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PubMed1987

Lamin B constitutes an intermediate filament attachment site at the nuclear envelope.

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.

Animals↗
PubMed1994

Axonal transport of type III intermediate filament protein peripherin in intact and regenerating motor axons of the rat sciatic nerve.

Slow axonal transport of peripherin has been studied in the motor axons of both intact and regenerating rat sciatic nerves 7 days post-crush. The studies were done by two-dimensional gel electrophoresis after intraspinal injection of 35S-methionine. In the first experiment, the sciatic nerves were removed 3 weeks after the radiolabeling pulse and cut into 6 mm segments. Each nerve segment was submitted to two-dimensional gel electrophoresis and analyzed by an original procedure which allowed us to study the distribution along the nerve of the radioactivity associated with several proteins of the cytoskeleton, especially the intermediate filament proteins, peripherin, and the low molecular mass neurofilament protein, NF-L. Peripherin was transported at two main rates: 66% of the total radiolabeled peripherin moved at 1.42 mm/day and the remainder moved at 2.28 mm/day. The radioactivity associated with NF-L exhibited a similar pattern. In the second experiment, similar intraspinal injections were made 7 days after a unilateral crush of the sciatic nerve. Regenerating nerves exhibited a clear SCa wave. However, in contrast to the intact nerves, the SCb wave could not be precisely defined in the regenerating nerves. Thus, the changes in the amount of transported proteins were analyzed in the SCa wave only. Autoradiograms of 2D-PAGE revealed that in the regenerating axons, the quantity of transported peripherin in SCa was increased by 3.5-fold. In contrast, the quantity of transported NF-L was decreased by 1.6-fold. The regenerating motor axons conveyed significantly greater (approximately twofold) amounts of labeled tubulins and actin than did intact motor axons. Our results suggest that peripherin, although mainly conveyed by SCa, plays a role during the elongation process in addition to actin and tubulin.

Animals↗
PubMed2003

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity.

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.

Amino Acid Sequence↗
PubMed1994

Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly.

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.

Amino Acid Sequence↗
PubMed1985

Receptor-mediated phosphorylation of astroglial intermediate filament proteins in cultured astroglia.

Primary cultures of purified astroglia have been shown to exhibit a variety of membrane receptors that regulate intracellular cyclic AMP levels. The experiments described in this paper were completed to examine the effect of such receptor agonists on protein phosphorylation in intact astroglia. An analysis of 32P-labelled proteins derived from whole cell extracts and separated via two-dimensional gel electrophoresis indicated that increasing cyclic AMP levels in astroglia stimulated the phosphorylation of two distinct proteins that had apparent molecular weights/isoelectric points (pI) of 51K/6.0 and 57K/5.7. Similar experiments with cultured meningeal cells indicated that only the 57K/5.7 protein was phosphorylated in response to elevated levels of cyclic AMP. The 51K/6.0 protein was never observed in gels derived from meningeal cells. Immunoblot experiments indicated that the 51K/6.0 protein stained with antiserum to glial fibrillary acidic protein (GFAP) and the 57K/5.7 protein stained with antibodies to vimentin. Concentration-effect studies indicate that these proteins are maximally phosphorylated at concentrations of receptor agonists that only slightly elevate cyclic AMP levels. All receptor agonists that have been shown to increase cyclic AMP levels appear similarly efficacious with respect to increasing the phosphorylation of the two proteins. These experiments suggest that the membrane receptors present on astroglia function, in part, to regulate phosphorylation of the intermediate filament proteins GFAP and vimentin.

Alprostadil↗
PubMed1995

Immunocytochemical localization of protein kinases Yes and Src in amoeboid microglia in culture: association of Yes kinase with vimentin intermediate filaments.

Amoeboid microglia isolated from primary cultures of neonatal rat brain correspond to a transient form of activated microglia, a resident population of macrophage-like cells. In order to understand the molecular aspects of microglial activation, we have studied amoeboid microglia in primary culture for the presence of Yes and Src protein tyrosine kinases, two kinases which have been implicated in signal transduction process during monocyte/macrophage activation. Immunofluorescence with an antibody raised against the peptide from unique N-terminal domains of Yes and Src demonstrated that Yes and Src kinases are enriched in perinuclear areas of amoeboid microglia. In addition, the antibody to c-yes peptide had a cytoplasmic distribution which coincided with the distribution of vimentin-containing intermediate filaments. Preadsorption of anti-c-yes antibody with an excess of antigenic peptide inhibited anti-c-yes immunofluorescence, while vimentin immunofluorescence remained unchanged. Double immunofluorescence images analyzed with the two-dimensional intensity distribution program (2-D scattered histograms) on Zeiss confocal scanning laser microscope demonstrate the colocalization of c-yes with vimentin. The extent of colocalization was more prominent after exposure of intact cultured microglia to dibutyryl cyclic AMP (dBcAMP), or to phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) or to okadaic acid, an inhibitor of protein phosphatases. The findings suggest that vimentin might serve as molecular support for Yes kinase and, since previous studies have shown that vimentin in amoeboid microglia is one of the major protein substrates of serine/threonine protein kinases, this function could be regulated by phosphorylation.

Animals↗
PubMed1995

The assembly state of the intermediate filament proteins desmin and glial fibrillary acidic protein at low ionic strength.

The low ionic strength structures of the type III intermediate filament (IF) proteins desmin and glial fibrillary acidic protein (GFAP) have been studied by transient electric birefringence measurements. Flexible dimers with a length of around 45 nm, particles with a length of 68 +/- 6 nm (presumably tetramers and hexamers) and larger aggregates of 108 +/- 19 nm are found. GFAP has an increased tendency to aggregate upon lowering of the pH. The aggregation state of desmin does not change in the pH range studied. The results are compared with previous results on vimentin.

Animals↗
PubMed1994

Characterization of multiple oligomeric vimentin intermediate filament units by transient electric birefringence measurements.

In this work we have studied the structure of soluble vimentin units from which intermediate filaments (IFs) are built. Several oligomeric forms have been presented in the literature as IF "building blocks", but there is still no agreement on this matter. By comparing our data with various models as proposed in the literature we can favour certain models and reject others. Transient electric birefringence (TEB) measurements were performed from which information is obtained concerning electric and hydrodynamic properties of the particles under investigation. TEB decay analysis at pH 6.8 after 70 microseconds pulses (at 20 degrees C in aqueous solution) yielded three decay times: 1.1(+/- 0.3) microseconds, 4.0(+/- 1.0) microseconds and 20.0(+/- 5.0) microseconds, with amplitudes of 45% to 60%, 30% to 45%, and less than 10%, respectively. At pH 8.5 after 70 microseconds pulses, more than 90% of the TEB signal with the second decay time is obtained, while the remainder had a decay time of 15.0(+/- 4.0) microseconds. Only when the pulse duration was decreased, the fast decay time around 1 microsecond was observed, suggesting that only a minor fraction of the particles at this pH value causes such a short decay time. At both pH values, the TEB measurements indicated that, at least in part, the molecules are oriented by a permanent dipole moment. It will be shown that the shortest decay time originates from bent or flexible dimers, and the second decay time from particles with a length of 54 to 65 nm containing, at least in part, a relatively large overall dipole moment. The longest decay time is probably due to larger aggregates. These results are consistent with a model in which single dimers, antiparallel staggered tetramers and hexamers coexist. Alternatively, but less likely on the basis of literature data, a model of parallel in-register tetramers with a considerable length contribution of the head groups would fit our research.

Animals↗
PubMed1996

Pathways of glycosphingolipid biosynthesis in SW13 cells in the presence and absence of vimentin intermediate filaments.

We reported previously that the incorporation of sugars into glycosphingolipids (GSL) is diminished in SW13 cells that lack a vimentin intermediate filament (IF) network (vim-) compared to vim+ cells. To further analyze the nature of this abnormality, we double-labeled cells with 3H-serine and 14C-sugars. There was no difference between vim+ and vim- cells in the incorporation of serine into GSL, although the usual difference in sugar incorporation was observed. This indicated that the defect in vim- cells was not in the incorporation of sugars into ceramide synthesized de novo by acylation of sphinganine (pathway 1). Sugars can also be incorporated into ceramide synthesized from sphingosine that is derived from catabolism of sphingolipids (pathway 2), and into GSL that recycle through the Golgi apparatus from endosomes (pathway 3). The amount of galactose and glucosamine incorporated into GSL in these three pathways was analyzed by the use of two inhibitors of sphingolipid biosynthesis. beta-Chloroalanine inhibits the de novo synthesis of sphinganine (pathway 1), and fumonisin B1 inhibits the acylation of sphinganine and sphingosine (pathways 1 and 2). We were surprised to observe that in both vim+ and vim- cells only 20-40% of sugar incorporation into GSL took place in pathway 1, and 60-80% of sugar incorporation took place in the recycling pathways. Moreover, in contrast to larger GSL, GlcCer was not synthesized in pathway 3. Our observations indicate that vimentin IF facilitate the recycling of GSL and sphingosine, and that the differences between vim+ and vim- cells are predominantly in pathways 2 and 3. Furthermore, although it is generally believed that virtually all GSL are synthesized in the de novo pathway, these data indicate that the recycling pathways predominate in the incorporation of sugars into GSL in SW13 cells.

Acylation↗
PubMed1996

Adhesion properties, intermediate filaments and malignant behaviour of head and neck squamous cell carcinoma cells in vitro.

