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PubMed1984

Intermediate filaments in monkey kidney TC7 cells: focal centers and interrelationship with other cytoskeletal systems.

Two-dimensional gel electrophoresis of intermediate-sized filament-enriched cytoskeletons of epithelial monkey kidney TC7 cells has shown that they are composed of at least two keratins (isoelectric focusing 36, Mr = 48,500; IEF 46, Mr = 43,500; HeLa protein catalogue number) and vimentin. Indirect immunofluorescence as well as immunoelectron microscopy using antibodies directed against specific polypeptides sometimes revealed a discontinuous staining of keratin-containing filaments. Indirect immunofluorescence analysis of cells stained with keratin or vimentin antibodies also revealed a bright perinuclear staining in 58% of the cells in interphase. Of particular interest were focal centers from which filaments radiated. Double-label immunofluorescence using tubulin and keratin antibodies showed that these centers codistributed with focal arrays of microtubules (most likely centrosomes) in interphase cells but were not colocalized with centrioles in mitosis or, in many cases, with the microtubule organizing centers seen after release from nocodazole treatment. Treatment of TC7 cells with demecolcine (10 micrograms/ml, 20 hr) resulted in a drastic rearrangement of the keratin and vimentin filaments. Likewise, treatment with cytochalasin B (10 micrograms/ml, 1 hr) produced a star-like arrangement of the keratin and vimentin filaments and, in most cases, these codistributed with patches of actin. The results provide evidence for the interaction of intermediate filaments (keratins and vimentin) with both microtubules and microfilaments.

Animals↗
PubMed1983

The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole.

Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.

Alkaloids↗
PubMed1985

The coiled-coil molecules of intermediate filaments consist of two parallel chains in exact axial register.

Amino acid sequence studies of helical particles derived from proteolytic digests of mouse epidermal keratin intermediate filaments (IF) have shown that their coiled-coil molecules are heterodimers of Type I and Type II keratins, with a parallel arrangement of the two chains. From a reappraisal of published chemical cross-linking data, it is concluded that the coiled-coil molecules in all IF consist of pairs of parallel chains in precise axial register.

Amino Acid Sequence↗
PubMed1987

Induction and segregation of glial intermediate filament expression in the RT4 family of peripheral nervous system cell lines.

We have found glial fibrillary acidic protein (GFAP), the major component of astrocyte intermediate filaments, to be expressed in cell lines of the RT4 peripheral neurotumor family. The RT4 family is a "stem-cell-like" cell line, RT4-AC, that spontaneously undergoes differentiation in culture to three derivative cell types. This process, termed cell-type conversion, results in a segregation among the derivative cell types of parental cell phenotypes that have been described as neuronal-like or glial-like. We have identified a 50-kDa GFAP-immunoreactive cytoskeletal protein and GFAP mRNA in continuous RT4-AC and RT4-D (glial-type derivative) cell lines, but not in two presumptive neuronal-type cell lines. This result suggests that GFAP gene expression is coordinately coupled with the expression of other glial properties during cell-type conversion. In addition, the RT4-AC and RT4-D sublines were found to significantly express GFAP only at high cell densities and not during logarithmic growth and to express GFAP precociously during morphological differentiation following treatment with 1 mM N6, O2'-dibutyryladenosine 3',5'-cyclic monophosphate. These observations closely reflect reports of glial filament expression in astrocyte cultures, suggesting that a common regulatory mechanism is employed by central and peripheral nervous system glia.

Animals↗
PubMed2002

The intermediate filament protein keratin 8 is a novel cytoplasmic substrate for c-Jun N-terminal kinase.

Keratins 8 (K8) and 18 are the primary intermediate filaments of simple epithelia. Phosphorylation of keratins at specific sites affects their organization, assembly dynamics, and their interaction with signaling molecules. A number of keratin in vitro and in vivo phosphorylation sites have been identified. One example is K8 Ser-73, which has been implicated as an important phosphorylation site during mitosis, cell stress, and apoptosis. We show that K8 is strongly phosphorylated on Ser-73 upon stimulation of the pro-apoptotic cytokine receptor Fas/CD95/Apo-1 in HT-29 cells. Kinase assays showed that c-Jun N-terminal kinase (JNK) was also activated with activation kinetics corresponding to that of K8 phosphorylation. Furthermore, K8 was also phosphorylated on Ser-73 by JNK in vitro, yielding similar phosphopeptide maps as the in vivo phosphorylated material. In addition, co-immunoprecipitation studies revealed that part of JNK is associated with K8 in vivo, correlating with decreased ability of JNK to phosphorylate the endogenous c-Jun. Taken together, K8 is a new cytoplasmic target for JNK in Fas receptor-mediated signaling. The functional significance of this phosphorylation could relate to regulation of JNK signaling and/or regulation of keratin dynamics.

