Biomedical subjects

G Krohne

Publications and source records attributed to G Krohne.

At least 37 records · Page 2Linked to original sources

PubMed1996

An antibody against a glycosylated integral membrane protein of the Xenopus laevis nuclear pore complex: a tool for the study of pore complex membranes.

We have recently described a monoclonal antibody (X222) which localizes at pore complexes in Xenopus oocytes and immunoblots a protein under nonreducing conditions with an apparent molecular mass of 215 kDa (Cordes, Gajewski, Stumpp, Krohne, Differentiation 58, 307-312, (1995)). Under reducing conditions, this antigen decreased from the previously reported 215 kDa to 200 kDa. Since this protein binds to the lectin concanavalin A (Con A), we now refer to it as gp200. In addition to Con A binding, Xenopus gp200 has several other features in common with the mammalian pore membrane protein gp210. On nuclear envelopes extracted with Triton X-100, gp200 was localized on the lateral walls of pores, a subdomain covered in native nuclear envelopes by the lipid bilayer. In mitotic extracts of Xenopus eggs gp200 was exclusively recovered in the membrane fraction. Treatment of oocyte membranes with urea and proteases indicated that gp200 is an integral membrane protein with the bulk of its mass located within the membrane lumen. In Xenopus oocyte nuclei gp200 is the major Con A binding protein. The new monoclonal antibody X222 has allowed us to characterize the first integral membrane protein, gp200, of the Xenopus pore complex.

Animals↗

PubMed1995

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy.

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10203 nm3. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11987 nm3 was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.

Animals↗

PubMed1995

Immunocytochemistry of annulate lamellae: potential cell biological markers for studies of cell differentiation and pathology.

We have generated specific antibodies recognizing different proteins of the nuclear pore complex and have applied them for the biochemical and microscopical analysis of annulate lamellae (AL). Using the example of the Xenopus laevis oocyte we show that the combined application of such antibodies to different components of the nuclear pore complex allows the unequivocal identification of AL. We propose to use such antibodies in immunostaining light microscopy for detecting AL as a diagnostic marker in a diversity of cell types, including those with pathological alterations in AL frequency and distribution.

Animals↗

PubMed1995

Physical association of moesin and CD46 as a receptor complex for measles virus.

Recently, two cellular membrane proteins, the membrane cofactor protein CD46 and the membrane-organizing external spike protein, moesin, have been identified to be functionally associated with measles virus (MV) infectivity of cells. We investigated the functional consequences of binding of monoclonal antibodies to both molecules individually and combined on MV attachment, fusion, and plaque formation and the putative direct physical interaction of moesin and CD46. We found that antibodies to moesin or CD46 separately inhibited MV-cell interactions to a high percentage in the plaque test, by approximately 85 and 75%, respectively. The inhibition by combinations of antibodies was additive at low concentrations and complete at high concentrations. This indicates that similar sites of interaction were blocked by steric hindrance. Furthermore, antimoesin antibodies blocked the infection of CD46-negative mouse cell lines with MV. Chemical cross-linking of cell surface proteins indicated the close proximity of CD46 and moesin in the membrane of human cells, and coimmunoprecipitation of moesin with CD46 suggested their physical interaction. Immunohistochemically by electron microscopy, CD46 and moesin were found to be localized at sites of the cellular membrane where MV particles adsorbed. These data support a model of direct interaction of CD46 and moesin in the cellular membrane and suggest that this complex is functionally involved in the uptake of MV into cells.

Animals↗

PubMed1995

The nuclear membrane-associated honeycomb structure of the unicellular organism Amoeba proteus: on the search for homologies with the nuclear lamina of metazoa.

In the protozoon Amoeba proteus, a complex and highly organized structure with the morphology of a honeycomb is associated with the nucleoplasmic surface of the nuclear membrane. We have tested whether this structure exhibits similarity to the nuclear lamina of metazoic organisms. First, we have shown that the honeycomb layer is composed of 3 to 5 nm thick protein fibrils resistant to treatment with detergent, high salt, and digestion with nucleases, thus possessing properties typical for karyoskeletal elements. However, in contrast to the meshwork of lamin filaments in somatic cells of metazoic organisms, the honeycomb layer is not tightly anchored to the nucleoplasmic side of pore complexes, or to the inner nuclear membrane. Second, in microinjection experiments we investigated whether fluorescently labeled lamins of Xenopus laevis (lamins A and LI) and Drosophila melanogaster (lamin Dmo) were able to associate in vivo with the Amoeba proteus honeycomb structure. In microinjected amoeba these three lamins were efficiently transported into the nucleus, but did not associate with the nuclear envelope. Our results suggest that the Amoeba proteus nuclear envelope, including the honeycomb layer, does not contain proteins exhibiting high homologies to lamins of metazoan species thus preventing the localized assembly of microinjected lamins along the nuclear periphery.

