Nuclear Matrix-intermediate Filament Research Articles

Phylogenetic occurrence of coiled coil proteins: implications for tissue structure in metazoa via a coiled coil tissue matrix.

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.

Nuclear matrix-intermediate filament proteins of the dental follicle/enamel epithelium and their changes during tooth eruption in dogs

Tooth eruption activates a localized resorption and formation of alveolar bone and these activities depend upon the adjacent parts, coronal and basal, respectively, of the dental follicle-enamel epithelium. In this study the nuclear matrix-intermediate filament (NM-IF) proteins of these tissues were isolated in order to continue investigations into the molecular mechanisms underlying eruption. Dental follicles were removed from the third and fourth premolar of dogs at 13, 16 and 20 weeks (pre-, early, and mid-to-late eruption of these teeth) and NM-IF proteins were extracted from the coronal and basal halves. Most of the NM-IF protein profiles of these coronal and basal parts on one-dimensional, sodium dodecyl sulphate-polyacrylamide gel electrophoresis were remarkably constant, indicating an essentially uniform cellular composition. However, differences between these tissues were observed and some of these changed during eruption. Based on recent observations that nuclear matrix changes reflect and may even mediate cell-specific changes in gene expression, these findings suggest that changes in nuclear matrix proteins may be related to the molecular basis for some aspects of differential gene expression in the coronal and basal regions of the dental follicle and account for the ability of these tissues to activate bone resorption and formation during tooth eruption.

Interference image analysis of heat-shocked HeLa cells.

HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.

Prompt heat-shock and heat-shifted proteins associated with the nuclear matrix-intermediate filament scaffold in Drosophila melanogaster cells

Elevated temperatures induced the synthesis of several new proteins in Drosophila melanogaster cells. Besides the conventional heat shock (HS) proteins, another set of temperature-induced proteins has been found. These latter resemble the prompt HS proteins of mammalian cells. The prompt HS proteins of Drosophila differ from the well-known conventional HS proteins in the following properties: (1) synthesis of the prompt HS proteins is insensitive to the transcription inhibitor actinomycin D, which blocks the appearance of conventional HS proteins; (2) induction of the prompt HS proteins requires a significantly higher temperature than conventional HS proteins; (3) prompt HS proteins associate strictly with the nuclear matrix-intermediate filament complex (NM-IF), while the conventional HS proteins are found in all subcellular fractions; (4) prompt HS proteins of Drosophila are induced by high temperature alone while the conventional HS proteins are also produced by a variety of stress conditions. Resinless-section electron micrographs show an altered nuclear matrix morphology in heat-shocked cells. The nuclear matrix fibers are altered in spatial distribution and have much additional electron-dense material. This added material probably reflects the soluble proteins shifted into the nuclear matrix at high temperature. The prompt HS proteins can be distinguished clearly from heat-shifted proteins by several criteria. Also, the prompt HS proteins are distinct from the heat-insensitive viral proteins of a persistent virus (HPS-1).

Cellular distribution of cholesterogenesis-linked, phosphoisoprenylated proteins in proliferating cells

A set of isoprenylated proteins has been detected in rapidly proliferating, suspension-grown murine lymphoma cells. Our evidence indicates that all of these isoprenylated proteins are phosphorylated. Subsequent to a 24 h incubation with mevinolin to deplete the intracellular mevalonate (MVA) level, cells were incubated with [ 3H]MVA and/or 32P i and both total cell and subcellular fraction proteins were resolved via 1- and 2-D gel electrophoresis, then assessed via subsequent autoradiography. The phospho-isoprenylated proteins comprise a set spanning a molecular mass range of 21–69 kDa and all dispay acidic p I. MVA-derivatized proteins of 21–24 kDa, which consist of multiple isoforms, are present in both cytosolic and nuclear fractions. Larger phospho-isoprenylated protein species (44–69 kDa) are specifically localized within the nucleus, where applicable extraction protocols indicate that they are part of or closely affiliated with the nuclear matrix-intermediate filament (NM-IF) components. The localization of the 69 kDa prenylated species within the NM-IF fraction, together with evidence of its phosphorylation, supports recent indications that this protein is the nuclear matrix component lamin B.

Incorporation of a product of mevalonic acid metabolism into proteins of Chinese hamster ovary cell nuclei.

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. The more basic 74-kD isoprenylated nuclear protein is similar in molecular mass and isoelectric pH variants to the lamin A precursor polypeptide reported by others. Starving cells for mevalonate results in a dramatic accumulation of a polypeptide that comigrates on two-dimensional, non-equilibrium pH gradient electrophoresis (NEPHGE) gels with the 74-kD isoprenylated protein. The 70-kD isoprenylated protein, which is resolved on NEPHGE gels as being higher in molecular mass and slightly more basic than lamin B, has not yet been identified.

Nuclear matrix-intermediate filament system and its alteration in adenovirus infected Hela cell

The nuclear matrix-intermediate filament (NM-IF) of normal and adenovirus infected Hela cells were investigated by means of both electron microscopy and two-dimensional gel electrophoresis. After infection there were some changes in NM-IF, and the viral factory was suspended in nuclear matrix. Two new polypeptides appeared in 2-D gel of NM-IF after infection. They are probably not viral proteins but cellular polypeptides. During viral replication, all of vimentin were degraded and phosphorylated keratin 18 increased. These facts suggest that NM-IF plays a certain role in adenovirus replication.

