RESUMO
Most events of ribosome biogenesis--such as transcription of the ribosomal RNA (rRNA) genes, processing of their primary transcripts into mature rRNAs and assembly with ribosomal and nonribosomal proteins to form the preribosomes--are confined to a special nuclear compartment, the nucleolus. Immunogold labelling and in situ hybridization at the ultrastructural level are providing novel insights into structure-function relationships of the nucleolus, and in vitro systems are beginning to shed light on the molecular mechanisms involved in the reforming of nucleoli after mitosis.
RESUMO
We have investigated the DNA distribution within the rat oocyte nucleolus during the early stages of follicular growth by means of the in situ terminal deoxynucleotidyl transferase method. In the fibrillogranular nucleolus, label is visualized on small clumps of peri- and intranucleolar chromatin. Such labeled clumps are frequently observed inside the interstices surrounding the fibrillar centers. Label is also consistently found in the fibrillar centers whereas the dense fibrillar component and the granular component are devoid of gold particles. These results contradict earlier data but conform with other recent immunocytochemical observations, obtained in nucleoli of a variety of somatic cell types, concerning the correlation between structure and function in the nucleolus.
Assuntos
DNA/análise , Oócitos/crescimento & desenvolvimento , Animais , Nucléolo Celular/química , Nucléolo Celular/ultraestrutura , DNA Nucleotidilexotransferase , Feminino , Técnicas Imunoenzimáticas , Oócitos/química , RatosRESUMO
In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus.
Assuntos
Carcinoma de Ehrlich/patologia , DNA de Neoplasias/ultraestrutura , Mitose , Animais , Anticorpos Monoclonais/imunologia , Bromodesoxiuridina/imunologia , Carcinoma de Ehrlich/ultraestrutura , DNA de Neoplasias/análise , Dactinomicina/farmacologia , Interfase , Metáfase , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica/métodos , Prófase , TelófaseRESUMO
C57 BL/6J mouse spleen lymphocytes have been stimulated by a polyclonal mitogene, the lipopolysaccharide of E. coli (LPS). Depending on the LPS concentration, two pathways of B lymphocyte differentiation can be obtained. At low dose, the population is mainly composed of blast cells (85%) and at a high dose, the latter transforms into plasma cells (80%). Four types of nuclear bodies have been distinguished and quantitatively studied at several stages of cell differentiation. Only the simple nuclear bodies type A, which could be related to the nuclear matrix, show quantitative modifications in small lymphocytes. Connections between granular nuclear bodies (type D) and nucleolar material have been observed. Some granular nuclear bodies exhibit a morphological zone similar to the nucleolar fibrillar centre as well as fibrillar and granular components. Autoradiographic studies indicate that the granular nuclear bodies contain RNA synthesized elsewhere in the nucleus and that this RNA subsequently migrates to the cytoplasm. Furthermore connections between granular nuclear bodies and chromatin have also been observed.
Assuntos
Linfócitos B/fisiologia , Núcleo Celular/fisiologia , Lipopolissacarídeos , Ativação Linfocitária , Animais , Linfócitos B/imunologia , Diferenciação Celular , Núcleo Celular/ultraestrutura , Replicação do DNA , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia EletrônicaRESUMO
In the present review on the organization of the mammalian cell nucleolus, we report and discuss data obtained during the past 10 years by means of cytochemical and immunocytochemical ultrastructural techniques. Particular emphasis is placed on the following topics: location of the nucleolus organizer regions in interphasic nucleolar components, structure of nucleolar chromatin in situ, and the structure-function relationship of the nucleolar components. The cytochemical and immunocytochemical results are compared and the concordant data are stressed for each topic.
Assuntos
Nucléolo Celular/ultraestrutura , Animais , Nucléolo Celular/metabolismo , Histocitoquímica/métodos , Microscopia EletrônicaRESUMO
AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.
Assuntos
Nucléolo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Western Blotting , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/ultraestrutura , Nucléolo Celular/ultraestrutura , Camundongos , Microscopia Imunoeletrônica , Peso Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/isolamento & purificação , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Coloração pela Prata , Células Tumorais CultivadasRESUMO
The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.
