The nucleus and the nucleolus. The contribution of French electron microscopists.
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Animals↗
Biomedical subjects
E Puvion
Publications and source records attributed to E Puvion.
Rearrangements of intranuclear structures involved in RNA processing in response to adenovirus infection.
We have studied in HeLa cells at the electron microscope level the response to adenovirus infection of the RNA processing machinery. Components of the spliceosomes were localized by in situ hybridization with biotinylated U1 and U2 DNA probes and by immunolabeling with Y12 anti-Sm monoclonal antibody, whereas poly(A)+ RNAs were localized by specific binding of biotinylated poly(dT) probe. At early stages of nuclear transformation, the distribution of small nuclear RNPs was similar to that previously described in non-infected nuclei (Visa, N., Puvion-Dutilleul, F., Bachellerie, J.P. and Puvion, E., Eur. J. Cell Biol. 60, 308-321, 1993; Visa, N., Puvion-Dutilleul, F., Harper, F., Bachellerie, J. P. and Puvion, E., Exp. Cell Res. 208, 19-34, 1993). As the infection progresses, the large virus-induced inclusion body consists of a central storage site of functionally inactive viral genomes surrounded by a peripheral shell formed by clusters of interchromatin granules, compact rings and a fibrillogranular network in which are embedded the viral single-stranded DNA accumulation sites. Spliceosome components and poly(A)+ RNAs were then exclusively detected over the clusters of interchromatin granules and the fibrillogranular network whereas the viral single-stranded DNA accumulation sites and compact rings remained unlabeled, thus appearing to not be directly involved in splicing. Our data, therefore, suggest that the fibrillogranular network, in addition to being the site of viral transcription, is also a major site of viral RNA splicing. Like the clusters of interchromatin granules, which had been already involved in spliceosome assembly, they could also have a role in the sorting of viral spliced polyadenylated mRNAs before export to the cytoplasm. The compact rings, which contain non-polyadenylated viral RNA, might accumulate the non-used portions of the viral transcripts resulting from differential poly(A)+ site selection.
Adenoviruses, Human↗
Localization of cyclin A at the sites of cellular DNA replication.
Cyclin A is a nuclear protein which is part of a kinase complex with either p34cdc2 or p33cdk2. Cyclin A is required in higher eukaryotic cells at the G1/S and the G2/M transitions. To examine the relationship between cyclin A and DNA replication, we simultaneously labeled exponentially growing HeLa cells for the distribution of cyclin A and proliferating cell nuclear antigen (PCNA). We have now demonstrated, by means of immunoelectron microscopy, that cyclin A is located at the sites of DNA replication visualized by both BrdU and PCNA labeling. Thus cyclin A may play a significant role in the phosphorylation of proteins at or near the sites of DNA replication.
Bromodeoxyuridine↗
Intranuclear distribution of poly(A) RNA determined by electron microscope in situ hybridization.
We used a biotinylated poly(dT) probe to localize poly(A) RNA in HeLa cells at optical and electron microscope levels. We established that the fluorescent speckled staining pattern corresponds at the ultrastructural level to the labeling of perichromatin fibrils, at least part of the population of perichromatin granules, and clusters of interchromatin granules. Coiled bodies and the interchromatin granule-associated zones, a recently described subcompartment containing U1 but not U2 snRNA, were not labeled. The density of the labeling of interchromatin granule clusters exceeded by three to five times that of the surrounding extranucleolar area. These results are discussed in relation to the role of perichromatin fibrils in splicing of pre-mRNA and to the possible involvement of interchromatin granules in the assembly of mature spliceosomes as well as in sorting and/or coordination of RNA molecules to be transported to the cytoplasm.
Cell Compartmentation↗
Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells.
