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Institute
Autoantibodies to components of the nucleolus are a unique serological feature of patients with scleroderma. There are autoantibodies of several specificities; one type produces a speckled pattern of nucleolar staining in immunofluorescence. In actinomycin D and 5,6-dichloro-{j-D-ribofuranosylbenzimidazoletreated Vero cells, staining was restricted to the fibrillar and not the granular regions. By double immunofluorescence, specific rabbit anti-RNA polymerase I antibodies stained the same fibrillar structures in drug-segregated nucleoli as scleroderma sera. Scleroderma sera immunoprecipitated 13 polypeptides from (35S)methionine-labeled HeLa cell extract with molecular weights ranging from 210,000 to 14,000. Similar polypeptides were precipitated by rabbit anti-RNA polymerase I antibodies, and their common identities were confirmed in immunoabsorption experiments. Microinjection of purified IgG from a patient with speckled nucleolar staining effectively inhibited ribosomal RNA transcription. Autoantibodies to RNA polymerase I were restricted to certain patients with scleroderma and were not found in other autoimmune diseases.
High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody
(1987)
A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.
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.
RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110000, required Mg2+ but not ATP, and was recognized by anti-topoisomerase I antibodies. When added to RNA polymerase I preparations containing topoisomerase activity, anti-topoisomerase I antibodies were able to inhibit the DNA relaxing activity of the preparation as well as RNA synthesis in vitro. RNA polymerase II prepared by analogous procedures did not contain topoisomerase activity and was not recognized by the antibodies. The topoisomerase I: polymerase I complex was reversibly dissociated by column chromatography on Sephacryl S200 in the presence of 0.25 M (NH4hS04. Topoisomerase I was immunolocalized in the transcriptionally active ribosomal gene complex containing RNA polymerase I in situ. These data indicate that topoisomerase I and RNA polymerase I are tightly complexed both in vivo and in vitro, and suggest a role for DNA topoisomerase I in the transcription of ribosomal genes.
Certain autoimmune sera contain antibodies against a nucleolar ribonucleoprotein particle associated with 7-2-RNA (R. Reddy et al. (1983) J. Bioi. Chem . 258, 1383; C. Hashimoto and J. A. Steitz (1983) J. Bioi. Chem. 258, 1379). In this study, we showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-I-j3-D-ribofuranosylbenzimidazole (DRB)-segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7- 2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of Mr 40,000 from e5S] methionine-Iabeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation, and/or transport of preribosomes
Small nucleolus-related bodies which occur in the nUcleoplasm of " micronuclei" lacking nucleolar organizers have been studied by immunofluorescence microscopy. These bodies stained specifically with three different antibodies directed against proteins that are normally associated with the dense fibrillar component of functional nucleoli, but not with antibodies specific for certain proteins of the granular component or the fibrillar centers. Our data show that, in the absence of rRNA genes, the various constituent proteins characteristic of the dense fibrillar component spontaneously assemble into spherical entities but that the subsequent fusion of these bodies into larger structures is prevented in these micronuclei. The similarity between these nucleolus-related bodies of micronuclei and the prenucleolar bodies characteristic of early stages of nucleologenesis during mitotic telophase is discussed.
The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach , involving a monoclonal antibody directed against double- and single-stranded DNA. Immunolabelling was performed . either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The results seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus.
After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtKz) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.
Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtKz cells with WGA or antibody PIt and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1 . Although PtKz cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome- associated components.
A non-radioactive in situ hybridization method is described for the localization of transcription units of defined genes to lateral loops of Xenopus laevis lampbrush chromosomes. Two Xenopus cONA probes were used encoding the nucleolar protein N038/ B23 and cytokeratin 1(8). Both proteins are known to be synthesized in Xenopus oocytes, and Northern blot analysis revealed the presence of the corresponding mRNAs in different oogenic stages. The probes were enzymatically labeled with biotin-dCTP and hybridized to lampbrush chromosomes. The sites of hybridization were detected either by indirect immunofluorescence microscopy using rabbit antibodies against biotin and fluorescein-conjugated antirabbit IgG or enzymatically using peroxidase-conjugated streptavi din. The probe encoding the nucleolar protein hybridized to two sets of lateral loops on different bivalents, the cytokeratin probe to at least four. Our finding that each probe hybridized to more than one chromosomal locus may reflect the tetraploid nature of the Xenopus laevis genome or results from cross-hybridization to other transcriptionally active members of the N038/ B23-nucleoplasmin or the cytokeratin-Iamin gene families. The method described should facilitate further in situ hybridization studies with appropriate genomic clones in order to map specific DNA sequences to defined loop regions and to come to a better understanding of the relationship between loop organization and gene transcription unit.
PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68000 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 doublelayered 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.
Nucleoli are the morphological expression of the activity of a defined set of chromosomal segments bearing rRNA genes. The topological distribution and composition of the intranucleolar chromatin as well as the definition of nucleolar structures in which enzymes of the rDNA transcription machinery reside have been investigated in mammalian cells by various immunogold labelling approaches at the ultrastructural level. The precise intranucleolar location of rRNA genes has been further specified by electron microscopic in situ hybridization with a non-autoradiographic procedure. Our results indicate that the fibrillar centers are the sole nucleolar structures where rDNA, core histones, RNA polymerase I and DNA to po isomerase I are located together. Taking into account the potential value and limitations of immunoelectron microscopic techniques, we propose that transcription of the rRNA genes takes place within the confines of the fibrillar centers, probably close to the boundary regions to the surrounding dense fibrillar component.
Nuclei assembled in Xenopus egg extract from purified DNA or chromatin resemble their natural counterparts in a number of structural and functional features. However, the most obvious structural element of normal interphase nuclei, the nucleolus, is absent from the in vitro reconstituted nuclei. By EM, cytological silver staining, and immunofluorescence microscopy employing antibodies directed against various nucleolar components we show that nuclei assembled in vitro contain numerous distinct aggregates that resemble prenucleolar bodies (PNBs) by several criteria. Formation of these PNB-like structures requires pore complex-mediated nuclear transport of proteins but is independent of the genetic content of the in vitro nuclei as well as transcriptional and translational events. Our data indicate that nuclei assembled in vitro are capable of initiating early steps of nucleologenesis but that the resulting PNBs are unable to fuse with each other, probably due to the absence of a functional nucleolus organizer. With appropriate modifications, this experimental system should be useful to define and analyze conditions promoting the site-specific assembly of PNBs into a coherent nucleolar body.
Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (V ASP). V ASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMPdependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, V ASP is associated predominantly with the distal parts of radial micro filament bundles and with microfilaments outlining the periphery, whereas less V ASP is associated with a central microfilamentous ring. V ASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, V ASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of V ASP to F -actin is also presented. The data demonstrate that V ASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.