellular ultrastructure. The absence of nuclear transport did not affect the sequestration into daughter nuclei of components such as DNA, DNA topoisomerase I and the nucleolar protein fibrillarin that are carried through mitosis on chromosomes. On the other hand, lamins, snRNAs and the p68 pore complex glycoprotein, all cytoplasmic during mitosis, remained largely cytoplasmic in the telophase-arrested cells. Electron microscopy showed the nuclei to be surrounded by a double-layered membrane with some inserted pore complexes. In addition, however, a variety of membranous structures with associated pore complexes was regularly noted in the cytoplasm, suggesting that chromatin may not be essential for the postmitotic formation of pore complexes. We propose that cellular compartmentalization at telophase is a two-step process. First, a nuclear envelope tightly encloses the condensed chromosomes, excluding non-selectively all macromolecules not associated with the chromosomes. Interphase nuclear organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm.     

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.     

Effect of gamma irradiation on nucleolar activity in root tip cells of tetraploid Triticum turgidum ssp. durum L

Ionizing irradiation induces positive or negative changes in plant growth (M1) depending on the amount of irradiation applied to seeds or plant parts. The effect of 50–350 Gy gamma irradiation of kernels on nucleolar activity, as an indicator of metabolic activity, in root tip cells of tetraploid wheat Triticum turgidum ssp. durum L. cv. Orania (AABB) was investigated. The number of nucleoli present in nuclei and micronuclei as well as the mitotic index in the different irradiation dosages was used as an indicator of the cells entering mitosis, the chromosomes with nucleolar organizer regions that are active as well as chromosome doubling in the event of unsuccessful mitotic division. Nucleolar activity was investigated from 17.5 to 47.5 h after the onset of imbibition to study the first mitotic division and its consequences on the cells that were in G₂ and G₁ phases at the time of gamma irradiation. Untreated material produced a maximum of four nucleoli formed by the nucleolar organizing regions (NORs) on chromosomes 1B and 6B. In irradiated material, additional nucleoli were noted that are due to the activation of the NORs on chromosome 1A in micronuclei. The onset of mitosis was highly significantly retarded in comparison to the control due to checkpoints in the G₂ phase for the repairing of damaged DNA. This study is the first to report on the appearance of nucleoli in micronuclei as well as activation of NORs in the micronuclei that are inactive in the nucleus and the effect of chromosome doubling on nucleolar activity in the event of unsuccessful mitotic division.

     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.     

Nucleolar DNA organization in the interphase nuclei of mouse fibroblasts.

The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.