We have examined the intranuclear distribution of U1 and U2 small nuclear RNAs (snRNAs) in HeLa cells by electron microscope in situ hybridization using biotinylated DNA probes reacting at the surface of thin sections of Lowicryl-embedded cells. U1 and U2 snRNAs colocalized on perichromatin fibrils, clusters of interchromatin granules and coiled bodies. The perichromatin granules were just occasionally labeled. In addition, we identified a novel nuclear domain associated with the clusters of interchromatin granules which contains U1 but not U2 snRNA. This new compartment termed "interchromatin granule-associated zone" has a fibrillar texture, does not contain DNA and might be the equivalent of the A snurposomes described in germinal vesicles of amphibians (Wu et al., J. Cell Biol. 113, 465-483 (1991)). We propose that the interchromatin granule-associated zones might be sites of the final maturation of the U1-pre-snRNP particle before its transfer to interchromatin granules and its subsequent assembly in the spliceosome.
Cell Compartmentation↗
Nuclear localization of the Epstein-Barr virus/C3d receptor (CR2) in the human Burkitt B lymphoma cell, Raji.
Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the cell surface interacted with soluble and particle-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.
Antigens, Differentiation, B-Lymphocyte↗
Alterations of nucleolar ultrastructure and ribosome biogenesis by actinomycin D. Implications for U3 snRNP function.
We have studied, at the electron microscope level, the reorganizations of nucleolar ultrastructure induced by actinomycin D (AMD) in different conditions of drug treatment associated with an inhibition of rRNA synthesis. We have analyzed in parallel the localizations of ribosomal genes, of their transcripts, of various pre-rRNA intermediates, as well as of U3 RNA and fibrillarin by in situ hybridization with nucleic acid probes and immunocytological detection on thin sections of human and mouse cells. Consistent with previous observations, dense fibrillar component (DFC) and granular component (GC) appear to contain distinct pre-rRNA species at different stages of their processing. DFC appears as a major site of U3 RNA accumulation, but a very substantial fraction of nucleolar U3 RNA is also found in GC, colocalizing with partially processed pre-rRNAs. Remarkably, the major nucleolar components retain their ultrastructural appearance when extensively depleted of their pre-rRNA moiety, and ribosomal genes are always detected over fibrillar center (FC), even after extended AMD treatments which result in the characteristic segregation of nucleolar components. Moreover, while for GC the U3 RNA and pre-rRNA contents evolve in parallel following the cessation of rRNA synthesis, a dramatic uncoupling is observed for DFC. The persistent presence of U3 RNA and fibrillarin after pre-rRNA depletion suggests that DFC could represent an anchorage site for U3 snRNPs, before their entering another cycle of pre-rRNA processing reactions.
3T3 Cells↗
Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells.
The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both non-encapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.
Animals↗
Nucleolar organization of HeLa cells as studied by in situ hybridization.
The distribution of the ribosomal genes and their ribosomal RNA (rRNA) products in the different compartments of the nucleolus of HeLa cells was examined on thin sections of Lowicryl embedded material. The ribosomal nucleic acids were visualized after hybridization with a set of biotinylated double-stranded ribosomal DNA (rDNA) probes from different locations along the gene, followed by immunogold labelling of biotin. Ribosomal genes were detected over both the entire fibrillar centres (FCs) and some masses of intranucleolar condensed chromatin. As for the rRNA components, comparison of the signal levels obtained with the different probes provides some information about the compartmentalization of distinct stages of ribosome biogenesis. Thus a probe specific for the 5' external transcribed spacer (5'ETS) portion of pre-rRNA labels almost exclusively the dense fibrillar component (DFC) and the border of the FCs, while the interior of the FCs appears devoid of any kind of rRNA species. By contrast, probes recognizing either 18S or 28S mature rRNA sequences label both the DFC and the granular component (GC). Moreover, mature 18S rRNA sequences are markedly under-represented relative to mature 28S rRNA sequences in the GC, as compared with the other nucleolar compartments. Our observations are consistent with the view that DFCs contain elongating and 47S-45S precursor rRNA molecules whereas the subsequent various rRNA processing intermediates are mainly located within the GC. Since the border of FCs is the only site where both rDNA and newly synthesized pre-rRNA coexist, the transcription of ribosomal genes seems to take place at the periphery of the FCs, and not in the DFC, suggesting that elongating and newly completed transcripts are immediately transferred into the surrounding DFC where they transiently accumulate before undergoing processing reactions and transfer to the GC.
Cell Nucleolus↗
Sites of transcription of adenovirus type 5 genomes in relation to early viral DNA replication in infected HeLa cells. A high resolution in situ hybridization and autoradiographical study.