The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy.

The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged. Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E. G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles.(ABSTRACT TRUNCATED AT 400 WORDS)

An investigation of the molecular components of desmosomes in epithelial cells of five vertebrates

We have shown previously, by fluorescent antibody staining, that desmosomal antigens are widely distributed in the tissues of vertebrate animals. Furthermore, we have demonstrated mutual desmosome formation between cells derived from man, cow, dog, chicken and frog. In this paper we have studied the components of desmosomes in a tissue or a cell line from each of these animals by immunoblotting with antibodies raised against the desmosomal components isolated from bovine nasal epithelium. Blotting was carried out on bovine nasal epithelial desmosomal cores, desmosome-enriched fractions derived from chicken and frog epidermis, nuclear matrix-intermediate filament scaffolds derived from Madin-Darby bovine and canine cells (MDBK and MDCK), and unextracted cultured human foreskin keratinocytes. The results show that desmosomes from all these sources contain high molecular weight proteins (desmoplakins) of similar or identical molecular weights (250 000 and 215 000). Antibodies against the two lower molecular weight desmosomal proteins (83 000 and 75 000) always recognized one or two bands in very similar molecular weight regions of the gels. The desmosomal glycoproteins were found to be much more variable than the proteins: they vary between sources in molecular weight, heterogeneity and antibody cross-reactivity. For instance, antibody specific for a group of glycoprotein bands of 175 000, 169 000 and 164 000 (Mr) in bovine nasal epithelium recognizes three bands of 245 000, 230 000 and 210 000 in MDCK cells but only a single band of 190 000 in keratinocytes. In mammals, the 175 000-164 000 glycoproteins and the desmosomal adhesion molecules, the desmocollins (Mr 130 000 and 115 000 in cow's nose), are immunologically distinct. In chicken and frog, however, there are glycoproteins that react with both anti-175 000-164 000 and anti-desmocollin antibodies, but there are also distinct desmocollin bands. The significance of these results is discussed in relation to conservation of desmosomal components and adhesion mechanisms. It is suggested that adhesion may be performed by a well-conserved protein domain and that the variation between desmosomal glycoproteins from different sources may be due to differences in their carbohydrate composition.

Shape-dependent regulation of cytoskeletal protein synthesis in anchorage-dependent and anchorage-independent cells.

We examine changes in protein synthesis that accompany suspension (i.e. shape alteration) of anchorage-dependent and anchorage-independent cells using a newly developed cell fractionation procedure based on detergent extraction. Using this procedure, a cell can be divided into four distinct and independent fractions: soluble, cytoskeleton, chromatin and nuclear matrix-intermediate filament. This fractionation procedure is used to investigate protein synthetic events associated with the release from anchorage-dependent growth, characteristic of transformed cells. Suspension results in several unexpected events in both anchorage-dependent (3T3) cells and anchorage-independent (SVPy 3T3) cells. Suspension of 3T3 cells results in a reduction of total protein synthesis; however, two proteins are enhanced in their amount of synthesis. Suspension of SVPy 3T3 results in about 20 proteins proceeding through short and long-term alterations in the rate of synthesis. The synthesis of some of these proteins is inhibited, others undergo a transient decrease in synthesis upon suspension and then increase above their rate in the anchored state, while the synthesis of others steadily increases after suspension. Suspension of anchorage-dependent cells results in a fraction specific shift in the rates of protein synthesis. Possible roles for these fraction-specific proteins are considered.

Tumor promoters induce a specific morphological signature in the nuclear matrix-intermediate filament scaffold of Madin-Darby canine kidney (MDCK) cell colonies.

Tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA), mezerein, teleocidin, aplysiatoxin, and benzoyl peroxide, although structurally unrelated, induce similar, profound changes in morphology in differentiated epithelial Madin-Darby canine kidney (MDCK) cell colonies. The alteration is evident in the organization of intermediate filaments in intact cells and in whole mounts of the nuclear matrix-intermediate filament (NM-IF) scaffold of the epithelial sheet. This substructure, obtained by salt extraction of the cytoskeletal framework, represents only 5% of the total cell protein but contains all of the intermediate filaments, nuclear matrix, and desmosomal core proteins arranged essentially as in the intact cell. The NM-IF is profoundly reorganized after exposure to TPA and retains the morphological changes observed in intact cells. These include bundling of the intermediate filaments, disruption of cell-cell borders, and marked deformation of the polygonal geometry of epithelia. Thus, TPA and all other complete or second-stage tumor promoters examined have a characteristic morphological signature that is not induced by mitogens, metabolic inhibitors, or agents known to disrupt microtubules or microfilaments. This signature, characteristic of tumor promoters, occurs in the absence of both protein and RNA synthesis. These results suggest that this response is prior to and independent of other biochemical markers for tumor promoters. Of the major filament systems, the cytokeratin network is implicated as an early or possibly primary site of tumor-promoter action because characteristics of the promoted cytoskeletal signature are observed in epithelial colonies after prior exposure to colchicine or cytochalasin D. Despite the massive reorganization of cytoskeletal morphology induced by TPA, the distribution of prelabeled proteins into structural fractions (i.e., cytoskeletal, chromatin, and the NM-IF) remains essentially unchanged. The sensitivity and specificity of the epithelial cell response suggest its possible use as a screen for promoting compounds.

Epithelial structure revealed by chemical dissection and unembedded electron microscopy.

Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.

Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well-preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold.