Ultrastructural study of the relationships between the various nucleolar components in Ehrlich tumour and HEp-2 cell nucleoli after acetylation

In the present study, we analysed the relationships between various nucleolar components in Ehrlich tumour and HEp-2 cells, using acetylation. Under these conditions, we found contacts between the condensed intranucleolar chromatin and the fibrillar centre, illustrating the continuity between the DNA present inside the fibrillar centre and that of condensed associated chromatin. We also found that although the dense fibrillar component is usually situated at the periphery of the fibrillar centre, it is sometimes found inside the centre. On the other hand, the layer of dense fibrils bordering the fibrillar centre is interrupted by nucleolar interstices. In addition, in HEp-2 cell nucleoli with a reticulated appearance, the numerous small fibrillar centres are bound together by strands of dense fibrillar component. These observations are discussed in terms of relationships between nucleolar ultrastructure and function(s).     

Immunocytochemical and ultrastructural analyses of the function of the ubiquitin-proteasome system during spermiogenesis with the use of the inhibitors of proteasome proteolytic activity in the alga, Chara vulgaris

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.     

Ultrastructure and behavior of the nucleoli and fibrillar centers during cell differentiation of liver erythroblasts of 12-day-old mouse embryos

The transformation of nucleolus and its structural components in the main groups of erythroid cells (from pronormoblasts to reticulocytes and dividing ones) has been studied. It is shown that during inactivation of the nucleolus, the granular component is reduced, and the degree of chromatin condensation increases. Enlargement and "naking" of fibrillar centres are also observed. At the stage of basophilic and polychromatophilic erythroblasts, the nucleolus has a mushroom-like shape with well developed fibrillar centres, which lie at the border of the nucleolus. Nucleolar RNP components consist predominantly of a fibrillar component and forms "caps" of these mushroom-like structures. Therefore, at this stage "free" fibrillar centres are found on ultrathin sections, if the section plane runs only through the fibrillar centre, or through ring-shaped nucleoli, i.e. the fibrillar centre surrounded by sheet of nucleolar RNP fibrilles, when the mushroom-like nucleolus is cut tangentially. Using serial section technique, small round nucleoli with an extremely weakly developed RNP material or free fibrillar centres, resembling those in telophase nuclei, are shown on the terminal stage of nucleolus transformation. It is noted that the main groups of erythroid cells differ from each other not only in the chromatin condensation degree, but also in the development of nucleolus material and in the size of fibrillar centres. However, such differences exist in either cell group. Consequently, we can distinguish between cell populations being on different stages of maturation. On this basis, we described on intermediate population of cells, which possess signs of pronormoblasts and basophilic erythroblasts. In all the cases, strands of electron opaque material bound with the condensed chromatin are present in fibrillar centres.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nucleolar chromatin and the secondary constriction

We have traced the nucleolar chromatin from early prophase to the metaphase stage. In prophase this chromatin begins to condense and in metaphase it is fully condensed. In mitotic chromosomes, this chromatin remains surrounded by achromatic materials resembling the fibrillar centre. As such this region of the chromosomes appears as a gap or constriction at the light microscope level. The possible role of this achromatic material in relation to nucleologenesis and satellite association has been discussed.     

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.     

3-dimensional organization of the nucleolus and the nucleolus-organizing regions in differentiated cells. I. Ring-shaped nucleoli in the endotheliocytes, smooth-muscle cells and fibroblasts in the mouse

The method of ultra-thin serial sections was used to study the three-dimensional structure and to perform the quantitative analysis of ring-shaped nucleoli of kidney and liver endotheliocytes, smooth muscle cells of kidney arterioles and fibroblasts of mice. Spatial models of ring-shaped nucleoli with one fibrillar centre are given. For the quantitative analysis the following parameters were measured: the number and volumes of nucleoli, fibrillar centers, RNP-containing structures, the vacuolar system and the RNP-index (the latter is a ratio of RNP-part and fibrillar center volumes). Nucleoli of the same type of cells, occasionally in the same nucleus, were found to differ sharply in their fibrillar center shape. Differences in the mean volume values of nucleoli, fibrillar centers and the RNP-part between some cell populations are sufficiently well pronounced. Within the same population ring-shaped nucleoli have, as a rule, specific volume values of nucleoli, RNP and fibrillar centers. The comparison of quantitative data obtained on different cell types showed that the mean RNP-index values were the most stable parameter. The structural relation between fibrillar centers, intra- and perinucleolar chromatin and lacunar region is shown. The structural organization of intranucleolar chromatin and rRNA in the nucleolar body and in fibrillar centers is discussed.     

A possible skeletal substructure of the macronucleus of Tetrahymena

Upon removal of chromatin from isolated macronuclei of tetrahymena, residual structures are obtained, the organization of which faithfully reflects the distinctive architecture of the macronucleus. Macronuclei are isolated by a new procedure in which cells are lysed by immersion in citric acid and Triton X-100. This method is rapid and efficient and leaves the nuclear structures stripped of nuclear envelope and nucleoli. The remaining interconnected chromatin bodies are structurally differentiated into a dense outer shell and a fibrillar inner core. The fibrillar component is identified as chromatin because it is removed upon digestion with DNase and extraction with 2 M NaCl. The dense shell of the chromatin body is unaffected by the digestion procedure, which leaves a skeletal structure comprised of hollow spherical bodies. Analysis of the protein composition by SDS acrylamide gel electrophoresis before and after digestion with DNase and RNase and high-salt extraction shows that histones are diminished, whereas the nonhistone protein composition remains unchanged. It was found the DNase not only extracts chromatin but also protects the nonchromatin structure from the otherwise disruptive effects of high-salt extraction. The method used for isolating the nuclei also affects the structure remaining after the digestion procedure the citric acid/Triton X-100 method enhances the stability of the interconnected spherical bodies. The results indicate that the method for isolating nuclei and the procedure by which chromatin is extracted are both major factors contributing to the detection of a possible nonchromatin nuclear skeleton.     

