Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components

1995 ◽  
Vol 73 (9-10) ◽  
pp. 651-657 ◽  
Author(s):  
Marie Duval ◽  
Xiaoying Ma ◽  
Jean-Paul Valet ◽  
Michel Vincent
Keyword(s):  
Chick Embryo ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Specific Binding ◽  
Developmentally Regulated ◽  
Intermediate Filament Proteins ◽  
In Vitro Recombination ◽  
Associated Proteins ◽  
Structural Lattice

IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly–reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin–IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.Key words: cytoskeleton, intermediate filaments, intermediate filament associated proteins, vimentin, IFAPa-400.

Expression of a developmentally regulated cross-linking intermediate filament-associated protein (IFAPa-400) during the replacement of vimentin for desmin in muscle cell differentiation

1991 ◽  
Vol 98 (2) ◽  
pp. 251-260 ◽  
Author(s):  
L.J. Cossette ◽  
M. Vincent
Keyword(s):  
Chick Embryo ◽  
Muscle Cell ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cell Types ◽  
Complete Disappearance ◽  
Developmentally Regulated ◽  
Complete Replacement ◽  
Embryo Cells

Myogenic and neurogenic tissues of the chick embryo transiently express IFAPa-400, a high molecular weight protein that colocalizes and is copurified with intermediate filaments. Using monoclonal antibody F51H2 to identify it, we carried out immunoelectron microscopy experiments on whole-mount chick embryo cells and showed that IFAPa-400 was localized at crossing points of intermediate filaments. Also, immunoblot experiments with F51H2, anti-vimentin and anti-desmin antibodies demonstrated the complete disappearance of IFAPa-400 in those muscle cell types that change their vimentin content for desmin during embryogenesis. During in vitro myogenesis, the expression of IFAPa-400 was shown to be concurrent with the progressive replacement of vimentin by desmin in myoblasts. When long-term myotube cultures were maintained on a fibroblast-like cell layer, we observed the complete replacement of vimentin by desmin, followed by the disappearance of IFAPa-400 from the myotubes. These results suggest that IFAPa-400 might be involved in the reorganization of the intermediate filament network during muscle differentiation.

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

1988 ◽  
Vol 106 (3) ◽  
pp. 723-733 ◽  
Author(s):  
R Foisner ◽  
F E Leichtfried ◽  
H Herrmann ◽  
J V Small ◽  
D Lawson ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Solid Phase ◽  
Chinese Hamster ◽  
Structural Element ◽  
Intermediate Filament Proteins ◽  
Rat Glioma ◽  
Branching Points

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

Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc's

1994 ◽  
Vol 107 (7) ◽  
pp. 1833-1843 ◽  
Author(s):  
C.F. Chou ◽  
M.B. Omary
Keyword(s):  
Okadaic Acid ◽  
Intermediate Filament ◽  
Brefeldin A ◽  
Mitotic Arrest ◽  
Protein Glycosylation ◽  
Intermediate Filament Proteins ◽  
M Phase ◽  
Associated Proteins ◽  
Phosphopeptide Mapping

The two major intermediate filament glycoproteins in human simple epithelia are keratins 8 and 18 (K8/18). A dramatic increase in terminal N-acetylglucosamine (GlcNAc) residues in K8/18 was previously noted after arresting cells in G2/M using anti-microtubule agents. Here we use in vitro galactosylation to show that increased terminal GlcNAc's is a general phenomenon that occurs in glycoproteins isolated from nuclear and plasma membrane fractions after cells are arrested in mitosis using colcemid, nocodazole, or okadaic acid. All three agents also resulted in a hyperphosphorylated form of K8 as determined by phosphatase treatment and tryptic phosphopeptide mapping. The altered glycosylation was found to be independent of microtubule disassembly, and was not directly related to the G2/M phase of the cell cycle after aphidicolin synchronization. Staurosporine (1 microM) inhibited K8/18 phosphorylation in okadaic acid- or nocodazole-treated cells, and inhibited the increase in K8/18 glycosylation without inhibiting the increase in terminal GlcNAc's of membrane-associated glycoproteins. In contrast, brefeldin A resulted in a dramatic increase in terminal GlcNAc's of membrane-associated but not intermediate filament proteins. Golgi complex-related staining using anti-beta-COP antibody showed significant fragmentation under conditions associated with altered membrane protein glycosylation. Our results suggest that Golgi disruption may be involved in the observed increase in terminal GlcNAc's of membrane but not intermediate filament glycoproteins. The mechanism of increased glycoprotein terminal GlcNAc's in association with mitotic arrest appears to be distinct for intermediate filaments and membrane-associated proteins, and in the case of intermediate filament proteins, phosphorylation may play an important role. Some of the effects of agents that induce mitotic arrest may be mediated by glycosylation changes.

scholarly journals Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

1997 ◽  
Vol 138 (6) ◽  
pp. 1379-1394 ◽  
Author(s):  
Carlos Caulín ◽  
Guy S. Salvesen ◽  
Robert G. Oshima
Keyword(s):  
Intermediate Filaments ◽  
Cleavage Site ◽  
Uv Light ◽  
Tail Domain ◽  
Proteolytic Fragment ◽  
Intermediate Filament Proteins ◽  
Caspase Cleavage ◽  
Structural Cell ◽  
Caspase 6

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.

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

1983 ◽  
Vol 96 (1) ◽  
pp. 37-50 ◽  
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.

Tetrins: polypeptides that form bundled filaments in Tetrahymena

1990 ◽  
Vol 96 (2) ◽  
pp. 293-302
Author(s):  
J.E. Honts ◽  
N.E. Williams
Keyword(s):  
Intermediate Filaments ◽  
Tetrahymena Pyriformis ◽  
Ciliated Protozoan ◽  
Microtubule Associated Proteins ◽  
Fine Filament ◽  
Helical Content ◽  
In Vitro Assembly ◽  
Associated Proteins ◽  
Filament Bundles

The cortex of the ciliated protozoan Tetrahymena contains a number of fibrous elements, including a network of filaments that pervades the feeding organelle of this organism. The cluster of polypeptides (79–89K; K = 10(3) Mr) in Tetrahymena pyriformis GL-C that constitute these filaments has been purified by in vitro assembly after solubilization in 1.0 M KI. Four distinct sets of these polypeptides, designated ‘tetrins’, have been shown to be distinguishable from each other by immunochemical and biochemical criteria. The smallest filaments reassembled in vitro were 3–4 nm in diameter and these fine filaments were seen to be bundled together into thicker strands of varying diameters, similar to those within the cell. The thicker filament bundles were clearly distinguishable from intermediate filaments, but fine filaments in these bundles were superficially similar to the 2–5 nm filaments described as microtubule-associated proteins in other organisms. The ultrastructure of the tetrin filaments localized within the feeding organelle reveals a substantial presence of these filaments apart from microtubules. In addition, circular dichroism measurements indicate a relatively low alpha-helical content for these filaments and suggest that the tetrins may be substantially different from other fine filament proteins such as the tektins and giardins.

Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

2000 ◽  
Vol 113 (13) ◽  
pp. 2471-2483 ◽  
Author(s):  
I. Hofmann ◽  
C. Mertens ◽  
M. Brettel ◽  
V. Nimmrich ◽  
M. Schnolzer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intermediate Filament ◽  
Solid Phase ◽  
Specific Interaction ◽  
Binding Assays ◽  
Intermediate Filament Proteins ◽  
Amino Terminal ◽  
Vitro Analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins

1983 ◽  
Vol 3 (6) ◽  
pp. 1146-1156
Author(s):  
W J Nelson ◽  
P Traub
Keyword(s):  
Intermediate Filament ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphorylation Site ◽  
Limited Proteolysis ◽  
Complete Removal ◽  
Product Inhibition ◽  
Nucleic Acid Binding ◽  
Intermediate Filament Proteins

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2+-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.

A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix

1991 ◽  
Vol 98 (3) ◽  
pp. 281-291
Author(s):  
P. Belgrader ◽  
A.J. Siegel ◽  
R. Berezney
Keyword(s):  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Structural Integrity ◽  
Rnase A ◽  
Matrix Proteins ◽  
Intermediate Filament Proteins ◽  
Isolation And Characterization ◽  
Hela S3

Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.

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