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D Hernandez-Verdun

Publications and source records attributed to D Hernandez-Verdun.

At least 55 records · Page 3Linked to original sources
PubMed1992

Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state.

The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis.

We investigated the perichromosomal architecture established during mitosis. Entry into mitosis brings about a dramatic reorganization of both nuclear and cytoplasmic structures in preparation for cell division. While the nuclear envelope breaks down, nuclear proteins are redistributed during chromosome condensation. Some of these proteins are found around the chromosomes, but little is known concerning their nature and function. Ten autoimmune sera were used to study the microenvironment of chromosomes and, in particular, the chromosome periphery. They were selected for their anti-nucleolar specificity and were found to recognize three nucleolar proteins that coat the chromosomes during mitosis. The distribution of these antigens was followed through the cell cycle by confocal laser scanning microscopy. The antigens dispersed very early during prophase and simultaneously with the chromosome condensation suggesting a correlation between these two processes. The antigens have apparent molecular weights of 53, 66, and 103 kDa on SDS-PAGE migration. Elution of the antibodies and immunopurification showed that they are RNA-associated proteins. The coimmunoprecipitating RNA moiety involved in these RNPs appeared to be U3, but the antigens are not related to the fibrillarin family. Therefore, small nucleolar RNPs follow the same distribution during mitosis as that described for small nuclear RNPs. Possible functions for these antigens are discussed.

Antibodies, Antinuclear↗
PubMed1992

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.

Blotting, Western↗
PubMed1992

Relocation of nucleolar proteins around chromosomes at mitosis. A study by confocal laser scanning microscopy.

The behaviour of nucleolar antigens known to associate with chromosomes at mitosis was investigated in mammalian cells (HeLa, HEp-2, PtK1, CHO) by immunofluorescence and confocal laser scanning microscopy. Serial optical sections through mitotic cells, from prophase to telophase, were used to generate three-dimensional images of the antigen distribution. Our results indicate that, at the onset of mitosis, these antigens leave the nucleoli in a highly ordered manner to form a network extending from the nucleoli towards the nuclear envelope. The migration begins at very early prophase, when the condensation of the chromosomes is not yet visible. After completion of the migration at late prophase, the labelling is found at the chromosome periphery. The antigens remain distributed as a sheath surrounding the chromosomes from prophase to telophase. Therefore, the proteins involved in the formation of this perichromosomal layer have different behaviour than those of the prenucleolar bodies. The antigens appear to interact strongly with chromosomes, since they are not lost during chromosome isolation in hypotonic buffer. Each chromosome is entirely covered from one telomere to the other, except in the centromeric region. Thus the relocation of these nucleolar proteins does not appear to be the result of a passive accumulation at the chromosome periphery, but seems rather to be due to an active targeting to specific sites. Consequently, these proteins may have a determining function in the progression of the cells through mitosis, possibly by participating in the protection and stabilization of the chromosomes.

Animals↗
PubMed1991

Computer reconstruction of nucleolar architecture by interactive three-dimensional color display.

The first complete three dimensional ultrastructural reconstruction of pancreatic cell nucleoli, was done using EM and computer 3D-assisted reconstruction of serial sections with interactive 3D back-to-front and color display methods based on voxel representation. The purpose of the study was to depict the architecture of the nucleolar components. We obtained information about the location of the nucleolus within the nuclear volume and about the shape and polarity of the 3 main nucleolar territories.

Animals↗
PubMed1991

A novel 43-kDa glycoprotein is detected in the nucleus of mammalian cells by autoantibodies from dogs with autoimmune disorders.

We have characterized a new antibody specificity in a panel of sera from dogs developing systemic lupus erythematosus (SLE) or clinically related autoimmune disorders. This antibody stains in a speckled fashion the nucleus of cells of different mammalian origins. The target antigen is a basic (pI 9.2) nuclear polypeptide with an apparent molecular weight of 43 kDa (p43) which is detected in various mammalian cell nuclei. p43, as studied in HeLa cells, appears to be cell cycle-independent. It is released from nuclei by salts (0.5 M NaCl or 0.25 M ammonium sulfate). Upon subfractionation of nuclear components, p43 is found in the fraction containing HnRNPs and is recovered in immunoprecipitates obtained with 4F4 monoclonal antibody to HnRNP C proteins. Immunoelectron microscopy revealed that p43 is concentrated over the dense chromatin periphery and interchromatin granule clusters. Another important feature of p43 is its ability to specifically bind wheat germ agglutinin lectin but not concanavalin A nor Ulex europaeus I, supporting the notion that p43 is a glycoprotein bearing an N-acetyl-glucosamine moiety. Consistent with this result, a radio-active p43 band is specifically immunoprecipitated by canine anti-p43 autoantibodies from HeLa cells metabolically labeled with [14C]glucosamine. Finally, anti-p43 antibodies do not immunoprecipitate SnRNA, indicating that p43 has no apparent association with SnRNPs.