Assuntos
Cátions/farmacologia , Cromatina/metabolismo , Eritrócitos/análise , Timo/análise , Animais , Cátions Bivalentes/farmacologia , Bovinos , Precipitação Química , Galinhas , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Eletroquímica , Microscopia Eletrônica , Nefelometria e Turbidimetria , Concentração Osmolar , Conformação Proteica/efeitos dos fármacos , SolubilidadeRESUMO
The Jo-1 antigen is a specific marker for autoimmune myositis with an excellent correlation with associated interstitial lung disease. Using an electron microscopy immunogold technique, we were able to show that the antigen was predominantly cytoplasmic.
Assuntos
Aminoacil-tRNA Sintetases/análise , Citoplasma/imunologia , Histidina-tRNA Ligase/análise , Núcleo Celular/imunologia , Citoplasma/ultraestrutura , Feminino , Humanos , Técnicas Imunológicas , Microscopia Eletrônica , Pessoa de Meia-Idade , Miosite/imunologia , Escleroderma Sistêmico/imunologiaRESUMO
The current study has documented changes in the ultrastructure as well as in the intranucleolar distribution of rDNA and rRNA in RT4 (human transitional cell bladder carcinoma) cell nucleoli following a 3-h exposure to toxic doses of 50 microM ametantrone (AMT), 200 microM poly (adenylate-uridylate) (poly r(A-U) or an AMT/poly r(A-U) combination with an AMT/polyribonucleotide ratio of 1:4 and a poly r(A-U) concentration of 200 microM. While the main nucleolar components (fibrillar center (F), dense fibrillar component (D), granular component (G) and interstices (I) can be discerned following all treatments, the nucleoli exhibit: compaction, segregation, a decrease in the number of F, an increase in the size of remaining F, margination of intranucleolar chromatin and retention of intranucleolar pre-rRNA and rRNA. The relative abilities of the test agents to induce nucleolar compaction are AMT/poly r(A-U) > poly r(A-U) > AMT > sham-treated, while the abilities of the test agents to induce the remaining nucleolar changes are AMT/poly r(A-U) > or = AMT > poly r(A-U) > sham-treated cells. Poly r(A-U) and the induced interferon induce nucleolar compaction, while AMT produces nucleolar segregation. These results are consistent with a model in which the poly r(A-U) and/or the AMT inhibit DNA transcription and rRNA processing as well as the release of nascent preribosomes from the nucleolus.
Assuntos
Antineoplásicos/farmacologia , Nucléolo Celular/química , Mitoxantrona/análogos & derivados , Poli A-U/farmacologia , RNA Neoplásico/análise , Carcinoma de Células de Transição , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/ultraestrutura , DNA de Neoplasias/análise , DNA Ribossômico/análise , Humanos , Microscopia Imunoeletrônica , Mitoxantrona/farmacologia , RNA Ribossômico/análise , RNA Ribossômico/ultraestrutura , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/ultraestrutura , Neoplasias da Bexiga UrináriaAssuntos
Nucléolo Celular/ultraestrutura , Animais , Cátions , Ciclo Celular , Diferenciação Celular , Nucléolo Celular/análise , Nucléolo Celular/enzimologia , Nucléolo Celular/fisiologia , Sobrevivência Celular , Cromatina/análise , Cromatina/ultraestrutura , Ritmo Circadiano , DNA/análise , Embrião de Mamíferos/fisiologia , Embrião não Mamífero , Eritrócitos/fisiologia , Interfase , Lipídeos/análise , Linfócitos/fisiologia , Meiose , Proteínas de Membrana/análise , Metais/análise , Mitose , Neoplasias/patologia , Região Organizadora do Nucléolo/análise , Células Vegetais , RNA/análise , Ribonucleoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Estações do Ano , TelófaseAssuntos
Antineoplásicos/farmacologia , Carcinoma de Ehrlich/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Carcinoma de Ehrlich/ultraestrutura , Galinhas , DNA/análise , DNA/biossíntese , Histocitoquímica , Camundongos , RNA/análise , RNA/biossíntese , RatosRESUMO
By means of diaminobenzidine staining method, the relations between fibrillar centres and nucleolus-associated chromatin are analyzed in Ehrlich tumour cell nucleoli. There is a continuity between fibrillar centres and condensed intra-nucleolar chromatin. The meaning of these connections is discussed.
Assuntos
Carcinoma de Ehrlich/ultraestrutura , Nucléolo Celular/ultraestrutura , Cromatina/ultraestrutura , Animais , Histocitoquímica , Camundongos , Coloração e RotulagemRESUMO
A technique based on the use of silver solutions, which selectively stains the nucleolus-organising regions (NORs) in chromosomes, was applied to interphase Ehrlich tumour cells. The results indicate that nucleolar fibrillar centres correspond to the NORs.
Assuntos
Carcinoma de Ehrlich/ultraestrutura , Cromossomos/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Animais , Técnicas Citológicas , Camundongos , Prata , Coloração e RotulagemRESUMO
Nucleoli were studied in chick fibroblasts cultured in vitro, under normal or under experimental conditions, and in several mammalian cell types in vivo. All these cells frequently contain nucleoli with fibrillar centres. The nucleolar fibrillar centres are composed of fibrous material of low electron density and are always intimately associated with the dense fibrillar component. Their morphology is very similar to that analysed cytochemically in Ehrlich tumour cells. It therefore appears that they could be related to the nucleolar organizers as suggested in Ehrlich tumour cells.
Assuntos
Nucléolo Celular/ultraestrutura , Animais , Carcinoma de Ehrlich/ultraestrutura , Nucléolo Celular/análise , Embrião de Galinha , DNA/análise , Camundongos , Microscopia Eletrônica , RNA/biossíntese , RatosRESUMO
The precise location of transcribing rRNA genes within Ehrlich tumor cell nucleoli has been investigated using two approaches: high-resolution autoradiography of cells pulse-labelled with tritiated uridine, varying the exposure time, and in situ-in vitro transcription coupled with an immunogold labelling procedure. When autoradiographic preparations are exposed for a short time, silver grains are found associated almost exclusively with interphasic cell nucleoli. Labelling of extranucleolar areas requires longer exposure. Within the nucleolus, the first sites to be revealed are in the dense fibrillar component. Prolonging exposure increases labelling over the dense fibrillar component, with label becoming more and more apparent over the fibrillar centers. Under these conditions, however, labelling does not extend into the granular component, and no background is observed. Initiation of transcription on ultrathin cell sections occurs preferentially at the borders of condensed chromatin blocks and in their close vicinity. The condensed chromatin areas themselves remain unlabelled. Inside most nucleoli, gold-particle clusters are mainly detected in the fibrillar centers, especially at their periphery, whereas the dense fibrillar component and the granular component remain devoid of label. These results, together with previous observations made on the same cell type, clearly indicate that the fibrillar centers are the sites of rRNA gene transcription in Ehrlich tumor cell nucleoli, while the dense fibrillar component is the site of pre-rRNA accumulation.
Assuntos
Nucléolo Celular/metabolismo , DNA Ribossômico/genética , Precursores de RNA/biossíntese , Transcrição Gênica , Animais , Autorradiografia , Carcinoma de Ehrlich , Cromatina/metabolismo , Cromatina/ultraestrutura , Imuno-Histoquímica , Camundongos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Células Tumorais CultivadasRESUMO
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).
Assuntos
Nucléolo Celular/ultraestrutura , Cromatina/ultraestrutura , Acetilação , Animais , Carcinoma de Ehrlich , Carcinoma de Células Escamosas , Linhagem Celular , Nucléolo Celular/fisiologia , Células Cultivadas , Humanos , Interfase , Camundongos , Microscopia EletrônicaRESUMO
The effects of MPB, a strong inhibitor of RNA synthesis, have been analysed at the ultrastructural level by means of stereological methods. After treatment, an increase in the nucleolar volume is observed. This enlargement is due to vacuolization of the nucleoli. However, the relative volumes of the nucleolar components are modified in various directions: the volume of the granular component decreases whereas the fibrillar centres increase in size. These results are discussed in terms of relations between morphology and function of the nucleolus.
Assuntos
Benzimidazóis/farmacologia , Nucléolo Celular/efeitos dos fármacos , Animais , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/ultraestrutura , Nucléolo Celular/ultraestrutura , Camundongos , Microscopia Eletrônica , RNA Neoplásico/biossínteseRESUMO
Nucleolar ultrastructure has been studied in absorptive cells of the intestinal villi and in undifferentiated cells of the crypts of Lieberkühn. In the top of the villi, the nucleoli are composed of fibrillar centres associated with dense chromatin. These observations seem to be related to RNA synthesis inhibition.