Biomedical subjects

R Foisner

Publications and source records attributed to R Foisner.

At least 37 records · Page 2Linked to original sources

PubMed1997

Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells.

The intermediate filament-binding protein plectin and cytokeratin were localised at the cellular periphery of fully polarised Madin-Darby canine kidney (MDCK) cells, whereas vimentin was primarily found in a perinuclear network. Confocal and immunoelectron microscopy revealed that plectin was restricted to areas underlying the lateral plasma membrane. It colocalised with fodrin, a component of the submembrane skeleton, and was closely associated with desmosomal plaque structures. Biochemically, plectin was shown to interact directly with immunoprecipitated desmoplakin in vitro. Upon loss of cell polarity in low calcium medium, plectin redistributed to a cytoplasmic vimentin- and cytokeratin-related network, clearly distinct from diffusely distributed fodrin and internalised desmoplakin structures. The structural reorganisation of plectin was also reflected by an increased solubility of the protein in Triton X-100/high salt, and a decrease in its half-life from approximately 20 to approximately 5 hours. Furthermore, unlike cytokeratins and vimentin, desmoplakin and fodrin did not associate with plectin attached to magnetic beads in cell lysates of unpolarised cells, while all proteins formed a stable complex in polarised cells. Altogether, these data indicate that plectin is involved in the anchorage of intermediate filaments to desmosomes and to the submembrane skeleton in polarised MDCK cells.

Animals↗

PubMed1996

Identification of plectin as a substrate of p34cdc2 kinase and mapping of a single phosphorylation site.

Plectin is an in vitro substrate for various kinases present in cell lysates from mitotic and interphase Chinese hamster ovary cells. Sensitivity of plectin kinase activity to the inhibitor olomoucine, and two-dimensional tryptic peptide mapping of plectin phosphorylated by various kinase preparations suggested that the major plectin kinase activity in mitotic extracts is related to the cell cycle regulator kinase p34cdc2. Bacterial expression of various truncated plectin mutant proteins comprising different domains of the molecule and their phosphorylation by purified p34cdc2kinase revealed that the target site of this kinase resided within plectin's C-terminal globular domain. Among the subdomains of the C-terminal region (six repeats and a short tail sequence), only repeat 6 and the tail were phosphorylated by p34cdc2 kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation site targeted by p34cdc2 kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34cdc2 recognition sequence motif within the repeat 6 domain, threonine 4542 was identified as the major target for the kinase. Protein kinase A, phosphorylating plectin also within repeat 6, targeted sites that were clearly different from those of p34cdc2 kinase.

Animals↗

PubMed1996

M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

Plectin, a widespread and abundant cytoskeletal cross-linking protein, serves as a target for protein kinases throughout the cell cycle, without any significant variation in overall phosphorylation level. One of the various phosphorylation sites of the molecule was found to be phosphorylated preferentially during mitosis. By in vivo phosphorylation of ectopically expressed plectin domains in stably transfected Chinese hamster ovary cells, this site was mapped to the C-terminal repeat 6 domain of the polypeptide. The same site has been identified as an in vitro target for p34cdc2 kinase. Mitosis-specific phosphorylation of plectin was accompanied by a rearrangement of plectin structures, changing from a filamentous, largely vimentin-associated state in interphase to a diffuse vimentin-independent distribution in mitosis as visualized by immunofluorescence microscopy. Subcellular fractionation studies showed that in interphase cells up to 80% of cellular plectin was found associated with an insoluble cell fraction mostly consisting of intermediate filaments, while during mitosis the majority of plectin (> 75%) became soluble. Furthermore, phosphorylation of purified plectin by p34cdc2 kinase decreased plectin's ability to interact with preassembled vimentin filaments in vitro. Together, our data suggest that a mitosis-specific phosphorylation involving p34cdc2 kinase regulates plectin's cross-linking activities and association with intermediate filaments during the cell cycle.

Amino Acid Sequence↗

PubMed1996

Cytoskeleton architecture of C6 rat glioma cell subclones whole mount electron microscopy and immunogold labeling.

Whole mount electron microscopy of extracted cells combined with immunogold labeling techniques can be used to characterize the cytoskeletal architecture of cultured cells. As shown with subclones of the C6 rat glioma cell line, heavy metal shadowing was suitable for getting basic information concerning the arrangement of the various filament types within the networks. Pure carbon shadowing combined with immunogold double labeling proved to be optimal to identify linkages between filaments, to localize filament associated proteins and to follow the arrangement of filaments in dense arrays such as lamellipodiae and cell margins. Thin connecting filaments which interact with actin as well as with vimentin filaments and can be labeled with antibodies to the intermediate filament associated protein plectin may play a major role in the structural organization of the cytoskeleton of these cells.

