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PubMed1983

"Prompt" heat shock proteins: translationally regulated synthesis of new proteins associated with the nuclear matrix-intermediate filaments as an early response to heat shock.

The response of mammalian cells, such as HeLa cells, to prolong exposure to increased temperature (termed heat shock) has been well characterized. In these studies new mRNA is synthesized for several proteins whose translation is best seen after a return to 37 degrees C. We show here another response to increased temperature of a distinctively different character. A set of at least 50 newly detectable proteins, exclusively associated with the nuclear matrix-intermediate filaments (NM-IF) fraction, is synthesized immediately upon exposure to high temperature. These are of very low abundance or nonexistent in the unstressed cell and none appear to correspond to the "classic" heat shock proteins produced after new transcription. Prior treatment with actinomycin D has little effect on these "prompt" proteins, and they appear to be made from preexisting mRNAs that are activated at the increased temperature. The protein synthesis in the soluble, cytoskeletal, and chromatin fractions is strongly reduced by the increased temperature, while the labeling of the prompt proteins associated with NM-IF complex rapidly rises severalfold above that in control cells. Additionally these results suggest that the four cell fractions are not arbitrary cell divisions; rather they represent physiologically significant compartments in the cell.

Autoradiography↗
PubMed1990

Neuronal intermediate filaments in rat dorsal root ganglia: differential distribution of peripherin and neurofilament protein immunoreactivity and effect of capsaicin.

Two major neuronal populations were revealed in rat dorsal root ganglia, immunoreactive for either peripherin, or neurofilament triplet proteins (adult L2 ganglia: 66.2% and 25.6%, mainly small and large diameter cells, respectively), together with a minor, double-immunostained population (L2: 8.1%, mainly intermediate-size neurons). After capsaicin treatment, a striking expansion in the latter population was seen (L2: 22.0%) together with a significant increase in size, restricted to the same population and the (remaining) peripherin-only immunoreactive neurons. Calcitonin gene-related peptide (CGRP) immunoreactivity was revealed in neurons of all 3 groups, in both normal and capsaicin-treated rats.

Animals↗
PubMed2003

Keratin 20 helps maintain intermediate filament organization in intestinal epithelia.

Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80-->His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia.

Amino Acid Sequence↗
PubMed2001

Molecular interaction between human tumor marker protein p150, the largest subunit of eIF3, and intermediate filament protein K7.

The human tumor marker protein p150 was identified as the largest subunit of eukaryotic translation initiation factor 3 (eIF3) (also known as p170/p180). Its expression level is not only upregulated in many transformed cell lines, but also in several human cancers including breast, cervical, esophageal, and stomach carcinomas. The function of p150 in cancer and initiation of translation are not well understood. Using the yeast two-hybrid genetic screen, we found that a portion of p150 interacts with hPrt1, another subunit of eIF3, and cytokeratin 7, an intermediate filament protein. The interactions between p150 and hPrt1, and between p150 and cytokeratin 7 were verified both in vivo and in vitro. The interaction site for hPrt1 was mapped to the carboxyl half of the coiled-coil region of the p150 protein between amino acids 664-835. The expression of hPrt1 was clearly upregulated in cancer tissue, similarly to that of p150. By contrast, no substantial difference in the expression level of cytokeratin 7 was observed between cancer and normal breast tissue, suggesting that cytokeratin 7 expression is not co-regulated with p150. Taken together, our studies suggest a new role for p150 in translation initiation, possibly by acting as an adapter molecule between the translation initiation apparatus and the cytoskeleton structure in the cell.

Binding Sites↗
PubMed1983

Involvement of Tetrahymena intermediate filament protein, a 49K protein, in the oral morphogenesis.

To study the biological function of Tetrahymena intermediate-type filament protein (a 49K protein), we examined the immunofluorescence localization of 49K protein within Tetrahymena cells. The results showed that the immunofluorescence was localized in the oral apparatus, mitochondria and mucocysts. Among them, the fluorescence in the oral apparatus was of high interest in its unique region and vicissitude in the cell cycle: a tau-shaped region of the oral apparatus intensely fluoresced during interphase, but the fluorescence completely disappeared during dividing phase. The tau-shaped region corresponded to 'posterior connectives' and the root part of 'deep fiber', to the conjunction parts of microtubule bundles. In the those parts, there was electron-dense material in the microtubule bundles. Hence, it is conceivable that 49K protein corresponds to the dense material and has a function of microtubule bundle conjunction. On the other hand, disappearance of immunofluorescence from the old oral apparatus of most dividing cells reflected the oral apparatus regression and remodelling which have been known as necessary sequential events in the cell cycle. We observed that oral fluorescence disappeared concurrently with the onset of oral regression and of constriction of division furrow, whereas at a late dividing stage immunofluorescence began to appear simultaneously in both new and old oral apparatus. Thus, the 49K protein may play a crucial role(s) not only in the morphogenesis of oral primordia but also in the transient morphogenesis in the old oral system.

Animals↗
PubMed1984

Hagfish slime gland thread cells. II. Isolation and characterization of intermediate filament components associated with the thread.