The distribution of viral RNA molecules in HeLa cells infected with adenovirus type 5 (Ad5) was determined by in situ hybridization at the ultrastructural level at an intermediate stage of nuclear transformation, when viral DNA synthetic activities were maximal but progeny viruses were still sparse. Transcription sites of the viral DNA were localized by short pulse, high resolution autoradiography. Nascent viral RNA was found mainly within the nuclear compartment identified at the peripheral replicative zone, which is known to be the main replicative site of Ad5 viral genomes. Viral RNA molecules also were present, but to a markedly lesser extent, within the contiguous single-stranded (ss) DNA accumulation site, another intranuclear virus-induced structure in which some replication of viral genomes also occurs. Two other virus-induced nuclear structures contained viral RNA, the occasional exceptionally enlarged clusters of interchromatin granules and the compact rings, both DNA-free structures of unknown significance but which might play a role in the process of maturation of the Ad5 primary transcripts. Viral messenger RNA molecules were localized over the large areas of the cytoplasm which contain numerous ribosomes. Our analysis of the effects of various enzymatic pretreatments of the sections of infected cells on the revelation of nascent RNA by in situ hybridization is reviewed.
Adenoviruses, Human↗
Analysis by in situ hybridization and autoradiography of sites of replication and storage of single- and double-stranded adenovirus type 5 DNA in lytically infected HeLa cells.
The distribution in the different compartments of infected nuclei of double-stranded (ds) and single-stranded (ss) adenovirus type 5 (Ad5) DNA and of the sites of viral DNA replication were examined on thin sections of Low-icryl-embedded material. The DNA is visualized with a biotinylated viral probe and immunogold labeling of biotin, and its replication is monitored by high-resolution autoradiography after short pulses with tritiated thymidine. The first detectable sites of viral DNA, named early replicative sites, contained all the ss and ds viral DNA and viral replicative activity. At a later stage of nuclear transformation, they gave rise to two new structures. The compact fibrillar ssDNA accumulation sites enlarged greatly and became transformed functionally to become a transient site of accumulation of large numbers of ss replicative intermediates. Double-stranded viral DNA and its replicative activity shifted primarily into immediately surrounding fibrillogranular peripheral replicative zones. Ad5 DNA replication continues in the ssDNA accumulation sites but it is intermittent, whereas in the peripheral replicative zones it is continuous. Still later in infection, a single, large, centrally located mass of dense fibrils, the viral genome storage site, developed in each nucleus which proved to be the main site of storage of nonreplicating, nonencapsidated, ds viral genomes. We discuss the possible distribution of the various viral DNA replicative intermediates among these virus-induced intranuclear structures.
Adenoviruses, Human↗
Replicating single-stranded adenovirus type 5 DNA molecules accumulate within well-delimited intranuclear areas of lytically infected HeLa cells.
The intranuclear compartments which contain adenovirus type 5 (Ad5) genomes during productive infection of HeLa cells were determined by high resolution when the rate of viral DNA replication is at its maximum. In situ hybridization was performed with and without denaturation of Ad5 DNA to reveal those portions of both double-stranded and single-stranded viral DNA and of single-stranded viral DNA alone, respectively, which were present at the surface of Lowicryl thin sections. Autoradiography after short pulse incorporation of tritiated thymidine revealed sites of active DNA replication whereas a pulse-chase experiment visualized the migration of the synthesized DNA. Immunocytochemistry determined distribution of the Ad5 72 kDa protein. Viral DNA was contained within multiple, well-delimited, compact fibrillar structures and their immediately surrounding, less well-defined, fibrillo-granular areas of the cell nucleoplasm. The compact fibrillar masses were primarily storage sites of single-stranded viral DNA and its accompanying 72 kDa protein, so they were named ssDNA accumulation sites. They contained intermittent viral replicative activity. Conversely, viral ssDNA and 72 kDa protein were rare in the surrounding fibrillo-granular areas in which Ad5 replication DNA is continuous, so we referred to them as peripheral replicative zones. Well-delimited subnuclear structures induced by viruses, therefore, represent accumulations of specific components of their replicating genomes.
