Nucleolar Remnants Research Articles

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum.

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

Localization by high resolution in situ hybridization of the ribosomal minichromosomes during the nucleolar cycle of Physarum polycephalum

We have used biotinylated rDNA probes to localize by in situ hybridization the extrachromosomal genes for ribosomal RNA in the slime mold Physarum polycephalum. We established conditions that allow for highly specific hybridization at the ultrastructural level and determined that the 60-kb palindromic rDNA molecules are confined to the nucleolus in interphase. Our study definitively locates these extrachromosomal genes in mitosis in the form of thin DNA fibers contained within nucleolar remnants. We further show that these rDNA minichromosomes do not condense and that they segregate as entities independent of the condensed chromosomal DNA. In telophase, these minichromosomes migrate from the poles toward the equatorial region of the nucleus in a direction opposite that of the chromosomes. Our results illustrate the discontinuous nature of the nucleolar organizing region in Physarum.

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10 −2 μg/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10 −2 μg/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10 −1 μg/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.

The 180-kDa isoform of topoisomerase II is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant

Monoclonal antibodies raised against two isoforms (170 and 150 180 kDa) of DNA topoisomerase II showed distinct fluorescence patterns in HeLa cells in different moments of the cell cycle ( C. Negri et al., 1992 , Exp. Cell Res. 200, 452–459). The ultrastructural distribution of the 150 180 -kDa isoform, which in immunofluorescence showed a localization into the nucleolar region, has been analyzed by electron microscopy with a gold-conjugated secondary antibody in HeLa and K562 cells. The results indicate that this isoform of the enzyme is exclusively localized in the nucleolus, mainly in the dense fibrillar component, while the nucleoplasm of interphase cells and the chromosomes of mitotic cells are completely negative. The antibody also reacts with the nucleolus of isolated nuclei and with the nucleolar remnant of purified nuclear matrices. A quantitative evaluation of the label distribution indicates that the percentage of label in the nucleolar remnant of isolated matrix is almost identical to that of the nucleolus in whole cells. The interaction with the insoluble proteins of the isolated nuclear matrix is also demonstrated by quantitative immunoblotting in which the MoAb specifically stains a unique band corresponding to the 150 180 -kDa isoform of topoisomerase II. The localization of the 150 180 -kDa isoform of topoisomerase II in the nucleolar remnant strongly suggests that it represents a structural element for the spatial organization and for the regulation of transcription of the ribosomal genes.

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells

The morphology and the polypeptide composition of the nuclear matrix obtained from 37°C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH 4) 2SO 4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non- equilibrium gel electrophoresis.

Nucleologenesis: Possible function for the nucleolar remnant during mitosis

Nucleologenesis: Possible function for the nucleolar remnant during mitosis

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.

Heat-induced stabilization of the nuclear matrix: A morphological and biochemical analysis in murine erythroleukemia cells

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 °C or from cells heat shocked in vivo at 43 or 45 °C. At the ultrastructural level it was possible to see that in the absence of a 37 °C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 °C nuclear incubation, very electrondense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electrondense residual nucleoli were present only when isolated nuclei had been exposed to 37 °C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electrondense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 °C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on onedimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.

Estimation of RNA Content of the Nucleolus and Nucleolus-derived Structures in <italic>Vicia faba</italic> Mitotic Cells Embedded in Lowicryl Resin Using RNase-gold Labeling

RNase-gold labeling was applied to Vicia faba meristematic cells embedded in hydrophilic Lowicryl K4M resin, and the label was examined over various cell structures with special focus on the nucleolus and nucleolus-derived structures. RNA-containing structures or regions like the nucleolus and cytoplasm were most heavily labeled whereas the vacuoles, mitochondria, and cell walls almost entirely lacked gold particles. The gold labeling ratio of the nucleolus to condensed chromatin reached about 5.4. Treatment with RNase-gold labeling remarkably reduced the presence of gold particles in the nucleolus and cytoplasm. During prophase, nucleolar remnants were specifically labeled, while the space between chromosomes was covered with very few gold particles. Prenucleolar material accumulated on late anaphase chromosomes possessed far fewer gold particles than the nucleolus. Interestingly, the prenucleolar material remained poorly labeled, while the nucleoli constructed from it contained large amounts of gold particles

Immunoelectron microscopic localization of the nucleolar protein B‐36 (fibrillarin) during the cell cycle of Physarum polycephalum

Monoclonal antibodies raised against the 34-kD nucleolar protein, B-36, from the slime mold Physarum polycephalum have been used to examine the electron microscopic localization of B-36 during the cell cycle in Physarum. During interphase, B-36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B-36, fibrillarin. During mitosis, B-36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA-specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that in Physarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the "NOR" equivalents do not separate during mitosis as in mammalian cells. In addition, the B-36-enriched nucleolar remnants appear to be recycled from one cell cycle to the next.

Formation of cytoplasmic nucleolus-like bodies from pre-existing nucleolar material in the mitotic cells of Vicia faba

The origin and behaviour of cytoplasmic nucleolus-like bodies was examined and correlated with the fate of the nucleolar material during mitosis of Vicia faba. The minute cytoplasmic nucleolus-like bodies first appeared in late anaphase. Relatively large bodies more than 1 μm in diameter were detected almost exclusively in early telophase but did not persist long in mid or late telophase. Ultrastructural survey revealed that the minute cytoplasmic nucleolus-like bodies in late anaphase consisted of ribosome-like granules and/or a fibrillogranular substance similar to the nucleolar remnants in prometaphase. Two types of the nucleolus-like bodies, however, were discerned in early telophase; one was composed of ribosome-like granules and/or the fibrillogranular substance and the other was exclusively fibrous in texture.

Nucleolar meiotic cycle in orthoptera

The evolution of nucleolar material was analyzed during spermatogenesis of two grasshopper species by using “in vivo” visualization and the silver staining method. Both nucleoli and nucleolar remnants are detectable during prophase I and absent from metaphase I until telophase I. During telophase I a great number of small silver positive masses which correspond to prenucleolar bodies (PBs) are observed covering the chromatin surface. At interkinesis these PBs coalesce to form nucleoli, which are dispersed at prophase II. Silver dots at NOR position were observed on metaphase II chromosomes. PBs reappear at telophase II and give rise to the nucleoli detected in early spermatids. This cycle is compared with those reported in plants and in some other animal species.

Monoclonal antibody to a nucleolar antigen of human B-lymphoblastoid cells

An anti-nucleolar monoclonal antibody reacting with human B-lymphoblastoid cells but not with normal periferal blood lymphocytes has been isolated. The antibody recognized in Namalwa cells an antigen with molecular mass 41 kDa and pI 5.6, different from all previously described nucleolar antigens. Inhibition of rRNA transcription with Actinomycin D caused redistribution of the 41 5.6 antigen, but even at long term drug action it remains associated with the nucleolar remnants.

Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading

Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.

Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.

Peculiar behavior of the nucleolus and appearance of cytoplasmic nucleolus-like bodies in the roottip meristems ofBrodiaea uniflora Engl. Grown at low temperature

Cytoplasmic nucleolus-like bodies, which showed vast variation both in size and in number per cell, were sometimes found in the telophase cells ofBrodiaea uniflora. When the plants were grown at low temperature, the frequency of telophase cells bearing cytoplasmic nucleolus-like bodies increased with the lapse of days. In contrast, growth at moderate temperature reduced this frequency. Prolonged exposure of the plants to low temperature caused peculiar phenomena concerning the behavior of the nucleolus and nucleolar material: 1. retention of nucleolar remnants at high frequency in metaphase, 2. pulverization of the nucleolar remnants into a great number of minute, fluffed fragments during metaphase, 3. appearance of dot-like nucleolar material in anaphase, and 4. appearance of nucleolus-like bodies, sometimes more than 10 Μm in diameter, in telophase. All these structures were strongly impregnated with silver. Electron microscopy revealed that both the nucleolar remnant and the nucleolus-like body consisted primarily of fibrils. Our observations clearly demonstrate that the nucleolus-like bodies are derived from the fibrillar component of the nucleolus and are formed by fusion of dot-like nucleolar material during anaphase.

The nucleolar cycle in the myxomycete Physarum polycephalum. II. Mode of formation of the nucleolus

The previous ultracytochemical investigation has revealed that the numerous nucleolar remnants observed during the mitotic stages in Physarum polycephalum possess the same characteristics as chromatin and originate from the fibrillar zones of the mature nucleolus. In the present study these nucleolar minichromosomes are shown to be incorporated as well-defined structural elements within the two daughter nuclei. At very early interphase these minichromosomes are first scattered throughout the nuclear cavity, but within a few minutes they aggregate to form several highly irregular masses corresponding to prenucleolar bodies. By the time these latter bodies fuse into typical horseshoelike nucleoli, pervading granular material starts accumulating in more noticeable amounts. Following labeling for 10 min with uridine radioautographic grains are already detected in very early interphase nuclei over dense regions undoubtedly corresponding to the minichromosomes. Incorporation of this RNA precursor increases as the minichromosomes fuse into prenucleolar bodies. By the time large nucleoplasmic zones have formed, radio-autographic grains are present in still larger quantity over the irregularly shaped nucleolus as well as over diffuse chromatin thoughout the nuclear cavity. Such early RNA synthesis is interpreted to indicate that the early stages of nucleolar formation do not merely involve fusion of the preexisting minichromosomes but also imply synthesis of new material.

The nucleolar cycle in the myxomycete Physarum polycephalum. I. Ultracytochemical characteristics of the mature nucleolus and of the persisting nucleolar material during the mitotic stages

The interphase nucleolus, in the myxomycete Physarum polycephalum, consists of granular and fibrillar zones, as in all eukaryotes studied so far. Each of these latter regions, however, is observed to be composed of a coarse filamentous element which is generally folded into a globular or cup-shaped structure characterized by a lighter core portion. These globular structures are bleached by EDTA and also react with both bismuth and phosphotungstic acid, thus showing that they contain chromatin. Similarly shaped nucleolar components are observed till late prophase and these show cytochemical properties identical to those seen during interphase. Following dispersion of the nucleolus at prometaphase, these globular nucleolar regions persist as characteristic remnants which contain DNA, RNA, and basic proteins. At anaphase, numerous similar bodies migrate with the chromosomes and can be demonstrated to become part of the chromatin masses which occupy the two poles of the dumbbell-shaped telophase nucleus. It is concluded from these observations that these nucleolar remnants originate from the fibrillar zones of the mature nucleolus and that these zones in turn correspond to well-defined structures possessing the general characteristics of chromatin. These structures are consequently referred to as minichromosomes.

Étude ultrastructurale du cycle nucléolaire de Physarum polycephalum après mise en évidence préférentielle des RNP par la réaction régressive à l'EDTA

Nucleolar cycle in Physarum polycephalum was studied using EDTA preferential stain for RNP. We have been able to establish the following. 1. 1. The nucleolar remnants that are observed during mitosis correspond to the fibrillar zones of the interphase nucleolus. 2. 2. In prometaphase and metaphase the nucleolar remnants lie peripherically around the chromosomal mass with which they are only superficially associated. 3. 3. Preribosomal granules appear 10–15 min after metaphase in the prenucleolar bodies. The results shown that the interphase nucleolus is an assembly of multiple subunits. The relationship between the nucleolar remnants and chromosomes is discussed in the light of the recent understanding of the extrachromosomal nature of rDNA.