Search PubMed⌕ Search

Biomedical subjects

U Scheer

Publications and source records attributed to U Scheer.

At least 37 records · Page 2Linked to original sources
PubMed1995

Localization of the Ran-GTP binding protein RanBP2 at the cytoplasmic side of the nuclear pore complex.

A partial cDNA clone coding for the mouse homologue of the human Ran-GTP binding protein, RanBP2, has been isolated by screening of a murine expression library with antibodies to nup180, a previously identified nuclear pore complex protein (nucleoporin). Whether the antibodies cross-reacted with the polypeptide encoded by the cDNA clone or, alternatively, nup180 is proteolytically related to RanBP2, has not been determined. The 3795-bp open reading frame of the cDNA encodes a polypeptide consisting of 1265 amino acids with three Ran-GTP binding domains (RanBD) that are almost identical with published partial amino acid sequences of human RanBP2 as deduced from several partial cDNA clones of other authors. Sequence analysis further revealed that murine RanBP2 contains tandemly repeated zinc fingers of Cys2-Cys2 type and multiple copies of the FXFG nucleoporin "signature" motif clustered in regions preceding the RanBDs. Antibodies raised against a synthetic peptide of the derived amino acid sequence decorated the cytoplasmic rings of nuclear pore complexes (NPCs) as shown by immunogold electron microscopy. We suggest that the cytoplasmically disposed nucleoporin RanBP2 provides docking sites for import substrate-receptor complexes and, further, that the affinity of these sites to the transport substrate is modulated in a Ran-dependent fashion.

Amino Acid Sequence↗
PubMed1994

The nucleolus.

The transcriptionally active rRNA genes have the remarkable ability to organize and integrate the biochemical pathway of ribosome production into a structural framework, the nucleolus. The past year has seen numerous advances in our understanding of the relationships between nucleolar substructures, the site of ribosomal RNA (rRNA) gene transcription and the pathway of ribosome maturation. Progress has also been made both in the molecular identification of nucleolar constituents and in our understanding of the interactions between these components and their assembly into higher order structures.

Animals↗
PubMed1993

Absence of somatic histone H1 in oocytes and preblastula embryos of Xenopus laevis.

Available data on the occurrence and expression of somatic histone H1 during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone H1A in oocytes is difficult to reconcile with the high transcriptional activity of all gene classes in this specific cell type. In the present study we have used polyclonal antibodies raised against somatic Xenopus histone H1 (H1A and H1A/B) for combined immunoblotting experiments to quantitate H1 pools and immunolocalization studies to visualize chromosome-bound H1. Both approaches failed to detect soluble or chromosomal histone H1 in vitellogenic oocytes, eggs, and cleavage-stage embryos up to early blastula. In addition, chromatin assembled in Xenopus egg extract was also negative for histone H1 as revealed by immunofluorescence microscopy. Lampbrush chromosomes not only lacked histone H1 but also the previously identified histone H1-like B4 protein (Smith et al., 1988, Genes Dev. 2, 1284-1295). In contrast, chromosomes of eggs and early embryos fluoresced brightly with anti-B4 antibodies. Our results lend further support to the view that histone H1 expression is developmentally regulated during Xenopus oogenesis and embryogenesis similar to what is known from other species.

Animals↗
PubMed1993

DNA topoisomerase II is not detectable on lampbrush chromosomes but enriched in the amplified nucleoli of Xenopus oocytes.

In somatic cells DNA topoisomerase II (topo II) is thought to be involved in the domain organization of the genome by anchoring the basis of chromatin loops to a chromosomal scaffold. Lampbrush chromosomes of amphibian oocytes directly display this radial loop organization in cytological preparations. In order to find out whether topo II may play a role in the organization of these meiotic chromosomes, we performed immunofluorescence studies using antibodies against Xenopus topo II. Our results indicate that topo II is apparently absent from lampbrush chromosomes and is hence unlikely to act as a "fastener" of the numerous lateral chromosomal loops. Topo II was, however, enriched in the amplified nucleoli of Xenopus oocytes.

Animals↗
PubMed1993

Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils.

Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of approximately 6.2 which we have designated as nup180. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGA-Sepharose demonstrated that nup180 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nup180 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nup180 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nup180 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.