Animals↗
PubMed1991

Behaviour of nucleolar proteins in nuclei lacking ribosomal genes. A study by confocal laser scanning microscopy.

The behaviour of nucleolar proteins in cycling PtK1 cells and in micronuclei with or without NORs was investigated by immunofluorescence using antibodies from autoimmune sera and confocal laser scanning microscopy. These antibodies were shown by electron microscopy to recognize antigens confined to only one of the three basic nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). Serial optical sections allowed us to determine the three-dimensional organization of these components in the nucleolus of cycling cells. Furthermore, clear differences were found in the distribution of the various antigens in micronucleated cells. Three patterns could be observed: (1) the FC antigens were found mainly in the nucleoli, but also in varying amounts in the dots; (2) surprisingly, the DFC antigens were found to accumulate preferentially in the dots; (3) the GC-specific marker stained intensively the nucleoli as well the dots. The results are interpreted with regard to possible mechanisms for targeting nucleolar proteins to the site of nucleolar formation.

Biomarkers↗
PubMed1990

Distribution of rDNA and 28S, 18S, and 5S rRNA in micronuclei containing a single chromosome.

Investigating genes and their transcription products in nuclear compartments corresponding to one mammalian chromosome, the ribosomal genes 18S-28S and 5S were localized in PtK1 micronucleated cells and rRNA was characterized in sorted micronuclei containing single identified chromosomes. In situ hybridization revealed the presence of 18S-28S rRNA genes in two micronuclei per cell and 5S rRNA genes in four micronuclei per cell. Flow cytometry histograms of isolated micronuclei stained with Hoechst 33342 exhibited five peaks (a-e) in which peaks b and c, respectively, corresponded to chromosomes 4 and X. Restricted genomic DNA from sorted peak c micronuclei showed the presence of 28S gene sequences. Direct sorting of the micronuclei from each peak on nitrocellulose and their hybridization with the 18S-28S rDNA probe revealed that the rRNA genes were exclusively located in micronuclei containing X chromosomes. Northern blotting showed the presence of 18S-28S and 5S rRNAs in peak c micronuclei and their absence from peak b micronuclei. Consequently, these procedures allowed us to show the presence of ribosomal genes and the corresponding rRNA in micronuclei containing single X chromosomes, and the absence of rRNA from micronuclei that do not contain the ribosomal genes. In regards to the transcription of these genes, the micronuclei from peak c can be considered as functional interphase X chromosomes.

Animals↗
PubMed1990

A 116,000 Mr nucleolar antigen specific for the dense fibrillar component of the nucleoli.

In ATT, a human autoimmune serum, we found anti-nucleolar antibodies that recognized nucleolar antigens confined to a single nucleolar compartment, the dense fibrillar component (DFC). We localized these antigens by immunoelectron microscopy in DFC of HeLa cell nucleoli both on Lowicryl sections and cryoultrathin sections without embedding. The antigens were solubilized by incubation with 2M NaCl but not by RNase or DNase treatment. The ATT serum crossreacted with rat liver nucleoli and PtK1 cell nucleoli in which immunofluorescence labelling displayed a clumpy pattern. During mitosis, the antigens dispersed in the cytoplasm until late telophase, when they gathered in the prenucleolar bodies. In human peripheral lymphocytes, or HeLa cells treated with actinomycin D, the antigens were still present but the fluorescence intensity decreased. By immunoblotting using human nuclear extracts, the ATT serum bound to a 116,000 Mr protein at dilutions up to 1:2000. The reactivity of this band diminished with actinomycin D-treated nuclear extracts. Two minor bands were also observed at 97 and 70K (K = 10(3) Mr). Immunopurification by competition or elution demonstrated that the 116K antigens were at the origin of the nucleolar labelling. This DFC marker appeared to be different from the NOR-silver-stained proteins, which in our preparations exhibited apparent molecular weights of 105, 80 and 38-40K. In addition, these 116K antigens did not exhibit the characteristics described for DNA topoisomerase I, fibrillarin or nucleolin. We propose the 116K antigen as a new marker of the DFC of the nucleoli.