Amino Acid Sequence↗
PubMed1990

Specific interaction of the intermediate filament protein vimentin and its isolated N-terminus with negatively charged phospholipids as determined by vesicle aggregation, fusion, and leakage measurements.

The interaction of the intermediate filament protein vimentin and its non-alpha-helical N-terminus with phosphatidylserine and phosphatidylinositol small unilamellar vesicles was investigated by measuring vesicle aggregation, fusion, and leakage. While the N-terminus suppressed Ca2(+)-induced fusion of phosphatidylserine vesicles, it caused their rapid aggregation in the absence of Ca2+; at a molar ratio of lipid to polypeptide of 25:3, the polypeptide/lipid complexes precipitated from the reaction mixture. This aggregation was efficiently diminished by NaCl. The phosphatidylinositol vesicles, on the other hand, became leaky when interacting with the N-terminus of vimentin, even at a molar ratio of lipid to polypeptide of 500:1. The leakage of phosphatidylinositol vesicles was suppressed by the addition of Ca2+ or NaCl to the reaction mixture. Intact vimentin also caused leakage of phosphatidylinositol vesicles, at low and high salt concentration. The results indicate specific and differential interactions of the N-terminus of vimentin with various negatively charged lipid species, although there is an electrostatic component common to these interactions.

Amino Acid Sequence↗
PubMed2000

Dissection of protein linkage between keratins and pinin, a protein with dual location at desmosome-intermediate filament complex and in the nucleus.

Pinin is a cell adhesion-associated and nuclear protein that has been shown to localize in the vicinity of intermediate filament (IF) convergence upon the cytoplasmic face of the desmosomal plaque as well as in the nucleus. The localization of pinin to the desmosomes has been correlated with the reinforcement of intercellular adhesion and increased IF organization. In this study, keratins 18, 8, and 19 were identified to interact with the amino end domain of pinin in a two-hybrid screening. Further truncation analyses indicated that the 2B domain of keratin contains the sequence responsible for interacting with pinin. The amino end of pinin (residues 1-98) is sufficient to bind to keratin. Point mutation analyses revealed two essential residues within the pinin fragment 1-98, leucine 8 and leucine 19, for the interaction with keratin. Finally, in vitro protein overlay binding assays confirmed the direct interaction of the amino end domain of pinin with keratins, while pinin mutant L8P GST fusion protein failed to bind to keratins in the overlay assay. Coupled with our previous morphological observations and transfection studies, these data suggest that pinin may play a role in epithelial cell adhesion and the IF complex through a direct interaction with the keratin filaments.

Cell Adhesion Molecules↗
PubMed1995

A complex of intermediate filament protein-DNA: a target for autoantibodies in systemic lupus erythematosus?

Autoantibodies in systemic lupus erythematosus which cross-react with double stranded DNA and intermediate filament proteins are frequently reported. However, little is known about the origin and the target of these antibodies. In this paper, a polyspecific monoclonal antibody, XY12, produced by the immunization of genetically non-autoimmune mice with a DNA-protein complex is detailed. Its antigen binding patterns are very similar to the autoantibodies. The data suggest that these autoantibodies may be triggered by a circulating nucleoprotein.

Animals↗
PubMed1999

Characterization of the nucleic acid binding region of the intermediate filament protein vimentin by fluorescence polarization.

Employing deletion mutant proteins and fluorescein-labeled oligodeoxyribonucleotides in a fluorescence polarization assay, the nucleic acid binding site of the intermediate filament (IF) subunit protein vimentin was localized to the middle of the arginine-rich, non-alpha-helical, N-terminal head domain. While deletion of the first few N-terminal residues (up to amino acid 17) had almost no effect, deletions of residues 25-64 or 25-68 essentially abolished the binding of nucleic acids by the respective proteins. Proteins with smaller deletions, of residues 25-39 or 43-68, were still able to bind nucleic acids quite well at low ionic strength, but only the proteins containing the first DNA-binding wing (residues 27-39) retained the ability to stably bind nucleic acids at physiological ionic strength. These results were confirmed by data obtained with two synthetic peptides whose sequences correspond to the smaller deletions. Nitration experiments showed that one or more of the tyrosines in the head domain are responsible for the stable binding by intercalation. Interestingly, the residues responsible for binding nucleic acids can be deleted without major influence on the in vivo polymerization properties of the mutant proteins. Only the protein with the largest internal deletion, of residues 25-68, failed to form filaments in vivo. Since the N-terminal head domains of IF proteins are largely exposed on the filament surface, but nevertheless essential for filament assembly, these results support the model that the middle of the head domain of vimentin may loop out from the filament surface and thus be available for interactions with other cellular structures or molecules.

