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PubMed2003

Effect of propionic and methylmalonic acids on the in vitro phosphorylation of intermediate filaments from cerebral cortex of rats during development.

In this study we investigated the in vivo and in vitro effects of methylmalonic (MMA) and propionic acids (PA), at concentrations usually found in methylmalonic acidemia and propionic acidemia respectively, on the phosphorylation of intermediate filament proteins in cerebral cortex of rats during development. Rats of 9, 12, and 17 days were acutely injected with the acids and sacrificed 90 min after injection. The cerebral cortex was dissected, and slices were incubated with 32P-orthophosphate. The cytoskeletal fraction was extracted and the radioactivity incorporated into intermediate filament subunits was measured. In addition, cortical slices from nontreated rats of 9, 12, 15, 17, 21, and 60 days of life were incubated with the acids in the presence of 32P-orthophosphate, the cytoskeletal fraction was extracted and the radioactivity was measured. Results demonstrated that MMA and PA significantly decreased the radioactivity incorporated into intermediate filament proteins at day 12, both in vivo and in tissue slices. In contrast, PA increased the in vitro phosphorylation of the cytoskeletal proteins in slices of 21-day-old animals. It acts through PP2A and PP2B in 12-day-old rats and through PKA and PKCaMII in 21-day-old animals. We propose that alteration of cytoskeletal protein phosphorylation caused by methylmalonic and propionic acids may be related to the neurological dysfunction characteristic of propionic and methylmalonic acidemia.

Animals↗

PubMed1990

[Cytoskeleton--function and pathology: studies on the pathology of the intermediate filament cytoskeleton of liver cells].

Microfilaments, microtubules and intermediate filaments (IF) are major filamentous components of the cytoskeleton and play a role in the modulation of cell shape, in cellular movements, cellular stability, intracellular organisation as well as cell-to-cell and cell-to-stroma interactions. Particular interest was concentrated in the last few years on IF because of their cell type-specificity and, consequently, their suitability as cell markers in diagnostic pathology. Despite their apparent stability, IF are dynamic structures which may be modified under pathologic conditions. In recent years, pathologic alterations related to the IF cytoskeleton have been described in a diversity of chronic and degenerative disorders, including alcoholic hepatitis and neurologic diseases (e.g. M. Alzheimer, M. Parkinson). Our studies were particularly devoted to the elucidation of the pathogenesis of severe alcoholic liver injury (alcoholic hepatitis), which is associated with inflammation, liver cell degeneration and necrosis and morphologically characterized by the appearance of cytoplasmic hyaline inclusions (i.e., Mallory bodies). In the present review morphologic, immunologic and biochemical studies on nature and pathogenesis of Mallory bodies are summarized. Moreover, similarities between Mallory bodies and other cytoskeleton-related inclusion bodies suggest common routes of pathogenesis. Consequently, studies along these lines may not only lead to the understanding of mechanisms involved in alcoholic injury but may also provide information on general principles of cell damage as well as on regulation and function of the IF cytoskeleton.

Animals↗

PubMed1994

Eight genes and alternative RNA processing pathways generate an unexpectedly large diversity of cytoplasmic intermediate filament proteins in the nematode Caenorhabditis elegans.

Cytoplasmic intermediate filament (IF) proteins of Caenorhabditis elegans are encoded by a dispersed multigene family comprising at least eight genes which map to three linkage groups. Exon sequences and intron patterns define three distinct subfamilies. While all eight IF genes display the long coil 1b subdomain of nuclear lamins, only six genes (a1-a4, b1 and b2) retain a lamin-like tail domain. Two genes (c1 and c2) have acquired entirely novel tail domains. The overall sequence identity of the rod domains is only 29%. The gene structures show a strong drift in number and positions of introns, none of which are common to all genes. Individual genes share only one to four intron locations with the Helix aspersa IF gene, but all eight nematode genes together account for nine of the 10 introns of the gastropod gene. All C.elegans IF genes are transcribed and all except gene c2 produce trans-spliced mRNAs. Alternatively spliced mRNAs arise from genes a1, b2 and c2 through several mechanisms acting at the transcriptional and posttranscriptional levels. These involve the alternative use of distinct promoters, polyadenylation sequences and both cis and trans RNA splice sites. The resulting sequence variations are restricted to the non-helical end domains. Minimally 12 distinct IF proteins are encoded by the various mRNAs. Different abundances in mixed-stage nematode populations suggest cell type- and/or stage-specific expression of individual mRNAs.

Alternative Splicing↗

PubMed1994

Expression of intermediate filaments in normal and neoplastic exocrine pancreas.

The intermediate filament (IF) proteins present in the normal and pathological exocrine human pancreas were studied by immunolocalization using antibodies to cytokeratins (CKs) and vimentin. Acinar cells of normal pancreas showed a presence of simple CKs 8 and 18. Duct epithelium consistently expressed CKs 7, 8, 18 and 19 whereas centroacinar cells were rather low in CK 7. A subpopulation of CK 4 cells was detected in inter-intralobular ducts. In addition, some ducts contained individual cells or groups of cells that were positive for the stratification-related CKs (CKs 4, 5, 13, 15, 16). All pancreatic ductal adenocarcinomas regularly expressed CKs 7, 8, 18, 19 and were also positive for the 34 beta E12 antibody. Cytokeratin 4 was detected in a minor population of tumor cells. Pancreatic carcinoma also contained minor amounts of stratification-related CKs in variable combinations. Mucinous cystoadenocarcinoma showed the presence of CKs 7, 8, 18, 19 and was also positive for 34 beta E12, whereas the serous microcystic tumor presented CKs 8, 18, 19 and a variable amount of CKs 4 and 7. The duct-ductular alterations of the exocrine pancreas contained a different combination and distribution of CK isoforms similar to normal pancreatic ductal system. Mucinous hypertrophy and pyloric gland metaplasia reacted with antibodies to CKs 7, 8, 18 and 19. Vimentin was focally present both in normal and neoplastic tissue. Our results indicate that pancreatic ducts are characterized by an intrinsic "biliary-pancreatic duct type" immunoprofile (CKs 7, 8, 18 and 19), in contrast to acinar cells expressing exclusively CKs 8 and 18. We also detected a subpopulation of ducts regularly expressing CK 4. Surprisingly, several stratification-related CKs were detected both in normal and neoplastic exocrine pancreas. Moreover, the differentiation phenotypes of pancreatic tumors were reminiscent of normal cellular compartments.

Adenocarcinoma↗

PubMed1993

The mechanism of interaction of filaggrin with intermediate filaments. The ionic zipper hypothesis.

Filaggrins of mammalian epidermis represent archetypical examples of intermediate filament-associated proteins that can bind large numbers of intermediate filaments in vitro (and keratin filaments in vivo) into macrofibrils. To explore the mechanism of this interaction, the secondary structures of filaggrins were analyzed. As much as 80% of mouse and human filaggrins consist of multiple repeating elements. The first level consists of a tetrapeptide beta-turn motif in which about 35% of the turns are positively charged and about 10% are negatively charged. At the next level, triplets of this motif form segments 13 to 14 residues in length, which in turn are repeated two to six times into blocks separated by short hydrophobic sequences to constitute a complete filaggrin molecule. Thus, filaggrins evolved by frequent duplications of a primordial repeat unit of about 13 to 14 residues with subsequent retention of the conserved beta-turn and charge characteristics. To test how these features bind filaments, two approaches were used. Of a series of synthetic peptides, those of 20 to 26 residues (about 2 segments) containing at least five beta-turns with a net charge of +2 (that is, about 40% of the turns are positively charged) were as effective as full length filaggrin in binding large numbers of both type I/II keratin and type III vimentin/desmin filaments, as judged by electron microscopy. Secondly, macrofibrils formed from unlabeled filaggrin and keratin filaments labeled in vivo with [1-13C]glycine or L-[4,4,5,5-2H4]lysine were probed by nuclear magnetic resonance. The effective isotropy and time scale of mobilities of the glycine-labeled end domains were essentially identical in keratin filaments alone and those bound in macrofibrils, suggesting that filaggrins do not bind filaments by way of their end domains. However, the lysine-labeled rod domains of the filaments in macrofibrils were considerably more constrained than in filaments alone. These data support the hypothesis that filaggrins bind filaments by way of simple ionic and/or H-bonding interactions between the conserved positive and negative charges on the beta-turns of filaggrins and the conserved distributions of negative and positive charges along the packed rod domains of intermediate filaments, as in an ionic zipper.

Amino Acid Sequence↗

PubMed1989

Phosphorylation of the peripherin 58-kDa neuronal intermediate filament protein. Regulation by nerve growth factor and other agents.

Peripherin, a recently described member of the intermediate filament multigene family, is present in peripheral and certain central nervous system neurons as well as in cultured neuron-like cell lines, including PC12 pheochromocytoma cells. In PC12 cells, peripherin appears to be the major intermediate filament protein and its relative levels and synthesis are specifically increased during nerve growth factor (NGF)-promoted neuronal differentiation. The present study examines the phosphorylation of peripherin and the regulation thereof by nerve growth factor and other agents in cultured PC12 cells. Immunoblotting experiments using a peripherin-specific antiserum show five distinct isoforms of this protein in whole cell and cytoskeletal extracts resolved by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of these isoforms incorporate detectable quantities of [32P]phosphate during metabolic radiolabeling. The small proportion (approximately 6%) of total cellular peripherin that is extractable with 1% Triton X-100, does not appear to incorporate phosphate. NGF increases peripherin phosphorylation by 2-3-fold within 1-2 h of treatment. Epidermal growth factor and insulin have no effect. The relative levels of phosphorylated peripherin are markedly elevated (17-fold) by long term NGF exposure, and peripherin becomes a major cytoskeletal phosphoprotein. Activators of protein kinases A and C and treatment with depolarizing levels of K+ also enhance peripherin phosphorylation by 2-3-fold, in cultures both with and without prior long term NGF treatment. Evidence is presented that NGF regulates peripherin phosphorylation by a mechanism independent of protein kinases A and C and of depolarization. The large increase in phosphorylated peripherin brought about by NGF treatment suggests that this neuronal filament protein may play a role in the elaboration and maintenance of neurites. The presence of multiple independent pathways that acutely enhance peripherin phosphorylation indicates that this role is subject to modulation by extrinsic signals.

Adrenal Gland Neoplasms↗

PubMed1984

Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin.

Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.

Animals↗

PubMed1980

Unusual familial cardiomyopathy with storage of intermediate filaments in the cardiac muscular cells.

Unusual histological and ultrastructural changes in cardiac muscle cells have been found in 3 brothers with progressive myocardial deficiency. Histologically, this cardiomyopathy was characterized by massive storage of PAS-negative proteinaceous material in most cardiac muscle cells. The electron microscope showed that this material consisted of sinuous filaments, 7-10 nm in diameter, similar to the intermediate filaments normally present in cardiac muscle cells. Filament storage coincided with the disintegration of neighbouring myofibrils, with particular change in Z bands giving rise to rod-like bodies and more complex structures formed by the association of Z band material and sarcoplasmic reticulum (SR) tubules. Filament storage and myofibrillar disintegration always occurred in areas where the SR developed and involuted extensively. Relatively high glycogen accumulation also occurred, in close relation to the SR changes. Discrete SR proliferation, glycogen overload and filament deposits were observed in a few skeletal fibres. These observations suggest that disturbance in the metabolism of desmin (protein subunit of intermediate filaments and a fundamental component of Z bands) might be involved in this type of cardiomyopathy. The influence of a chronic defect in calcium regulation might also be envisaged in view of the marked SR abnormalities.

Adult↗

PubMed1987

Discrimination between the nuclear lamin and intermediate filament (cytokeratin/vimentin) proteins of rat hepatic tumor cells by differential solubility and electrophoretic criteria.

1. The major proteins which comprise the high salt/detergent-insoluble cytoskeletal matrix of rat hepatic tumor cells containing abnormal (Mallory body-like) aggregates of intermediate filaments were distinguished on the basis of electrophoretic mobility and differential solubility. 2. Gel electrophoresis of the intermediate filament-enriched cytoskeletal fraction of Mallory body hepatic tumor cells revealed the presence of: (a) intermediate filament proteins typical of cultured liver epithelial cells (cytokeratins A and D, vimentin), (b) some residual actin and, (c) two peptides of Mr = 68,000-72,000. 3. Analysis of the products of filament disassembly/reassembly mixtures indicated that the two Mr = 68,000-72,000 peptide species had the solubility characteristics of nuclear lamins. 4. The presence of nuclear lamin proteins in the high salt/detergent-resistant fraction of cultured liver cells was consistent with the resolution of residual nuclear-like structures in extracted cell monolayers. 5. Thus, while cytokeratin/vimentin-class intermediate filament proteins and nuclear lamins co-isolate from rat liver cells under conditions of high salt/detergent extraction, these two types of cytoskeletal proteins could be distinguished on the basis of their differential solubility and molecular weight.

Animals↗

PubMed1987

[Immunohistological demonstration of the intermediate filaments in a laryngeal carcinosarcoma: considerations on its histogenesis].

A carcinosarcoma of the larynx was analysed by means of a light microscope and by immunohistological staining technique using antibodies against various intermediate filament proteins. Whereas tumour areas of an epithelial character reacted with antibodies against keratin, the spindle cells demonstrated a positive immunofluorescence with vimentin antibodies. Inside some tumour cells a coexpression of keratin and vimentin (intermediate filaments of mesenchymal cells) could be demonstrated. It is likely that these double stained cells represent the primitive stem cell of the carcinosarcoma, differentiating during further development either into epithelial or mesenchymal tumour cells.

Carcinosarcoma↗

PubMed1996

The cytoskeleton and disease: genetic disorders of intermediate filaments.

Specialized cytoskeletons play many fascinating roles, including mechanical integrity and wound-healing in epidermal cells, cell polarity in simple epithelia, contraction in muscle cells, hearing and balance in the inner ear cells, axonal transport in neurons, and neuromuscular junction formation between muscle cells and motor neurons. These varied functions are dependent upon cytoplasmic networks of actin microfilaments (6 nm), intermediate filaments (10 nm) and microtubules (23 nm), and their many associated proteins. In this chapter, I review what is known about the cytoskeletons of intermediate filaments and their associated proteins. I focus largely on epidermal cells, which devote most of their protein-synthesizing machinery to producing an extensive intermediate filament network composed of keratin. Recent studies have shown that many of the devastating human disorders that arise from degeneration of this cell type have as their underlying basis either defects in the genes encoding keratins or abnormalities in keratin IF networks. I discuss what we know about the functions of IFs, and how the link to genetic disease has enhanced this understanding.

Amino Acid Sequence↗

PubMed1998

Estrogen regulates the association of intermediate filament proteins with nuclear DNA in human breast cancer cells.

In a previous study we showed that the levels of the intermediate filament proteins, cytokeratins 8, 18, and 19, in the nuclear matrix-intermediate filament (NM-IF) fraction from the hormone-dependent and estrogen receptor (ER)-positive human breast cancer cell line T-47D5 were regulated by estrogens. In contrast, estrogens did not regulate the cytokeratins in the NM-IF fraction of the hormone-independent and ER-positive cell line, T5-PRF. In this study, human breast cancer cells were treated with cis-diamminedichloroplatinum to cross-link protein to nuclear DNA in situ, and proteins bound to DNA were isolated. We show that cytokeratins 8, 18, and 19 of T-47D5 and T5-PRF were associated with nuclear DNA in situ. The levels of the cytokeratins 8, 18, and 19 bound to nuclear DNA or associated with the cytoskeleton of T-47D5 human breast cancer cells decreased when estrogens were depleted or the pure antiestrogen ICI 164,384 was added. In contrast, the cytokeratin levels associated with nuclear DNA or cytoskeleton were not significantly affected by estrogen withdrawal or antiestrogen administration in T5-PRF cells. These observations suggest that estrogen regulates the organization of nuclear DNA by rearrangement of the cytokeratin filament network in hormone-dependent, ER-positive human breast cancer cells and that this regulation is lost in hormone-independent, ER-positive breast cancer cells.

Breast Neoplasms↗

PubMed1994

[The localization of intermediate filaments in polykaryons in the interphase and mitosis].

Localization of vimentin filaments has been investigated in polykaryons formed by cell fusion in culture of Chinese hamster cells (line Ag17). A network of vimentin filaments was observed around each nucleus of the interphase polykaryons. This compartmentalization of the network persists after beginning of mitoses up to metaphase, both in bipolar and multipolar mitotic cells. The picture of localization of intermediate filaments is most clear in polykaryons with mitotic asynchrony of the nuclei at early stages of mitosis. In anaphase, the central part of the network disappears, and in the polykaryon only peripheral part of the network is observed. The compartmentalization of the network of intermediate filaments is disrupted by such a way. In telophase, bright peripheral fluorescence may by the result of the beginning of network reorganization, and this process will be finished in daughter cells. The role of intermediate filaments in distribution of chromosomes and centrioles between daughter cells during mitosis is discussed.

Animals↗

PubMed1989

Autoantibodies to intermediate filaments in sera of patients with Schistosoma mansoni infection.