Aged↗
PubMed1989

Three-dimensional organization of micronuclei induced by colchicine in PtK1 cells.

In PtK1 cells micronucleated by colchicine, we previously demonstrated that some micronuclei contain a single chromosome. Here, we investigated interphase chromosome organization in micronucleated PtK1 cells using conventional electron microscopy and three-dimensional computer reconstruction. The distribution of micronuclei was not always polarized, but in some cells they formed a ring. When this occurred, centrioles and Golgi apparatus were located inside the ring. On freeze-fracture replicas, we observed that nuclear pore distribution among the micronuclei was heterogeneous, and on thin sections some micronuclei displayed an incomplete nuclear envelope, with gaps in the double membrane and areas without lamina or condensed chromatin. By autoradiography, we showed that the fibrillar dots were not sites of active transcription. We applied three-dimensional reconstruction to one micronucleated cell containing 22 micronuclei whose size indicated that each micronucleus probably contained one chromosome. In this cell we demonstrated that only the smallest micronuclei had an incomplete nuclear envelope. The presence in micronuclei of either nucleoli or fibrillar dots was found to be mutually exclusive. These dots might constitute stores of nucleolar proteins which migrate into micronuclei possessing no ribosomal genes. In NOR-bearing micronuclei, the structural organization was similar to that of diploid nuclei: the nucleoli were attached to the nuclear membrane and a nucleolar canal was seen, even in single-chromosome spherical micronuclei. Taken together, these findings indicate that in the diploid nuclei of PtK1 cells, the three-dimensional organization of the nucleolar domain seems to be directly controlled by the X-chromosome.

Animals↗
PubMed1988

Autoimmune serum containing an antibody against a 94 kDa nucleolar protein.

Little information exists on how various nucleolar proteins function in ribosome biogenesis. Of special interest is that group of nucleolar proteins which are not incorporated into mature ribosomes because they are candidates for a role in the regulation of ribosome construction. Non-ribosomal nucleolar proteins can be analyzed using autoimmune sera from scleroderma patients which often contain antinucleolar antibodies. One such serum, designated ScBr, is shown by indirect immunofluorescence to react specifically with nucleoli in cells of 3 different mammalian species, indicating that the antigen is at least partly conserved evolutionarily. It is not RNase-sensitive, but is completely eliminated after incubation with pronase and 2 M NaCl. Immuno-electron microscopy was carried out on Lowicryl ultrathin sections to localize the antigen. The labeling was observed over both the granular and the dense fibrillar component but not the fibrillar centers, indicating that the antigen is associated with ribosomal RNA transcription sites and ribosome assembly into precursor particles. In addition, the antibody was localized to small nucleoplasmic entities, termed dense nuclear bodies. This could indicate a relationship between nucleoli and dense nuclear bodies. By immunoprecipitation, only a single protein of 94 kDa molecular weight was revealed. By immunoblotting, the band at 94 kDa was found to be the only positive band for high ScBr dilutions. Observation of the behavior of the antigen during mitosis revealed that it became dispersed into the cytoplasm after breakdown of the nuclear envelope, lining most of the chromosomes rather than remaining associated with the NOR-chromosomes. The antigen appeared to be restored to nucleoli only in late telophase; phase-dense prenucleolar bodies of early telophase cells did not show positive staining for the antigen. During actinomycin-D RNA synthesis inhibition as well as in non-stimulated lymphocytes the positive staining is greatly decreased. These results were consistent with a role for the 94 kDa nucleolar protein in the process of preribosome assembly.

Animals↗
PubMed1988

Replication bands and nucleoli in the macronucleus of Euplotes eurystomus: an ultrastructural and cytochemical study.

The principal structural compartments of the macronucleus of Euplotes eurystomus were examined by ultrastructural and cytochemical procedures. Interphase chromatin is condensed in highly compact granules that stain intensely with the DNA-specific osmium-amine procedure. Nucleoli react strongly with silver and with thiol-specific reagents, but are almost completely unstained by osmium-amine. The organelle of DNA synthesis, the replication band, is composed of 2 zones. The forward zone consists of highly ordered chromatin fibers, stains strongly with osmium-amine, with silver, and with thiol-specific reagents. The rear zone, which is the site of DNA synthesis, is impoverished in DNA, and is very sensitive to collapse induced by in vivo heat shock, or during nuclear isolation.