Amino Acid Sequence↗
PubMed1985

Antibodies to vimentin intermediate filaments in sera from patients with SLE and RA: quantitation by solid phase radioimmunoassay.

A solid phase radioimmunoassay was used to quantify levels of antibodies to vimentin intermediate filaments (VIF) of the cytoskeleton in sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Significantly higher levels of these antibodies were found in 52% of the SLE sera studied, compared to 6% in RA sera and 10% of sera from healthy controls. These antibodies include IgG and IgM classes of immunoglobulins. Our results show that high levels of anti-VIF antibodies are more prevalent in SLE sera, whereas no significant differences are found between sera from patients with RA and healthy controls.

Adult↗
PubMed1983

Complement-mediated binding of monocytes to intermediate filaments in vitro.

An in vitro model for complement (C)-mediated monocyte binding to injured cells is described. This model is based on the property of cytoskeletal intermediate filaments (IMF) to directly and independently activate serum C via the classic pathway. Fixed monolayers of cultured fibroblasts, endothelial cells, and L 132 epithelial cells were each used as targets. The authors first subjected the target cells to limited detergent extraction in order to make permeable the plasma membrane, and then exposed them to normal human serum. Immunohistologic analysis demonstrated that this process permitted binding of clearly demonstrable amounts of C1q, C4, and C3 to cytoplasmic loci containing IMF. The target cells were then incubated for various lengths of time with peripheral blood mononuclear cells containing about 20% monocytes. Preferential binding of monocytes was demonstrated by histochemical and immunologic staining methods. Only 5% of the attached cells were B lymphocytes. By transmission electron microscopy the attached cells exhibited typical monocyte morphology. Attachment to target cells involved contact with areas rich in IMFs and led to signs of activation; both an increased number of cytoplasmic organelles and phagocytosis of target cell material were observed. Attachment of monocytes was clearly dependent on serum treatment of the targets; the ratio was 10:1 when compared with control experiments using target cells that had not been incubated with serum. It is reasonable to consider that bound monocyte-derived macrophages associate with activated C components, most likely C3b fragments or C1q, for which monocyte surface receptors have been established. This mechanism may be triggered in various inflammatory reactions involving cell and tissue injury and as an event in the final stages of macrophage-mediated removal of injured or nonviable cells.

Animals↗
PubMed1999

The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Animals↗
PubMed1985

Incidence of anti-intermediate filament antibody in serum samples of students with suspected glandular fever.

Serum samples from 40 students with suspected infectious mononucleosis were tested for the presence of antibodies to intermediate filaments (AIFA) of the cytoskeleton. Twenty had antibodies to the Epstein-Barr virus capsid antigen before their illness, and during it their sera remained negative by the Paul-Bunnell test. The other 20 patients did not have antibodies to the Epstein-Barr virus capsid antigen before their illness and seroconverted during the illness. These patients (true infectious mononucleosis group) developed positive Paul-Bunnell tests. Sera from normal subjects (blood donors) were also tested for AIFA. AIFA was present in titres greater than 1/10 in 80% of the infectious mononucleosis group (mean titre 1/40-1/80), 10% of the Paul-Bunnell negative glandular fever group, and 8.5% of the normal blood donors.

Adult↗
PubMed1985

Use of monoclonal antibodies for analyzing the distribution of the intermediate filament protein vimentin in human non-Hodgkin's lymphomas.

A series of human non-Hodgkin's lymphomas was examined for immunoreactivity with monoclonal antibodies to the intermediate filament protein vimentin with the use of an avidin-biotin immunoperoxidase method. The lymphoid cell nature of each tumor was established with the use of a panel of monoclonal antibodies to lymphoid cell differentiation antigens. There were 28 B-cell and 2 T-cell lymphomas in the series; of the 30 tumors, 11 (37%) were immunoreactive for vimentin. There was no correlation between vimentin immunoreactivity and the histopathologic type of lymphoma. In some tumors, there was nonspecific stromal immunoreactivity for vimentin, but the neoplastic lymphocytes were not immunoreactive. The selective expression of vimentin in non-Hodgkin's lymphomas may be due to masking of the appropriate epitopes or to selective expression of the vimentin gene in certain tumors. On the basis of these results, monoclonal antibodies to vimentin appear to be of limited usefulness in establishing the diagnosis of non-Hodgkin's lymphoma.

Antibodies, Monoclonal↗