Autoantibodies to the intermediate filament proteins vimentin and keratin were studied in sera of 50 Caribbean patients with Schistosoma mansoni infection and 50 control subjects. Autoantibodies were detected by indirect immunofluorescence on HEp-2 cells pretreated with colchicine. The incidence of anti-vimentin antibodies in patients' sera was 94% for IgM, 12% for IgG, and 4% for IgA; in the control subjects incidence was 52%, 0%, and 4%, respectively. Anti-keratin antibodies were found in 82%, 4%, and 4% of patients' sera and 42%, 0%, and 2% in controls, respectively. The difference between the geometric means of titres for patients (1:150) and controls (1:26) was highly significant (P less than 0.001). The possible role and genesis of autoantibodies to intermediate filaments is discussed.

Adolescent↗

PubMed1994

Cytoplasmic desmosomes and intermediate filament disturbance following acrylamide treatment in cultured rat keratinocytes.

The present paper describes disturbances in the organization of tonofilaments and desmosomes of rat lingual and epidermal keratinocytes after treatment of the cells with acrylamide in culture. This treatment induced changes in cell shape, reduction of intercellular adhesion and a perinuclear accumulation of cytoplasmic organelles. Using specific antibodies for cytokeratins, the filaments were disorganized particularly in the perinuclear region. In untreated cells, keratin filament labelling was very weak or absent above and below the nucleus thus leaving a black nuclear space in fluorescine microscopy. Following acrylamide treatment, the keratin filament labelling covered the nuclear space which indicated the accumulation of these filaments all around the nucleus. Furthermore, the desmosomal junctions were often associated with thick keratin bundles. Antibodies for desmoplakins revealed a reduction in intercellular labelling and stronger cytoplasmic labelling. Ultrastructurally, well-developed long tonofilaments were found to associate with large desmosomal junctions. Furthermore, small-sized desmosomal structures were identified within the cytoplasm. Morphologically, these were identical to cell surface desmosomes and were almost always associated with well-developed tonofilaments. The effect of acrylamide on the protein kinase A activity might be implicated in the disturbances of the desmosome-intermediate filament complex and in the initiation of contractile forces necessary for perinuclear accumulation of intermediate filaments and for the formation of intact cytoplasmic desmosomes. The acrylamide-induced intermediate filament and desmosomal changes may provide valuable information on the mechanism of intact cytoplasmic desmosome formation in several skin diseases and in squamous cell carcinoma.

Acrylamide↗

PubMed2000

Alpha-ketoisocaproate increases the in vitro 32P incorporation into intermediate filaments in cerebral cortex of rats.

In this study we investigated the effects of alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV) and alpha-keto-beta-methylvaleric (KMV) acids on the phosphorylation of intermediate filament (IF) proteins of cerebral cortex of rats. Tissue slices were incubated with [32P] orthophosphate in the presence or absence of the acids. The intermediate filament enriched cytoskeletal fraction was isolated and the radioactivity incorporated into neurofilament subunits, vimentin and glial fibrillary acidic protein was measured. Results demonstrated that KIC significantly increased phosphorylation of these proteins whereas the other acids had no effect. Experiments using protein kinase inhibitors indicated that the effect of KIC was mediated by Ca2+/calmodulin- and cAMP-dependent protein kinases. This study provides evidence that KIC, a key metabolite accumulating in maple syrup urine disease, increases phosphorylation of IF proteins.

Animals↗

PubMed1995

Abnormal expression of intermediate filament proteins in X-linked myotubular myopathy is not reproduced in vitro.

Expression patterns of the intermediate filament proteins (IFPs) desmin and vimentin, in biopsy material taken from a 1 day old boy with fatal neonatal X-linked myotubular myopathy (XLMTM) were compared with the expression of these proteins in cultured myotubes, from the same patient. Immunohistochemical studies revealed the persistence of high levels of desmin in virtually all, and vimentin in most, of the myofibres within the patient's biopsy. Analysis of intermediate filament expression in differentiating, cultured muscle cells did not reveal overt differences between XLMTM cultures and cultures of control muscle. Titin distribution patterns indicated a normal process of myofibrillogenesis in XLMTM myotubes. We conclude that the failure to properly regulate IFP-expression is not intrinsic to XLMTM muscle fibres. The possibility that this failure is due to a defective external, possibly neural factor, is discussed.

Cells, Cultured↗

PubMed1993

Intermediate filaments in ovarian tumors.

The availability in recent years of an ever increasing number of monoclonal antibodies to intermediate filaments which react with formalin-fixed, paraffin-embedded sections by the immunoperoxidase method, makes it possible to apply this technique in most surgical pathology laboratories. In addition to representing a major diagnostic tool that is often indispensable in the differential diagnosis, intermediate filaments typing of complex ovarian neoplasms has also shed some light on the histogenesis and differentiation potential of ovarian tumors.

Female↗