Animals↗
PubMed1992

The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.

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 (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent 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, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP 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 VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.

Actin Cytoskeleton↗
PubMed1992

In vitro assembly of prenucleolar bodies in Xenopus egg extract.

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.

Animals↗
PubMed1991

Assigning functions to nucleolar structures.

Nucleoli provide the fascinating possibility of linking morphologically distinct structures such as those seen in the electron microscope with biochemical features of the formation and stepwise maturation of ribosomes. Localization of proteins by immunocytochemistry and of rRNA genes and their transcripts by in situ hybridization has greatly improved our understanding of the structural-functional relationships of the nucleolus. The present review describes some recent results obtained by electron microscopic in situ hybridization and argues that this approach has the potential to correlate each step of the complex pre-rRNA maturation pathway with nucleolar structures. Evidence is accumulating that the nucleolus-specific U3 snRNPs (small nuclear ribonucleoprotein particles) participate in rRNA processing events, similar to the role played by the nucleoplasmic snRNPs in mRNA maturation. The intranucleolar distribution of U3 snRNA is consistent with the view that it is involved in both early and late stages of pre-rRNA processing.

Animals↗
PubMed1991

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level.

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 topoisomerase 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.

Animals↗
PubMed1991

Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.

Animals↗
PubMed1990

Functional and dynamic aspects of the mammalian nucleolus.

Nucleoli are the sites of ribosome biogenesis. Transcription of the ribosomal RNA genes as well as processing and initial packaging of their transcripts with ribosomal and non-ribosomal proteins all occur within the nucleolus in an ordered manner and under defined topological conditions. Components of the nucleolus have been localized by immunocytochemistry and their functional aspects investigated by microinjection of antibodies directed against the enzyme responsible for rDNA transcription, RNA polymerase I. The role of nascent transcripts in postmitotic formation of nucleoli will be discussed.

Animals↗
PubMed1990

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro.

We analysed the soluble form in which the nuclear pore complex protein p68 is stored in Xenopus laevis eggs and its involvement in pore complex assembly processes. We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein of nuclear pore complexes from Xenopus oocytes, is located in the pore channel and participates in mediated transport of karyophilic proteins. Using a monoclonal antibody directed against p68 (PI1) we removed this protein from Xenopus egg extract by immunoadsorption. On addition of lambda DNA the immuno-depleted extract supported reconstitution of nuclei which were surrounded by a continuous double-membrane envelope but lacked pore complexes and were unable to import karyophilic proteins such as nucleoplasmin or lamin LIII. Essentially identical results were obtained with extract depleted of WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extract necessary for pore complex assembly but did not interfere with nuclear membrane formation demonstrates that these processes are independent of each other. Analysis of the immunoprecipitate on silver-stained SDS-polyacrylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel filtration we showed that p68 together with associated protein(s) forms a stable, approximately globular complex with an Mr of 254,000, a Stokes radius of 5.2 nm and a sedimentation coefficient of 11.3 S. Our finding that p68 occurs in the form of larger macromolecular assemblies offers an explanation for the distinctly punctate immunofluorescence pattern observed in the cytoplasm of mitotic cells after staining with antibodies to p68.

Animals↗
PubMed1989

Functional role of newly formed pore complexes in postmitotic nuclear reorganization.

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 PtK2 cells with WGA or antibody PI1 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 PtK2 cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophase-like completed cytokinesis, their nuclei showed a telophase-like 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.

Animals↗
PubMed1989

Mapping of transcription units on Xenopus laevis lampbrush chromosomes by in situ hybridization with biotin-labeled cDNA probes.

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 cDNA probes were used encoding the nucleolar protein NO38/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 anti-rabbit IgG or enzymatically using peroxidase-conjugated streptavidin. 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 NO38/B23-nucleoplasmin or the cytokeratin-lamin 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.

Animals↗
PubMed1989

Nucleocytoplasmic sorting of macromolecules following mitosis: fate of nuclear constituents after inhibition of pore complex function.

PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68,000 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 double-layered 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.

Animals↗
PubMed1988

Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription.

RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110,000, 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 (NH4)2SO4. 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.

Animals↗