Paranemin and the organization of desmin filament networks

De novo expression of vimentin, GFAP or peripherin leads to the assembly of an extended intermediate filament network in intermediate filament-free SW13/cl.2 cells. Desmin, in contrast, does not form extended filament networks in either SW13/cl.2 or intermediate filament-free mouse fibroblasts. Rather, desmin formed short thickened filamentous structures and prominent spot-like cytoplasmic aggregates that were composed of densely packed 9–11 nm diameter filaments. Analysis of stably transfected cell lines indicates that the inability of desmin to form extended networks is not due to a difference in the level of transgene expression. Nestin, paranemin and synemin are large intermediate filament proteins that coassemble with desmin in muscle cells. Although each of these large intermediate filament proteins colocalized with desmin when coexpressed in SW-13 cells, expression of paranemin, but not synemin or nestin, led to the formation of an extended desmin network. A similar rescue of desmin network organization was observed when desmin was coexpressed with vimentin, which coassembles with desmin, or with keratins, which formed a distinct filament network. These studies demonstrate that desmin filaments differ in their organizational properties from the other vimentin-like intermediate filament proteins and appear to depend upon coassembly with paranemin, at least when they are expressed in non-muscle cells, in order to form an extended filament network.

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Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

The Journal of Cell Biology ◽

10.1083/jcb.96.1.37 ◽

1983 ◽

Vol 96 (1) ◽

pp. 37-50 ◽

Cited By ~ 93

Author(s):

E Schmid ◽

DL Schiller ◽

C Grund ◽

J Stadler ◽

WW Franke

Keyword(s):

Mammary Gland ◽

Epithelial Cell ◽

Cell Line ◽

Cell Lines ◽

Intermediate Filament ◽

Striking Difference ◽

Intermediate Filament Proteins ◽

Cell Clones ◽

Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

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Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras

Journal of Cell Science ◽

10.1242/jcs.111.13.1767 ◽

1998 ◽

Vol 111 (13) ◽

pp. 1767-1778 ◽

Cited By ~ 6

Author(s):

C.L. Ho ◽

J.L. Martys ◽

A. Mikhailov ◽

G.G. Gundersen ◽

R.K. Liem

Keyword(s):

Green Fluorescent Protein ◽

Dynamic Behavior ◽

Cell Lines ◽

Intermediate Filaments ◽

Intermediate Filament ◽

Fluorescent Protein ◽

Nih3t3 Cells ◽

Green Fluorescent ◽

Filament Network ◽

Perinuclear Area

In order to study the dynamic behavior of intermediate filament networks in living cells, we have prepared constructs fusing green fluorescent protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament network. We generated stable SW13 and NIH3T3 cell lines that express an enhanced green fluorescent protein fused to the N-terminus of full-length vimentin. We were able to observe the dynamic behavior of the intermediate filament network in these cells for periods as long as 4 hours (images acquired every 2 minutes). In both cell lines, the vimentin network constantly moves in a wavy manner. In the NIH3T3 cells, we observed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of nocodazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or ‘collapse’ of intermediate filaments. We injected anti-IFA antibodies into NIH3T3 cells stably expressing green fluorescent protein fused to vimentin and found that individual intermediate filaments move slowly towards the perinuclear area without obvious disassembly. These results demonstrate that individual intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tubulin antibodies disrupt the interactions between intermediate filaments and stable microtubules and cause the collapse of the vimentin network showing that these interactions play an important role in keeping the intermediate filament network extended. The nocodazole inhibition of intermediate filament extension and the anti-IFA microinjection experiments are consistent with a model in which intermediate filaments exhibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severed.

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Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin

Development ◽

10.1242/dev.105.2.279 ◽

1989 ◽

Vol 105 (2) ◽

pp. 279-298

Author(s):

H. Herrmann ◽

B. Fouquet ◽

W.W. Franke

Keyword(s):