3T3 Cells↗

PubMed1995

In vivo assembly kinetics of fluorescently labeled Xenopus lamin A mutants.

The assembly kinetics of Xenopus wild type lamin A and 7 mutants were analyzed by the microinjection of renatured 5-iodoacetamidofluorescein (5-IAF)-labeled protein into mouse 3T3 cells. This experimental system has the advantage that the masking of the mutant phenotype by the formation of heterooligomeric complexes with newly synthesized lamins of the microinjected cell is negligibly small. The wild type protein and all mutants containing a nuclear localization signal were transported within 10 min into the nucleus. The wild type protein exhibited a strong lamina fluorescence 30 min after microinjection, whereas mutant molecules showed a delayed but complete, a delayed and incomplete, or no lamina assembly at all. The lamin A mutant lacking the carboxy-terminal cysteine of the CxxM-motif exhibited a delayed but complete assembly, whereas previously performed transfection experiments demonstrated no significant interaction of this mutant wih the lamina. According to present knowledge this phenotype in transfected cells can be explained by an overexpression of this mutant. We have identified an additional domain in the carboxy-terminal tail of lamin A that promotes its assembly into the lamina. In vitro this domain is required for the chromatin binding of lamin A. Lamin A molecules lacking the non-helical amino-terminal head domain showed no significant lamina staining, whereas point mutations in conserved regions of the alpha helix resulted in an incomplete assembly.

3T3 Cells↗

PubMed1994

Imaging nuclear pores of aldosterone-sensitive kidney cells by atomic force microscopy.

In nuclei of renal target cells, aldosterone enhances transcriptional activity followed by the translocation of specific RNA molecules across the nuclear envelope. Trafficking between cell nucleus and cytoplasm occurs via nuclear pore complexes (NPCs) located in the double-layered nuclear envelope. We investigated the nucleocytoplasmic transport route by structure-function analysis at subcellular level in quiescent and aldosterone-stimulated cells. With atomic-force microscopy (AFM) we imaged individual pores of the nuclear surface of cultured kidney cells and related the number of pores per micron2 to nuclear envelope conductance (Gn, per micron2) evaluated electrically by current injection into the isolated nucleus. NPCs were equally distributed resembling "donut-like" structures with outer diameters of 134 +/- 12 nm (n = 50), each equipped with a central channel. Six hours of aldosterone exposure (0.1 microM) increased the number of NPCs per micron 2 of nuclear surface from 7.4 +/- 0.4 to 9.8 +/- 0.4 (n = 12; P < 0.01). At the same time Gn rose from 6900 +/- 520 to 9600 +/- 610 pS/micron2 paralleled by an increase of the intranuclear electrical potential from -2.8 +/- 0.2 to -6.2 +/- 0.4 mV (n = 18; P < 0.01). Assuming that NPCs represent the sole conductive pathway in the nuclear envelope, we calculate a mean single NPC conductance of 932 and 980 pS, in the absence and presence of aldosterone, respectively. We conclude that aldosterone facilitates nucleocytoplasmic transport by increasing the number of NPCs but not by modifying their biophysical properties. Possibly, aldosterone controls similar transport mechanisms in both plasma membrane and nuclear envelope.

Aldosterone↗

PubMed1994

Lamin-dependent nuclear envelope reassembly following mitosis: an argument.

During the division of most eukaryotic cells, the nuclear envelope disassembles and subsequently reassembles around the segregated chromosomes to form two new nuclei. With the use of cell-free systems, the molecular mechanisms that underlie this complicated process are beginning to be studied. The importance of nuclear lamins in nuclear envelope reassembly has been controversial for the past five years, but recent findings may help to clarify their role. In this article, we propose a working model for nuclear envelope reassembly.

Journal Article↗

PubMed1994

Mammalian cells transfected with the listeriolysin gene exhibit enhanced proliferation and focus formation.