Actins↗

PubMed1995

Integral membrane proteins associated with the nuclear lamina are novel autoimmune antigens of the nuclear envelope.

We have analyzed sera from 55 patients, most with rheumatic diseases, that all react with the nuclear envelope of human cells in immunofluorescence microscopy. The molecular targets of these autoantibodies were characterized by immunoblot analysis of fractions derived from rat liver nuclear envelopes. While numerous sera were found to react with previously characterized autoimmune antigens of the nuclear envelope including nuclear lamins and the pore complex glycoprotein gp210, a substantial number of the sera were found to recognize relatively minor integral membrane proteins of the nuclear envelope associated with the nuclear lamina (LAP 1A and LAP 2), which have not been previously identified as autoantigens. Autoantibodies to LAP 1A and LAP 2 are present in 9 and 29% of the patient sera, respectively. Only autoantibodies to lamins A/C were encountered more frequently (in 31% of the sera) than autoantibodies to LAP 2, suggesting that LAP 2 may be among one of the most prominent autoantigens of the nuclear envelope in rheumatic disease patients. Since recent studies have suggested that LAP 1A and LAP 2 may be involved in attaching lamins and chromosomes to the nuclear envelope, these findings could promoted understanding of nuclear envelope functions as well as autoimmunity.

Autoantibodies↗

PubMed1994

Integral membrane proteins and dynamic organization of the nuclear envelope.

The nuclear envelope is a complex structure consisting of nuclear membranes, nuclear pore complexes and lamina. Several integral membrane proteins specific to the nuclear pore membrane and the inner nuclear membrane are known. Pore membrane proteins are probably important for organization and assembly of the nuclear pore complex, while proteins of the inner nuclear membrane are likely to play major roles in the structure and dynamics of the nuclear lamina and chromatin. Biochemical studies are now identifying potential binding partners for some of these integral membrane proteins, and analysis of nuclear envelope assembly at the end of mitosis is providing important insights into their functions.

Journal Article↗

PubMed1994

A panel of monoclonal antibodies to rat plectin: distinction by epitope mapping and immunoreactivity with different tissues and cell lines.

A panel of twelve monoclonal antibodies (mAbs) to plectin was analyzed to localize molecular epitopes and assess their utility for various immunotechniques. Based on staining patterns obtained by immunoblotting of proteolytic plectin fragments five groups of mAbs with spatially closely linked epitopes were distinguished. In combination with previous results obtained with recombinant mutant proteins, the epitopes of all mAbs could be shown to reside within three separated domains of plectin's rod domain. Immunoblot analyses of several different rat tissues and a number of cultured cell lines derived from various species, including rat, hamster, cow, mouse and man, revealed considerable variations in immunoreactivity of antibodies. Differences in mAb immunoreactivities were also revealed by immunofluorescence microscopy of different tissues and cultured cell lines. One third of the mAbs examined produced staining patterns that were indistinguishable from those generated by conventional rabbit antisera, confirming the widespread and highly divers cellular localization of plectin previously reported. Microinjection of several monoclonal antibodies into Rat 1 cells had no detectable effects on the structure and organization of plectin arrays or of other cytoskeletal filaments. This new collection of mAbs provides a more reliable tool for histological studies than previously available antisera and should be useful for studies of tissue and cell type specific functions of plectin domains in vivo and in vitro.

Animals↗

PubMed1993

Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation.

Lamina-associated polypeptides (LAPs) 1A, 1B, 1C, and 2 are integral membrane proteins of the nuclear envelope associated with the nuclear lamina. Using in vitro assays, we show that LAPs 1A and 1B specifically bind to both lamins A and C and lamin B1, while LAP 2 associates only with lamin B1. LAP 2 also binds to mitotic chromosomes. The LAPs are phosphorylated during mitosis, and phosphorylation of LAP 2 by mitotic cytosol inhibits its binding to both lamin B1 and chromosomes. During late anaphase, LAP 2 associates with chromosomes prior to assembly of most lamins. Together, these data suggest that LAP 2 may have a key role in initial events of nuclear envelope reassembly, and that both LAP 2 and LAP 1 may be involved in attaching lamins to the nuclear envelope.