Amino Acid ◽

Xenopus Laevis ◽

Intermediate Filament ◽

De Novo ◽

Mesenchymal Cell ◽

Amino Acid Sequences ◽

Cytoskeletal Proteins ◽

Cdna Clones ◽

Mammalian Development ◽

Intermediate Filament Proteins

To provide a basis for studies of the expression of genes encoding the diverse kinds of intermediate-filament (IF) proteins during embryogenesis of Xenopus laevis we have isolated and characterized IF protein cDNA clones. Here we report the identification of two types of Xenopus vimentin, Vim1 and Vim4, with their complete amino acid sequences as deduced from the cloned cDNAs, both of which are expressed during early embryogenesis. In addition, we have obtained two further vimentin cDNAs (Vim2 and 3) which are sequence variants of closely related Vim1. The high evolutionary conservation of the amino acid sequences (Vim1: 458 residues; Mr approximately 52,800; Vim4: 463 residues; Mr approximately 53,500) to avian and mammalian vimentin and, to a lesser degree, to desmin from the same and higher vertebrate species, is emphasized, including conserved oligopeptide motifs in their head domains. Using these cDNAs in RNA blot and ribonuclease protection assays of various embryonic stages, we observed a dramatic increase of vimentin RNA at stage 14, in agreement with immunocytochemical results obtained with antibody VIM-3B4. The significance of very weak mRNA signals detected in earlier stages is discussed in relation to negative immunocytochemical results obtained in these stages. The first appearance of vimentin has been localized to a distinct mesenchymal cell layer underlying the neural plate or tube, respectively. The results are discussed in relation to programs of de novo synthesis of other cytoskeletal proteins in amphibian and mammalian development.

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Peripherin assembles into homopolymers in SW13 cells

Journal of Cell Science ◽

10.1242/jcs.108.10.3279 ◽

1995 ◽

Vol 108 (10) ◽

pp. 3279-3284 ◽

Cited By ~ 3

Author(s):

C. Cui ◽

P.J. Stambrook ◽

L.M. Parysek

Keyword(s):

Intermediate Filament ◽

Network Formation ◽

Full Length ◽

Intermediate Filament Proteins ◽

Filament Assembly ◽

Mature Neurons ◽

Carboxyl Terminal ◽

Relationship Of ◽

Filament Network ◽

Carboxyl Tail

The properties of full-length and mutant peripherins were studied in intermediate filament-less SW13 cells to define regions of peripherin that are essential for initiation of filament assembly. A full-length rat peripherin gene transfected into SW13 cells resulted in filament formation, consistent with the close structural relationship of peripherin to other type III intermediate filament proteins that readily form homopolymers. Translation of full-length rat peripherin is initiated predominantly at the second of two inframe AUGs. Deletions within the amino terminus of wild-type peripherin abolished its ability to form filaments in SW13 cells. In contrast, deletion of the entire carboxyl-terminal tail of peripherin did not affect its ability to form filamentous arrays in transfected SW13 cells. These results indicate that, of the intermediate filament proteins that are expressed in mature neurons, only peripherin and alpha-internexin are capable of making homopolymer intermediate filaments. In addition, mutations of the carboxyl tail of peripherin generally do not interfere with filament network formation.

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The association of prosomes with some of the intermediate filament networks of the animal cell.

The Journal of Cell Biology ◽

10.1083/jcb.107.4.1517 ◽

1988 ◽

Vol 107 (4) ◽

pp. 1517-1530 ◽

Cited By ~ 55

Author(s):

M F Grossi de Sa ◽

C Martins de Sa ◽

F Harper ◽

M Olink-Coux ◽

M Huesca ◽

...

Keyword(s):

Immunoelectron Microscopy ◽

Intermediate Filament ◽

Biochemical Composition ◽

Animal Cell ◽

Filament Networks ◽

Filament Network

The small RNP complexes of defined morphology and biochemical composition termed prosomes, first isolated from the cytoplasm associated with repressed mRNA (Martins de Sa, C., M.-F. Grossi de Sa, O. Akhayat, F. Broders, and K. Scherrer. J. Mol. Biol. 1986. 187:47-493), were found also in the nucleus (Grossi de Sa, M.-F., C. Martins de Sa, F. Harper, O. Coux, O. Akhayat, P. Gounon, J. K. Pal, Y. Florentin, and K. Scherrer. 1988. J. Cell Sci. 89:151-165). Immunofluorescence, immunoelectron microscopy, and immunochemical studies using mAbs directed against some of the prosomal proteins of duck erythroblasts indicate that in the cytoplasm of HeLa and PtK cells, prosome antigens are associated with the intermediate filament network of the cytokeratin type.

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Expression of intermediate filament-associated proteins paranemin and synemin in chicken development.