Mouse 3T6 and 3T3 fibroblasts and rat epithelial L2 cells were transfected with recombinant plasmids containing the listeriolysin gene (hly) of Listeria monocytogenes. This bacterial gene (with and without the 5' signal sequence) was cloned under the control of a murine metallothionein promoter, resulting in elevated transcription of both forms of the hly gene after induction with ZnSO4. However, the gene product could be observed only when the listeriolysin gene lacking the 5' signal sequence was used. Intact listeriolysin could not be detected in the cytoplasm or in the supernatant of the hly-transfected cells. 3T6 and L2 cells transfected with the intact hly gene exhibited significantly increased cell proliferation and increased formation of actin microfilaments upon induction of hly expression with ZnSO4. Both cell types are not contact inhibited and formed large piles of spherical cells after transfection with hly. In contrast, contact-inhibited 3T3 cells transfected with the hly gene showed increased proliferation but no formation of such cell aggregates. When 3T6 fibroblasts were transfected with the hly gene without the 5' signal sequence, inhibition of growth, lack of cell layer confluency, and altered (spherical) cell morphology were observed.

3T3 Cells↗

PubMed1994

Ultrastructural study of Listeria monocytogenes entry into cultured human colonic epithelial cells.

Evidence that Listeria monocytogenes enters Caco-2 cells through the apical surface is presented. Attachment of bacteria to host cells seems to induce modifications of microvilli which are either in direct contact with the bacterial surface or in close vicinity, resulting in the formation of lamellipodia involved in the cellular uptake of the bacteria. Such modifications are not induced by L. monocytogenes SLCC 53, which carries a deletion in the prfA gene, although attachment of this mutant to Caco-2 cells occurs. Listeria innocua does not attach well to Caco-2 cells and also fails to cause structural alterations of the microvilli. Treatment of confluent monolayers of Caco-2 cells with ethylene glycol-bis(beta-aminoethyl ether)- N,N,N1,N1-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly reduced the uptake of Listeria cells. Attachment and invasion of L. monocytogenes was not accompanied by accumulation of filamentous actin around the entering bacterial cell.

Bacterial Adhesion↗

PubMed1994

Properties of fluorescently labeled Xenopus lamin A in vivo.

Wild type Xenopus lamin A, a lamin A mutant lacking the carboxy-terminal cysteine (C662-->S), and human vimentin were expressed in bacteria and fluorescently labeled with 5-iodoacetamidofluorescein (5-IAF). In vitro reconstitution experiments and microinjection of both lamins into living cells revealed that they were indistinguishable from the non-fluorescently labeled proteins. When the 5-IAF lamin A was microinjected into the cytoplasm of 3T3 cells it was rapidly transported into the nucleus, giving rise within 1 h to a strong lamina fluorescence, whereas the lamin A mutant formed dotlike intranuclear aggregates. 5-IAF lamin A associated with the nuclear envelope of microinjected 3T3 cells and 5-IAF vimentin which was incorporated into the preexisting vimentin filaments of this cell line, were analyzed by photobleaching employing two different methods, (i) scanning microphotolysis using a modified laser scanning microscope, and (ii) the conventional photobleaching technique in which the integral fluorescence of a single spot was measured by photon counting. A low but significant fluorescence recovery was measured within 10 min for both 5-IAF-labeled intermediate filament proteins, lamin A and vimentin, in bleached areas of the nuclear envelope and the cytoplasmic intermediate filaments, respectively.

3T3 Cells↗

PubMed1993

Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations.

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Höger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.

Animals↗

PubMed1993

Listeria ivanovii is capable of cell-to-cell spread involving actin polymerization.

Listeria ivanovii has been considered to be pathogenic to animals but has rarely been found associated with human infections. It has been claimed that L. ivanovii lacks the actA gene, which in L. monocytogenes encodes a protein required for interaction with host cell actin. Using fluorescence microscopy and electron microscopy, we demonstrate that L. ivanovii can invade mammalian cells, lyse the phagosomal membrane, polymerize host cell actin, reorganize actin to form tails, and spread from cell to cell. However, no DNA homologous to the actA gene could be detected by polymerase chain reaction. Further, L. ivanovii lacks the 90-kDa surface protein which in L. monocytogenes is encoded by actA. Despite the ability to spread from cell to cell, L. ivanovii differed significantly from L. monocytogenes in being unable to form plaques on monolayers of 3T3 fibroblast cells.

Actins↗

PubMed1993

Presence of a nuclear lamina in pachytene spermatocytes of the rat.