The slime glands of hagfish have two major cell types, gland thread cells (GTCs) and gland mucous cells (GMCs), both of which upon contact with water contribute to the formation of an abundant quantity of viscous mucus. In previous studies we reported a method for the isolation of GTCs and showed that each ellipsoidal thread cell normally contains a single tapered thread which is uniquely coiled into a space-saving conformation and occupies most of the cell volume. Subsequently, the developing thread was found to consist mainly of intermediate filaments (IFs) aligned in parallel not only to one another but also to a far fewer number of interspersed microtubules (see accompanying paper). In the present report, urea extracts of GTCs were purified and characterized to establish the properties of thread components. One major (alpha) and two minor (beta, gamma) components prepared by anion exchange chromatography were shown to have similar apparent molecular weights of 63,500 +/- 500 daltons but different isoelectric pH values (alpha, 7.56; beta, 5.67; gamma, 5.31). Although the amino acid content of alpha differed significantly from beta and gamma, each of the three was highest in Gly, relatively high in Glx, Ser, Thr, Asx, Ala, Val, and Leu, and relatively low in Cys/2 and Trp. The amino acid compositions of beta and gamma were very similar, and only beta showed evidence of carbohydrate. The threonine content of the alpha component was higher than has been reported for IFs of different origin, and the high content of hydroxyamino acids (18, 19 residues per 100) in alpha, beta, and gamma has been approached only by several IF polypeptides from human or bovine epidermal keratins. Mixtures of the purified components formed 9-11-nm filaments in vitro. The results indicate that the hagfish thread cell is a rich source of IFs, which have a structure that facilitates formation of macrofibrils within the cell.

Amino Acids↗
PubMed1994

Familial cardiomyopathy, mental retardation and myopathy associated with desmin-type intermediate filaments.

The clinical and morphological findings of a familial case affected by mental retardation, severe biventricular hypertrophic cardiomyopathy and vacuolar myopathy are reported. The phenotype of this patient is similar to that described by other authors, in which a lysosomal glycogen storage disease with normal acid maltase levels was suspected. However, in our case the vacuoles were stained by several antibodies directed against various sarcolemmal proteins, such as dystrophin and spectrin, and therefore, were not of lysosomal origin. Some of these vacuoles were clearly derived from the splitting of the fibres and invagination of the extracellular space; autophagic vacuoles were not observed. The accumulation of desmin-type, intermediate filaments was demonstrated on immunocytochemistry both in the skeletal and cardiac muscles. A brother of the propositus was also affected by mental retardation, severe cardiomyopathy and died suddenly at the age of 24 yr. A cardiomyopathy and mental subnormality were also present in other male cousins of the proband, while sudden death occurred in several females relatives, whose intelligence was normal. None of these latter individuals was available for further investigation. This report expands the spectrum of desmin associated myopathy and cardiomyopathy to include a familial condition with associated mental retardation.

Adult↗
PubMed2005

Cell surface expression of intermediate filament proteins vimentin and lamin B1 in human neutrophil spontaneous apoptosis.

Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, alpha-tubulin, and beta-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein, vimentin.

The crossreactivity of antibodies against a renal autoimmune epitope of Streptococcus pyogenes M protein with glomerular mesangial cells was investigated. The antibodies directed against the amino acid sequence Ile-Arg-Leu-Arg of the nephritogenic type 1 M protein reacted in a fibrillar pattern with mesangial cells cultured from isolated glomeruli. In Western blots of urea-extracted mesangial proteins, the antibodies reacted with a 56-kD protein. Monoclonal and polyclonal antibodies identified the 56-kD mesangial protein as vimentin. Two synthetic peptides of human vimentin containing the sequence Arg-Leu-Arg reacted with the autoimmune antibodies raised against a streptococcal M protein peptide. These results provide evidence that the intermediate filament protein vimentin shares autoimmune epitopes with streptococcal M protein.

Amino Acid Sequence↗
PubMed1997

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath.

Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

Actin Cytoskeleton↗
PubMed2001

A new marker for cup cells in the rabbit small intestine: expression of vimentin intermediate filament protein.

Cup cells in the intestinal lining in mammals are largely limited to the ileum, suggesting a specific but undetermined function. The investigation of these cells has been hindered by the lack of a suitable marker for immunohistochemistry. The small intestine of rabbits was examined for a potential molecular marker. Segments of New Zealand white rabbit small intestine, with and without Peyer's patches, were removed, rinsed, fixed with periodate-lysine-2% paraformaldehyde (PLP), and frozen for immunohistochemistry, or fixed in 2.5% glutaraldehyde solution for electron microscopy. The sections were stained with monoclonal antibodies (V9, 3B4) against vimentin, which is known to be absent in enterocytes other than antigen-sampling M cells, and observed by immunohistochemistry and immunoelectron microscopy. Epithelial expression of vimentin intermediate filament proteins was limited to a subpopulation of villus-associated cells and to cells over Peyer's patch domes. Immunoreactivity with vimentin antibodies was selectively found in cells with a cup-like indentation in the brush border in villous epithelium and in M cells in Peyer's patch follicle epithelium. Immunoelectron microscopy revealed that the labeled cells in villous epithelium had shorter microvilli than the adjacent absorptive cells, and small, sparse, mitochondria, which are ultrastructural characteristics of cup cells. Vimentin filaments were recognized in the area surrounding the nucleus and extending beneath the plasma membrane of cup cells. In rabbit small intestine, vimentin is useful for the detection of cup cells, as well as M cells, and may facilitate the investigation of these little understood cells.