The Journal of Cell Biology ◽

10.1083/jcb.97.6.1860 ◽

1983 ◽

Vol 97 (6) ◽

pp. 1860-1874 ◽

Cited By ~ 59

Author(s):

M G Price ◽

E Lazarides

Keyword(s):

Smooth Muscle ◽

Intermediate Filament ◽

Muscle Cells ◽

Myocardial Cells ◽

Adult Chicken ◽

Intermediate Filament Proteins ◽

Adult Muscle ◽

Associated Proteins ◽

Visceral Smooth Muscle ◽

Chicken Development

The expression of two intermediate filament-associated proteins, paranemin (280,000 mol wt) and synemin (230,000 mol wt), was investigated with respect to the expression of two core intermediate filament proteins, desmin and vimentin, in various embryonic and adult chicken muscle and nonmuscle cells. All developing muscle cells, regardless of their type, simultaneously express desmin, vimentin, paranemin, and synemin. However, a difference is observed in the expression of paranemin in adult muscle. This protein is removed during differentiation of both fast and slow skeletal muscle, visceral smooth muscle, and the smooth muscle of muscular arteries, but remains in mature myocardial cells, cardiac conducting fibers, and the smooth muscle cells of elastic arteries. Some of these cells express vimentin, others desmin, and still others a mixture of the two. On the other hand, synemin is expressed in all the above types of adult muscle cells except myocardial cells. Adult myocardial cells also lack vimentin, and its presence is gradually reduced after hatching. Since in adult striated muscle all expressed intermediate filament proteins are found predominantly in association with the peripheries of myofibrillar Z discs, these results suggest that a change in the composition of skeletal and cardiac muscle Z discs occurs during chicken development and maturation. Erythrocytes that express synemin and vimentin do not express paranemin, while both embryonic and adult Schwann cells co-express paranemin and vimentin, but not synemin. Endothelial cells of muscular vessels express paranemin, while those of elastic vessels do not, and neither contains synemin. Paranemin and synemin are not expressed in neurons, epithelial, and most glial cells, suggesting that these two polypeptides are expressed only in conjunction with desmin or vimentin. These results suggest that the composition of intermediate filaments changes during chicken development, not only with respect to their core subunit proteins but also with respect to two associated polypeptides, particularly in muscle cells.

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Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.3049 ◽

1990 ◽

Vol 111 (6) ◽

pp. 3049-3064 ◽

Cited By ~ 85

Author(s):

P A Coulombe ◽

Y M Chan ◽

K Albers ◽

E Fuchs

Keyword(s):

Intermediate Filament ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Filament Assembly ◽

Keratin Filaments ◽

Keratin Filament ◽

Filament Networks ◽

10 Nm Filaments

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

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Alterations of intermediate filaments in various histopathological conditions

Biochemistry and Cell Biology ◽

10.1139/o95-069 ◽

1995 ◽

Vol 73 (9-10) ◽

pp. 627-634 ◽

Cited By ~ 5

Author(s):

Monique Cadrin ◽

Maria-Grazia Martinoli

Keyword(s):

Intermediate Filaments ◽

Intermediate Filament ◽

Lewy Bodies ◽

Degenerative Diseases ◽

Intermediate Filament Proteins ◽

Mallory Bodies ◽

Filament Structure ◽

Cytoskeletal Component ◽

Filament Networks ◽

Precise Role

Intermediate filament proteins belong to a multigene family and constitute an important cytoskeletal component of most vertebrate cells. Their pattern of expression is tissue specific and is highly controlled during embryonic development. Numerous pathologies are known to be associated with modifications of intermediate filament organisation, although their precise role has not yet been elucidated. The present review focuses on the most recent data concerning the possible causes of intermediate filaments disorganization in specific pathologic conditions affecting the epidermis, the liver, and the nervous system. We discuss the formation of abnormal intermediate filament networks that arise as a consequence of mutations that directly affect intermediate filament structure or are induced by multifactorial causes such as modifications of post-translational processes and changes in the levels of expression.Key words: intermediate filaments, phosphorylation, Mallory bodies, Lewy bodies, degenerative diseases.