The nuclear lamina is a karyoskeletal structure located at the periphery of cell nuclei. The major constituents are the lamins, which belong to the evolutionarily conserved multigene family of intermediate filament proteins. Lamins show a conspicuous cell type-specific expression pattern. The majority of somatic cells of vertebrates express A-type (lamins A and C) as well as B-type (lamins B1 and B2) lamins. Although a lamina structure has been demonstrated to be a ubiquitous component of somatic nuclei its existence in certain meiotic stages during spermatogenesis has been a matter of debate. In this study, we investigated the expression of lamins in rat spermatogenic cells using immunological and protein-chemical methods. We report on the presence of a nuclear lamina structure in rat pachytene spermatocytes. With the aid of a novel broad-reacting lamin antibody we have demonstrated the expression of a protein that is closely related, if not identical, to lamin B1.

Animals↗

PubMed1993

Sequential O-glycosylation of nuclear pore complex protein gp62 in vitro.

Gp62 is a nuclear pore complex glycoprotein of vertebrates containing multiple O-linked N-acetylglucosamine monosaccharides. We have recently shown (Cordes et al., Eur. J. Cell Biol. 55, 31-47 (1991)) that gp62 of mouse and Xenopus laevis is predominantly glycosylated in the amino-terminal half of the molecule. Here we describe in vitro glycosylation in rabbit reticulocyte lysate of gp62 and of individual segments of this protein expressed as recombinant polypeptides in E. coli. Competition experiments between purified polypeptides revealed that gp62 exhibits in vitro at least two types of binding sites for a cytosolic N-acetylglucosaminyltransferase resulting in a sequential glycosylation of the protein. High affinity binding sites for this glycosyltransferase were located in the amino-terminal domain between amino acids 248-341 of mouse gp62, whereas low affinity binding sites were scattered in the entire amino-terminal half. The segment containing amino acids 248-341 was able to compete with in vitro O-glycosylation of gp62 subdomains containing low affinity binding sites. This gp62 segment was also able to compete with O-glycosylation of other unrelated cellular proteins in vitro.

Amino Acid Sequence↗

PubMed1991

Interaction of Xenopus lamins A and LII with chromatin in vitro mediated by a sequence element in the carboxyterminal domain.

Morphological data suggest an interaction of the nuclear lamina with chromatin which markedly changes during the cell cycle. To study the molecular basis of this interaction we developed a novel lamin/chromatin binding assay that quantitated the binding of soluble, radiolabeled lamins to minichromosomes assembled in Xenopus laevis oocyte nuclear extracts. Lamins were derived from couple in vitro transcription and translation of the corresponding cDNAs. Chromatin binding was detected by monitoring the cofractionation with assembled minichromosomes in gel filtration and sucrose gradient centrifugation. Binding of lamins to chromatin increased with chromatin concentration and was accompanied by lamin polymerization. Lamins of the A-(Xenopus LA and human LC) as well as the B-type (Xenopus LI and LII) showed strikingly different chromatin binding capacities. Lamins A and LII bound efficiently of lamins LI and LC was detected. Using site-directed mutagenesis, we were able to define carboxy-terminal sequence elements of LA and LII required for the observed lamin/chromatin interaction that are rich in serine, threonine, and glycine residues. Competition experiments with a synthetic peptide containing the chromatin binding motif of lamin A corroborate the importance of these sequence elements in the lamin/chromatin interaction.

Amino Acid Sequence↗

PubMed1991

Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos.

To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.

Animals↗

PubMed1991

Gene structure and chromosomal localization of the murine lamin B2 gene.

The structure of the murine lamin B2 gene has been analyzed by cloning, sequencing and hybridization techniques, including in situ hybridization. The gene exists in single copy on the distal arm of chromosome 10 and comprises at least 15 kb, containing 12 exons and 11 introns. The transcriptional start point, as determined by primer extension analysis and RNase protection, was mapped to the region -264 to -254 upstream the ATG start codon. The 5' upstream region does not reveal any classical TATA box elements but typical features of genes encoding "housekeeping" proteins. The intron pattern is strikingly similar to those of the Xenopus laevis lamin LIII (Döring, V., R. Stick, EMBO J. 9, 4073-4081 (1990)) and of the intermediate filament protein of the invertebrate Helix aspersa (Dodemont, H., D. Riemer, K. Weber, EMBO J. 9, 4083-4094 (1990], particularly in the central rod and in the tail domains. Moreover, this lamin gene contains an additional intron in the region encoding the rod domain. Our data are compatible with the evolutionary hypothesis that IF proteins have evolved from a lamin-like ancestor molecule.

Amino Acid Sequence↗