Animals↗

PubMed1991

Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells.

We have previously described a gene delivery system based upon the receptor-mediated endocytosis of DNA complexed with transferrin-polycation conjugates. This delivery system has been found to be very effective for both the internalization and the expression of genetic material in cells that have many transferrin receptors. Upon scrutinization of the parameters involved in this method, which we have termed transferrinfection, we note two important features of the process: the polycation in polycation-transferrin conjugates, as expected, serves to attach the transferrin moiety to the DNA and, in addition, the polycation functions to condense the DNA into a doughnut structure. Electron microscopic analysis of a range of poorly active to highly active transferrinfection samples reveals a strong correlation between DNA condensation and cellular DNA uptake. Furthermore, we demonstrate that the transfection activity of the DNA complex can be increased by addition of free polycation as long as a sufficient quantity of polycation-transferrin conjugates remains in the complex to ensure its binding to the cellular receptor.

Amino Acid Sequence↗

PubMed1991

Protein kinase A- and protein kinase C-regulated interaction of plectin with lamin B and vimentin.

Solid-phase binding assays with protein species purified from cultured rat glioma C6 cells and Ehrlich ascites revealed that plectin bound specifically to lamin B but not to lamins A and C. Lamin B interaction was significantly decreased upon in vitro phosphorylation of either lamin B or plectin with protein kinase A or C. In contrast, phosphorylation of plectin with kinase A increased its binding to vimentin, suggesting a different regulation of plectin interactions by this kinase. 32P-radiolabeling of rat glioma C6 cells revealed plectin as a major in vivo target of protein kinase A and protein kinase C. Plectin, present in lysates of dibutyryladenosine 3',5'-cyclic monophosphate-treated cells, showed a 2.5 times higher binding affinity to vimentin than plectin from phorbol ester-treated cells. Furthermore, the relative amounts of plectin in 1% Triton X-100/high salt-insoluble cell fractions decreased to one-fourth of control values upon treating cells with phorbol esters, whereas vimentin was unaffected. This finding suggested a protein kinase C-dependent weakening of plectin interaction with intermediate filaments in vivo. Taken together, these results point to a role of plectin in interlinking cytoskeletal and nuclear elements and suggest that specific protein kinases are involved in regulating these interactions.

Animals↗

PubMed1991

Intermediate filament-associated proteins.

The term intermediate filament-associated proteins refers to a growing number of proteins whose ability to interact with intermediate filaments has been either directly demonstrated or inferred from indirect evidence. Here we discuss recently published data on the identification and characterization of such proteins, with emphasis on their tissue/cell type-specific expression, subcellular distribution and possible function(s).

Cell Membrane↗

PubMed1991

Monoclonal antibody mapping of structural and functional plectin epitopes.

To map structural and functional epitopes of the cytomatrix protein plectin, a set of mAbs was prepared by immunization of mice. Using immunoblot analysis of plectin fragments obtained after limited digestion with various proteases, two groups of mAbs were distinguished. The epitopes of one group (1) were located on a 130-kD terminal segment of the plectin 300-kD polypeptide chain, whereas those of the other group (2) bound within a 40kD segment confined to a central domain of the polypeptide chain. Domains containing the epitopes of group 2 mAbs were shown to include in vitro phosphorylation sites for kinase A, whereas kinase C phosphorylation sites were found on the same terminal segment that contained group 1 mAb epitopes. Rotary shadowing EM of mAb (Fab fragment) -decorated plectin molecules at various states of aggregation, ranging from characteristic dumbbell-shaped single molecules to highly complex multimeric structures, revealed that the epitopes of group 1 as well as those of group 2 mAbs were located on plectin's roughly 200-nm long rod domain interlinking its two globular end domains. Epitopes of group 1 mAbs were localized within a region near the center of the rod, those of group 2 in more peripheral sections near the globular end domains. Solid-phase binding assays carried out in the presence of Fab fragments of mAbs demonstrated an interference of certain group 1 mAbs in the interactions of plectin with vimentin and lamin B. On the other hand, plectin's self-interaction was inhibited mainly by Fab fragments with epitopes in the peripheral rod domain (group 2 mAbs). Together, these results suggested that the molecular binding sites of plectin for vimentin and lamin B, as well as the phosphorylation sites for kinase C, were confined to a defined central section of plectin's rod domain. In addition, they suggest an involvement of peripheral rod sections in plectin self-association.