Animals↗
PubMed1985

Intermediate filaments and the initiation of desmosome assembly.

The desmosome junction is an important component in the cohesion of epithelial cells, especially epidermal keratinocytes. To gain insight into the structure and function of desmosomes, their morphogenesis has been studied in a primary mouse epidermal (PME) cell culture system. When these cells are grown in approximately 0.1 mM Ca2+, they contain no desmosomes. They are induced to form desmosomes when the Ca2+ level in the culture medium is raised to approximately 1.2 mM Ca2+. PME cells in medium containing low levels of Ca2+, and then processed for indirect immunofluorescence using antibodies directed against desmoplakins (desmosomal plaque proteins), display a pattern of discrete fluorescent spots concentrated mainly in the perinuclear region. Double label immunofluorescence using keratin and desmoplakin antibodies reveals that the desmoplakin-containing spots and the cytoplasmic network of tonofibrils (bundles of intermediate filaments [IFB]) are in the same juxtanuclear region. Within 1 h after the switch to higher levels of Ca2+, the spots move toward the cell surface, primarily to areas of cell-cell contact and not to free cell surfaces. This reorganization occurs at the same time that tonofibrils also move toward cell surfaces in contact with neighboring cells. Once the desmoplakin spots have reached the cell surface, they appear to aggregate to form desmosomes. These immunofluorescence observations have been confirmed by immunogold ultrastructural localization. Preliminary biochemical and immunological studies indicate that desmoplakin appears in whole cell protein extracts and in Triton high salt insoluble residues (i.e., cytoskeletal preparations consisting primarily of IFB) prepared from PME cells maintained in medium containing both low and normal Ca2+ levels. These findings show that certain desmosome components are preformed in the cytoplasm of PME cells. These components undergo a dramatic reorganization, which parallels the changes in IFB redistribution, upon induction of desmosome formation. The reorganization depends upon both the extracellular Ca2+ level and the establishment of cell-to-cell contacts. Furthermore, the data suggests that desmosomes do not act as organizing centers for the elaboration of IFB. Indeed, we postulate that the movement of IFB and preformed desmosomal components to the cell surface is an important initiating event in desmosome morphogenesis.

Animals↗
PubMed1981

Expression of glial and vimentin type intermediate filaments in cultures derived from human glial material.

Several cultures established from biopsies of apparently normal adult human glial material showed no cells positive for glial fibrillary acidic protein (GFA) when examined after seven or more cumulative population doublings (CPD), although the established glioma line U251 MG showed approximately 3% GFA-positive cells, and U333 CG/343 MG clone 3 showed greater than 98% GFA-positive cells. Both the human glia delivered cultures and the glioma lines were positive when assayed with sera specific for vimentin. We therefore investigated the expression of GFA as a function of cumulative population doublings after the establishment of primary cultures. Under our experimental conditions, although GFA-positive cells were clearly present in the primary cultures accounting for some 3%-14% of the cells present, the GFA marker was subsequently lost, and the proliferating cultures expressed only the vimentin type of intermediate filament. Those cells that were GFA-positive also appeared to be vimentin-positive. GFA expression was not reinduced in cultures that had lost the GFA marker by treatment with dibutyryl cyclic AMP. We discuss two alternate hypotheses for the origin of the GFA-negative cells: (1) the cultures area of astrocyte origin but lost the ability to express GFA on culturing; (2) the cultures originate from cells of nonastrocyte origin present in the primary material.

Cell Division↗
PubMed1981

Binding of glycosomes to endoplasmic reticulum and to intermediate filaments in cardiac conduction fibers.

Canine cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of glycogen and protein) were found attached to membranes of endoplasmic reticulum (ER) and to intermediate filaments (IF). The bound glycosomes retained their integrity after treatment of tissue en block with uranyl acetate, a procedure that destroyed free glycosomes in the same cell. the coincident distributions of ER and IF and their association with glycosomes suggests a functional relation between there three components.

Acids↗
PubMed2004

Involvement of the intermediate filament protein cytokeratin-18 in actin pedestal formation during EPEC infection.

While remaining extracellular, enteropathogenic Escherichia coli (EPEC) establish direct links with the cytoskeleton of the target epithelial cell leading to the formation of actin-rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins; and recruitment of some is dependent on phosphorylation of Tyr 474. Using Tir as bait and HeLa cell cDNA library as prey in a yeast two-hybrid screen, we identified cytokeratin 18 as a novel Tir partner protein. Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and cytoskeleton reorganization. This study is the first to implicate intermediate filaments in microfilament reorganization following EPEC infection.

Actins↗