Nucleolar organizers and fibrillar centres in Triticum aestivum L., cv. Chinese spring

Serial sectioning of wheat roots prepared for electron microscopy was used to count the number of fibrillar centres per nucleus and nucleolus and to calculate their sizes. After growth at 35 ° C for two days the nucleoli became segregated and a one to one relationship was evident between chromosomal nucleolar organizers and fibrillar centres. This was confirmed using an aneuploid line carrying an additional pair of organizers. Quantitative studies showed that the fibrillar centres occupied a volume 0.24% of the total chromatin reticulum. From this it was calculated that only about 1/3 of the fibrillar centre material was likely to be nucleolar organizer chromatin. The other material was considered to be the protein revealed in silver staining studies. The importance of this was shown by its constant ratio to the size of all nucleoli in a given nucleus. — Evidence was found for the movement and fusion of organizer flanking regions during growth at 35 ° C. The number of junctions between chromatin and nucleolar organizers drops by about half giving one per organizer after segregation, and serial sectioning demonstrated such junctions in close proximity, an arrangement suggestive of incipient fusion.     

Intranuclear microfilament bundles in the ependymal cells of the third ventricle of the rat

Summary Intranuclear microfilament bundles were observed in ependymal cells of the third ventricle of the rat. They appeared as single, cylindricallyshaped fibrillar bands up to 4.9 m in length. In cross sections of bundles, the microfilaments exhibited an apparently regular arrangement. They were separated by a distance of 4 to 5 nm, measuring approximately 11 nm from centre to centre. One bundle consisted of 55 to 88 microfilaments. The intranuclear bundles terminated at the inner membrane of the nucleus. They exhibited close spatial relationship to perinuclear chromatin, chromatin centres, intrachromatin granules and fibrillar structures. The average ratio of nuclear sections with and without bundles was 115.Research fellow of the Alexander von Humboldt FoundationThis research was partly carried out at the Department of Histology and Embryology, Academy of Medicine at Poznan, Poland, and was supported in part by a grant from the Polish Academy of Science     

Histone H1 variants as sperm-specific nuclear proteins of Rana catesbeiana,and their role in maintaining a unique condensed state of sperm chromatin

Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization. Mol. Reprod. Dev. 47:181–190, 1997. © 1997 Wiley-Liss, Inc.     

Complete displacement of somatic histones during transformation of spermatid chromatin: a model experiment.

Displacement of histones from calf thymus chromatin has been studied in an attempt to postulate the mechanisms involved in the total removal of somatic-type histones during transformation of spermatid chromatin. When chromatin is saturated with protamine (protamine/DNA, 0.5), histone I becomes displaceable at 0.15-0.3 M NaCl, suggesting that direct replacement by highly basic sperm histone could be a mechanism for its removal. While histone I is the only histone which is extensively degraded upon incubation of chromatin and, therefore, proteolysis might provide an additional mechanism for the removal of this histone, acetylation of chromatin by acetic anhydride greatly increases suscpetibility of histones IIb1, IIb2, and III to the chromosomally associated protease. These histones are extensively degraded and displaced from the DNA upon incubation of the acetylated chromatin. Although histone IV is not appreciably degraded, the proteolytic removal of acetylated histone III from chromatin weakens the interaction of acetylated histone IV to the DNA, and this histone becomes dissociable at 0.3 M NaCl. A comparison of the extent of chemical acetylation of individual histones observed in this investigation with that of enzymatic acetylation which can be achieved in vivo suggests that acetylation and proteolysis could be a mechanism for the removal of histone IIb2 and III. The displacement of histones IIb1 and IV could be explained on the basis of decreased binding to DNA as a result of their acetylation together with the proteolytic removal of their respective partner histones, IIb2 and III.     

The presence of F3-F2a1 dimers and F1 oligomers in chromatin.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic ¹²⁵I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.     

Histone hyperacetylation has little effect on the higher order folding of chromatin

HeLa cells were grown in the presence of 10 mM sodium butyrate and soluble chromatin containing hyperacetylated histones was prepared by mild micrococcal nuclease digestion and sucrose gradient fractionation. Sedimentation and electric dichroism were used to study the cation-induced folding of this acetylated chromatin from the 10 nm filament to the 30 nm solenoid conformation. Although under some conditions acetylated chromatin appears slightly less condensed than control chromatin, the major conclusion is that hyperacetylation of histones does not in itself prevent the formation of the higher order chromatin solenoid.     

Electron microscopic study of chromatin in maturing lymphocyte nuclei using ammoniacal silver]

By means of the ammoniacal silver reaction, cytochemical properties of the chromatin in white rat lymph node lymphocytes were investigated at different stages of their maturation. Electron dense granules of the reaction product are shown to be localized over the compact chromatin region. The number of granules increases as the amount of compact chromatin enlarges. A possible role of arginine histones in the process of chromatin condensation is suggested, this suggestion being based on the assumption that ammoniacal silver binds with active arginine groups of histones.