Animals↗
PubMed1987

Structure of ribosomal genes of mammalian cells in situ.

The structural and functional organization of ribosomal genes was investigated in situ in human circulating lymphocytes and a human tumour cell line, TG cells, Stereo-pair electron micrographs revealed that this ribosomal chromatin is not structured into nucleosomes, but composed of completely extended filaments, 2-3 nm thick. Despite its homogeneous morphological structure only a small portion of ribosomal chromatin present in the dense fibrillar component is transcriptionally active. This was demonstrated in TG cells by exclusive autoradiographic labelling on serial sections of the dense fibrillar component with 3H-uridine and by the distribution of RNase-gold particles in all the ribonucleoprotein (RNP) structures but not in the fibrillar centres. The extended, non-nucleosomal configuration of both transcriptionally inactive and active ribosomal chromatin could be explained by the peculiar protein composition of this chromatin. Staining with the acrolein-silver-methenamine technique for basic proteins indicated that all the completely extended ribosomal chromatin is devoid of histones, even after inactivation of transcription by actinomycin D. Stereo-electron-microscopical visualisation of the Ag-NOR proteins revealed a thread-like structural organization of these proteins with a spatial distribution superimposable on that of the ribosomal chromatin filaments.

Cell Line↗
PubMed1987

RNA polymerase activity in PtK1 micronuclei containing individual chromosomes. An in vitro and in situ study.

Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.

Animals↗
PubMed1987

Identification and sorting of micronuclei containing individual chromosomes.

Micronuclei are diploid genome subfractions which can be induced by colchicine treatment. The purpose of the present study was to identify the chromosome content of micronuclei. Accordingly, PtK1 cells were micronucleated with colchicine and their DNA was labeled by the Hoechst 33342 fluorochrome. The fluorescence intensity of the micronuclei was measured by flow cytometry and compared to that of metaphase chromosomes of the same species. We observed histograms exhibiting five distinct peaks for both micronuclei and chromosomes. Each class of micronuclei corresponded to a peak in the flow karyotype with the same values. As the smallest micronuclei contained the same amount of DNA as one chromosome, it seemed possible to identify the DNA content of the micronuclei containing a single chromosome. This identification was confirmed by fluorescence microscope observations of the micronuclei and chromosomes sorted from their respective peaks. This was clear for peak number 3, which exhibited fluorescent markers both in micronuclei and in chromosomes. These markers, called chromocenters, were connected with the nucleoli in micronuclei and were specific for the nucleolar organizer regions of the X chromosomes. As we paid particular attention to maintaining the transcriptional activity of the micronuclei throughout the procedure used to sort and identify them, it is concluded that such an approach might be very suitable for investigating the expression of reduced nuclear domains corresponding to individual chromosomes.

Animals↗
PubMed1986

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells.

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK1 cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In 'early restoration' cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK1 cells in G1 and those in G2/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1-2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.

Animals↗
PubMed1986

A protein of Mr 80,000 is associated with the nucleolus organizer of human cell lines.

A rabbit serum which had previously been reported to have an immunological affinity for centrosomes of human cell lines was shown also to be specific for the nucleus. Optical and ultrastructural immunolocalization in HeLa cells showed that this specificity is restricted to the fibrillar centre of nucleoli either in untreated or actinomycin D treated interphase cells. In mitotic cells discrete labelling was observed on chromosomes and shown to correspond, on spread metaphase plates, to the short arms of acrocentric chromosomes, i.e. to the nucleolar organizer regions (NORs). Using independent cell fractionation procedures in the human T-lymphoblastic KE 37 cell line and purification of immunoglobulins by affinity to antigens detected by electrophoresis and blotting, a strict correlation between immunoreactive proteins and cytological staining was established. The nucleolar specificity was shown to correspond to a protein with an Mr of 80,000 while the centrosomal specificity corresponded principally to a protein doublet of 60,000-65,000. These antigens share common epitopes as shown by the staining of both NOR and centrosome by immunoglobulins purified by affinity to either type of protein.

Animals↗