Animals↗

PubMed1991

Immunolocalization and molecular properties of a high molecular weight microtubule-bundling protein (syncolin) from chicken erythrocytes.

A protein of apparent molecular weight 280,000 (syncolin), which is immunoreactive with antibodies to hog brain microtubule-associated protein (MAP) 2, was purified from chicken erythrocytes. Immunofluorescence microscopy of bone marrow cells revealed the presence of syncolin in cells at all stages of erythrocyte differentiation. In early erythroblasts syncolin was diffusely distributed throughout the cytoplasm. At later stages it was found along microtubules of the marginal band, as confirmed by immunoelectron microscopy. The association of syncolin with the marginal band was dependent on the integrity of microtubules, as demonstrated by temperature-dependent de- and repolymerization or marginal band microtubules. Syncolin cosedimented in a saturable manner with microtubules assembled in vitro, and it was displaced from the polymer by salt. Brain as well as erythrocyte microtubules, reconstituted with taxol from MAP-free tubulin and purified syncolin, were aggregated into dense bundles containing up to 15 microtubules, as determined by electron microscopy. On the ultrastructural level, syncolin molecules were visualized as globular or ringlike structures, in contrast to the thin, threadlike appearance of filamentous MAPs, such as brain MAP 2. According to ultrastructural measurements and gel permeation chromatography, syncolin's molecular weight was approximately 1 x 10(6). It is suggested that syncolin's specific function is the cross-linking of microtubules in the marginal band and, by implication, the stabilization of this structure typical for nucleated (chicken) erythrocytes.

Animals↗

PubMed1988

Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins.

The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.

Animals↗

PubMed1988

Penetrability of White Rot-Degraded Pine Wood by the Lignin Peroxidase of Phanerochaete chrysosporium.

The penetration of enzymes into wood cell walls during white rot decay is an open question. A postembedding immunoelectron microscopic technique was the method of choice to answer that question. Infiltration of pine wood specimens with a concentrated culture filtrate greatly improved the labeling density and, thereby, reproducibility. Characterization of the concentrated culture filtrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) revealed three closely spaced proteins of molecular weights about 42,000 showing immunoreactivity against anti-lignin peroxidase serum. It was shown by immunogold labeling that lignin peroxidase of Phanerochaete chrysosporium is located on the surface of the wood cell wall or within areas of heavy attack. It did not diffuse into undecayed parts of the cell wall. The reasons for preventing lignin peroxidase from penetrating wood cell walls during white rot decay are discussed.

Journal Article↗

PubMed1987

Structure and hydrodynamic properties of plectin molecules.

Plectin is a cytoskeletal, high molecular weight protein of widespread and abundant occurrence in cultured cells and tissues. To study its molecular structure, the protein was purified from rat glioma C6 cells and subjected to chemical and biophysical analyses. Plectin's polypeptide chains have an apparent molecular weight of 300,000, as shown by one-dimensional sodium dodecyl sulfate/polyacrylamide electrophoresis. Cross-linking of non-denatured plectin in solution with dimethyl suberimidate and electrophoretic analyses on sodium dodecyl sulfate/agarose gels revealed that the predominant soluble plectin species was a molecule of 1200 X 10(3) Mr consisting of four 300 X 10(3) Mr polypeptide chains. Hydrodynamic properties of plectin in solution were obtained by sedimentation velocity centrifugation and high-pressure liquid chromatography analysis yielding a sedimentation coefficient of 10 S and a Stokes radius of 27 nm. The high f/fmin ratio of 4.0 indicated a very elongated shape of plectin molecules and an axial ratio of about 50. Shadowing and negative staining electron microscopy of plectin molecules revealed multiple domains: a rigid rod of 184 nm in length and 2 nm in diameter, and two globular heads of 9 nm diameter at each end of the rod. Circular dichroism spectra suggested a composition of 30% alpha-helix, 9% beta-structure and 61% random coil or aperiodic structure. The rod-like shape, the alpha-helix content as well as the thermal transition within a midpoint of 45 degrees C and the transition enthalpy (168 kJ/mol) of secondary structure suggested a double-stranded, alpha-helical coiled coil rod domain. Based on the available data, we favor a model of native plectin as a dumb-bell-like association of four 300 X 10(3) Mr polypeptide chains. Electron microscopy and turbidity measurements showed that plectin molecules self-associate into various oligomeric states in solutions of nearly physiological ionic strength. These interactions apparently involved the globular end domains of the molecule. Given its rigidity and elongated shape, and its tendency towards self-association, plectin may well be an interlinking element of the cytoskeleton that may also form a network of its own.