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Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin

Journal of Cell Science ◽

10.1242/jcs.62.1.129 ◽

1983 ◽

Vol 62 (1) ◽

pp. 129-147

Author(s):

U.E. Traub ◽

W.J. Nelson ◽

P. Traub

Keyword(s):

Acetic Acid ◽

Electrophoretic Mobility ◽

Cell Lines ◽

Intermediate Filament ◽

Gel Electrophoresis ◽

Degradation Products ◽

Mammalian Species ◽

Polyacrylamide Gel ◽

Intermediate Filament Proteins

A total of 53 different cell lines originating from a variety of mammalian species were cultured in vitro and analysed for the presence of vimentin, employing polyacrylamide gradient slab gel electrophoresis in urea/acetic acid as buffer system. Irrespective of the cell culture conditions, and the growth potential and morphology of the cells, vimentin was expressed in all cell lines examined, with two exceptions: MPC-11 mouse myeloma and MOPC-31C mouse plasmacytoma cells. Immunoblotting with the monoclonal antibody alpha-IFA, which is directed against an antigenic determinant shared by all classes of intermediate filaments, did not detect any other of the known intermediate filament proteins in MPC-11 and MOPC-31C cells. Vimentin synthesized by various cell lines was characterized by four different criteria: (1) its extractability with Triton X-100 under various ionic conditions; (2) its behaviour in (NH4)2SO4 fractionation of cellular extracts; (3) its electrophoretic mobility in polyacrylamide gel electrophoresis in urea/acetic acid; and (4) the co-isolation of polypeptides of higher electrophoretic mobility, which, by comparison with degradation products of vimentin obtained with the Ca2+-activated proteinase specific for intermediate filament proteins in vitro, were identified as products of Ca2+-dependent proteolysis of vimentin. Although the degradation products occurred in different ratios in extracts of different cell lines, they constituted the same characteristic set of proteins whenever degradation of vimentin was observed. The formation of proteolytic breakdown products could be partially to totally suppressed when the cells were harvested, washed and processed in the presence of EGTA and proteinase inhibitors. The experimental data show that: (1) vimentin, as well as the Ca2+-activated proteinase specific for intermediate filament proteins, is highly conserved during the evolution of mammalian species; (2) the proteolytic breakdown products of vimentin, which give rise to a characteristic ‘staircase’ in two-dimensional gel electrophoresis, are probably artefacts of isolation; (3) the expression of vimentin is neither a prerequisite for nor necessarily indicative of rapid cell proliferation in vitro; and (4) the techniques described can be used for the routine identification of vimentin in cells and tissues in case vimentin-specific antibodies are not available.

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Intermediate filament proteins in classic and variant types of small cell lung carcinoma cell lines: a biochemical and immunochemical analysis using a panel of monoclonal and polyclonal antibodies

Journal of Cell Science ◽

10.1242/jcs.83.1.37 ◽

1986 ◽

Vol 83 (1) ◽

pp. 37-60

Author(s):

J.L. Broers ◽

D.N. Carney ◽

M. Klein Rot ◽

G. Schaart ◽

E.B. Lane ◽

...

Keyword(s):

Lung Cancer ◽

Cell Lines ◽

Intermediate Filament ◽

Polyclonal Antibodies ◽

Small Cell ◽

Intermediate Filament Protein ◽

Small Cell Lung ◽

Cell Lung Carcinoma ◽

Intermediate Filament Proteins ◽

Variant Type

The intermediate filament protein (IFP) characteristics of a panel of lung cancer cell lines including adenocarcinoma (two cell lines) and small cell lung cancer (SCLC, three classic and three variant cell lines) were examined using one- and two-dimensional gel electrophoretic techniques, immunocytochemical techniques and immunoblotting assays. A panel of 28 monoclonal and polyclonal antibodies to the five different types of IFP were used. The results of our studies indicate that these human lung adenocarcinoma, classic SCLC and variant SCLC cell lines can be differentiated on the basis of their pattern of IFP. The main conclusions from this study can be summarized as follows. The two adenocarcinoma cell lines contain cytokeratins 7, 8, 18, and sometimes 19, next to vimentin intermediate filament (IF). The three classic-type SCLC cell lines contain only cytokeratin IFs but not vimentin IF or neurofilaments (NFs). Cytokeratin polypeptides 7, 8, 18 and 19 could be detected. All three variant-type SCLC cell lines do not contain detectable amounts of cytokeratins. In contrast, two out of three variant SCLC cell lines contain neurofilament proteins. All three variant-type SCLC cell lines contain vimentin IF. Using immunoblotting assays with monoclonal and polyclonal antibodies to defined NF proteins the presence of the 68 X 10(3) Mr and the 160 X 10(3) Mr NF polypeptide could be demonstrated in two variant SCLC cell lines. As patients with SCLC-variant phenotype have a poorer prognosis after cytotoxic therapy than patients with ‘pure’ SCLC, the use of antibodies to IFP in staining fresh lung tumours, especially anaplastic ones, may differentiate the two subtypes of SCLC. Such a distinction would have a major impact on therapy selections and may be of prognostic importance.

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