Adenoviruses, Human↗
Replication timing of 10 developmentally regulated genes in Physarum polycephalum.
We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.
Blotting, Southern↗
Rapid effect of heat shock on two heterogeneous nuclear ribonucleoprotein-associated antigens in HeLa cells.
During severe heat shock, which known to interrupt both splicing of RNA transcripts and nucleocytoplasmic transport, it is to be expected that the substructure of heterogeneous nuclear ribonucleoproteins (hnRNP) is altered in some way. Recently, we have shown that such a stress actually induces rapid alterations at the level of individual proteins (Lutz, Y., M. Jacob, and J.-P. Fuchs. 1988 Exp. Cell Res. 175:109-124). Here we report further investigations on two related 72.5-74-kD hnRNP proteins whose behavior is also rapidly modified by a heat shock at 45 degrees C, whereas no effect is observed at 42 degrees C. Using a monoclonal antibody, we show that in situ the antigens are available only when the cells are heat shocked at 45 degrees C. Subcellular fractionation shows that in normal cells the antigens are associated with the bulk of hnRNP (50-200S). During heat shock, whereas the overall characteristics of the bulk of preexisting hnRNP are unchanged, these antigens rapidly switch to a subpopulation of hnRNP with larger average size (50 to less than 300S) and increased stability. Structural analysis of the associated hnRNP in normal and stressed cells shows that in both cases the antigens are associated with the nuclear matrix subcomplex of hnRNP, which in situ is part of the internal nuclear matrix. Such hnRNP antigens, which are rapidly redistributed during a heat shock at the upper temperature range of the stress response, might well be involved in splicing and/or transport control.
Antibodies, Monoclonal↗
Ultrastructural localization of viral DNA in thin sections of herpes simplex virus type 1 infected cells by in situ hybridization.
We have used in situ hybridization at the ultrastructural level to localize non-encapsidated and encapsidated herpes simplex virus type 1 (HSV-1) genomes in nuclei of infected rabbit fibroblasts. A biotinylated cloned subgenomic HSV DNA fragment was used as hybridization probe. The probe hybridized to the viral DNA accessible at the surface of Lowicryl sections was revealed by immunogold labeling. Non-encapsidated viral DNA was detected exclusively within the virus-induced central region of 4 h to 17 h infected nuclei. Localization of the probe either near the nuclear envelope or within marginated host chromatin was found only on HSV DNA which was packaged into viral nucleoids. The use in parallel of in situ hybridization with specific staining for DNA and autoradiography after tritiated thymidine incorporation, followed by either conventional fixation of the cells or the nucleoprotein loosening procedure, indicated that non-encapsidated viral DNA and marginated host chromatin formed two juxtaposed compartments without interpenetration even after experimentally produced mild dispersion of the nuclear components.
Animals↗
Cytochemical study of the localization and organization of parental herpes simplex virus type I DNA during initial infection of the cell.
Changes in the location and structural organization of parental herpes simplex virus type 1 (HSV-1) DNA during its migration from the extracellular space to the interior of the nucleus of the target cell were examined by in situ hybridization using an HSV-1 DNA probe, specific DNA staining, and autoradiography after infection of cells with tritium-labeled viruses. In situ hybridization was carried out on denatured DNA to reveal as much as possible of the HSV-1 sequence present at the surface of the sections, and also on non-denatured DNA which revealed the presence of single-stranded portions of parental DNA, both prior to and during its intracellular migration. The results from in situ hybridization and autoradiography demonstrated that a short interval of about 15 min separated the initial contact of the viruses with the cells from the entry of parental viral DNA into the nucleus. In transit, morphologically intact nucleoids were released into the cytoplasm, and swollen nucleoids which contained partially decondensed viral DNA became detectable in the juxtanuclear cytoplasm and the periphery of the nucleus among the cell chromatin fibers. Completely decondensed parental viral DNA fibers could not be distinguished structurally from cellular DNA, but their position could be revealed by the in situ hybridization label. The infective DNA became randomly distributed within all compartments of the nucleus except the matrix-associated clusters of interchromatin granules.
Animals↗
Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells.
The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.
Antibodies, Monoclonal↗
Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts.
A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.
Animals↗