Animals↗

PubMed1986

Molecular aspects of MAP-1 and MAP-2: microheterogeneity, in vitro localization and distribution in neuronal and nonneuronal cells.

We have studied various aspects of MAP-1 and MAP-2 from neuronal as well as nonneuronal sources. MAP-1 and MAP-2 polymerized from brain were resolved into a number of subcomponents upon electrophoresis on low percentage gels. Based on peptide mappings performed under a variety of different conditions, we conclude that the three major subcomponents of MAP-1 have very similar, though not identical structures. The two major MAP-2 subcomponents might have identical structure, because their peptide maps were hardly distinguishable. The apparent microheterogeneity of high Mr MAPs is not yet understood on a molecular basis. Proteolysis during isolation or a different degree of phosphorylation, however, seems to be an unlikely cause for microheterogeneity. When localized on microtubules polymerized in vitro by electron microscopy, both MAP-1 and MAP-2 polypeptides apparently form helical arrays on the polymer's surface with periodicities of 100 nm. In the presence of taxol, MAPs form irregular and bulky extensions. Both MAPs are found to be widespread in neuronal as well as nonneuronal cells. MAP-1- and MAP-2-related polypeptides, together with other high Mr proteins, such as plectin, were associated with microtubules polymerized by taxol from extracts of a nonneuronal cultured cell line. MAP-2 from cultured cells was found to be extremely sensitive to proteolysis, in particular in the presence of free Ca-ions. MAP-1 and MAP-2 generally were found associated with typical microtubule structures such as interphase and spindle microtubules and primary cilia. A differential distribution of MAP-1 and MAP-2 was clearly evident in neural tissues, where MAP-2 was restricted to cell bodies and dendrites, whereas MAP-1 was present also in axons. Moreover, a differential distribution of MAPs and tubulin was observed in de-and regenerating peripheral nerve, and in a few occasions, also with nonneuronal cells. A quite unexpected result was the identification of a protein in the extracellular matrix of cultured fibroblast cells, which has antigenic determinants in common with MAP-1 and MAP-2 from brain. As a whole, the data presented support a concept in which a family of structurally homologous, though not identical, high Mr polypeptides constitute the crosslinking elements between microtubules and various other cellular components. The structural diversity of these polypeptides might play a role in the development and dynamic changes in the cytoskeletal architecture.

Animals↗

PubMedDate not supplied

Distribution and ultrastructure of plectin arrays in subclones of rat glioma C6 cells differing in intermediate filament protein (vimentin) expression.

Histochemical and biochemical studies suggest that the functions of the intermediate filament (IF) binding protein plectin comprise the physical linkage of IFs to each other and to other cytoskeletal elements, and their anchorage at membrane-attached junctional complexes. To further evaluate this hypothesis the expression, cellular distribution, and ultrastructure of plectin arrays were studied in rat glioma C6 cell subclones differing in IF protein (vimentin) expression. Here we show that plectin is expressed in a vimentin-negative C6 cell subclone (C6-D10) at levels similar to those of the vimentin-positive control subclone C6-D8. However, the amount of cytoskeleton-associated plectin found after extraction of cells with Triton X-100 or Triton X-100/high salt was significantly reduced in IF-negative compared to IF-positive cells. Using immunofluorescence microscopy, plectin structures were detected throughout the cytoplasm of IF-deficient cells. Unlike in IF-containing cells, where plectin colocalized largely with the vimentin network, in the IF-negative subclone the protein was mainly associated with polymeric actin structures. The release of plectin from IF-deficient cytoskeletons upon treatment with heavy meromyosin argued for specificity of the plectin microfilament interaction. Whole mount electron microscopy in conjunction with immunogold labeling of cytoskeletons revealed that in both IF-positive and IF-negative cells, plectin label specifically associated with thin (3-nm) filamentous structures that were clearly distinct from the major cytoskeletal filament systems. In IF-containing cells these filaments were found to link IFs to actin filaments and to connect vimentin filaments to each other. In IF-deficient cells, filamentous plectin structures were found to form dense cytoplasmic networks together with actin filaments and actin filament bundles. These data support the hypothesis that filamentous plectin arrays play an important role in the structural organization and mechanical integration of the cytoskeleton, in particular IFs and microfilaments.

Actin Cytoskeleton↗