This study compared phenotype and behaviour of seven head and neck squamous cell carcinoma (HNSCC) cell lines and normal epithelial cells. Indications were found that in HNSCC cells: 1) loss of the cell-cell adhesion molecule E-cadherin was correlated with loss of epithelioid cell morphology but not with loss of cell-cell cohesion; 2) reduced expression of the cell-cell adhesion molecule desmoglein was unrelated to cell shape or motility; 3) high expression of the cell-substrate adhesion molecule alpha 6 beta 4, a receptor for laminin, corresponded with epithelioid colony formation while, in our adhesion assay, low expression of alpha 6 beta 4 paradoxically coincided with an increased adhesion to laminin; 4) presence of vimentin intermediate filaments coincided with loss of anchorage dependency. We propose that by simultaneous downregulation of E-cadherin, replacement of alpha 6 beta 4 by an aberrant laminin receptor and co-expression of vimentin a malignant phenotype arises of spindle-shaped, motile, anchorage-independent HNSCC cells with enhanced laminin-binding capacity.

Cadherins↗
PubMed1987

Application of antibodies to intermediate filament proteins in simple and complex tumors of the female genital tract.

Polyclonal antibodies to cytokeratins, vimentin, and desmin and monoclonal antibodies to vimentin and to individual cytokeratin polypeptides, specific for glandular epithelia (RGE 53) or kertinizing stratified squamous epithelia (RKSE 60), have been applied in gynecological tumors with simple or complex composition. In general, tumors with simple composition showed reaction patterns fitting their known epithelial or mesenchymal nature, i.e., cytokeratin positivity in epithelial tumors only, vimentin positivity in mesenchymal tumors, and expression of desmin and vimentin in muscle cell tumors. Rather frequently, coexpression of cytokeratins and vimentin was noted in endometrial adenocarcinomas. Tumors with complex composition, such as müllerian mesodermal mixed tumors (MMMTs), that may pose considerable problems in conventional histopathology revealed various reaction patterns, with either expression of only cytokeratins or coexpression of cytokeratins and vimentin in carcinomatous areas and expression of only vimentin in sarcomatous areas. However, in addition, some MMMTs contained cells that were also positive for desmin. Intermediate filament protein immunohistochemistry appeared to be helpful in establishing a diagnosis of MMMT and in characterizing the different tumor components, which may prove to be useful in the evaluation of gynecological treatment protocols.

Adenofibroma↗
PubMed2002

The parallel helices of the intermediate filaments of alpha-keratin.

Recent Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection technique (ATR) has been applied to alpha-keratin fibers (horse-hair) extended in water both at 21 and 95 degrees C. Infrared absorption bands in the Amide 1 region indicated that at extensions to 40-50% strain in water at 21 degrees C alpha-helices had completely disappeared and parallel beta-sheets were formed [Appl. Spectrosc. 55 (2001) 552]. However, when the hair fibers were extended to the same strain at 95 degrees C in water the result was the formation of anti-parallel beta-sheets. These results suggest that the relatively more stable anti-parallel beta-state [Polymer 10 (1969) 810] is only attained in extended alpha-keratin fibers at elevated temperatures and must result from major molecular rearrangement. It was concluded that the alpha-helices in the intermediate filaments (IFs) of alpha-keratin fibers must be parallel. This is in contrast to the previously accepted orientation of anti-parallel alpha-helices, based primarily on findings of X-ray diffraction studies of the structure of beta-keratin in highly extended fibers [Polymer 10 (1969) 810; Keratins, IL: Thomas Springfield (1972); Nature 316 (1985) 767].

Animals↗
PubMed1987

Regulation of human mesothelial cell differentiation: opposing roles of retinoids and epidermal growth factor in the expression of intermediate filament proteins.

We report here the discovery that retinoids are potent regulators of epithelial morphology and keratin expression in cultured human mesothelial cells. When LP-9 cells are cultured in medium supplemented with vitamin A-depleted serum, they grow with an extreme spindle-shaped morphology and synthesize abundant levels of vimentin, but very little keratin. When retinoic acid is added to the medium at 1 X 10(-8) to 1 X 10(-6) M, keratin synthesis is increased, vimentin synthesis is decreased, and the cells assume an epithelioid morphology. Keratin synthesis, but not epithelioid morphology, seems to be dependent on cell density as well: even when vitamin A is present, sparse cultures cannot fully maintain keratin synthesis. In contrast, epidermal growth factor (EGF) acts in an antagonistic fashion to suppress both keratin synthesis and epithelial morphology. The effects of vitamin A, EGF, and cell shape on intermediate filament (IF) expression seem to occur in a growth-independent manner, and they appear to be at the level of transcription or mRNA stability. Even so, their effects on IF expression do not appear to be rapid ones, and hence it is unlikely that these agents interact directly at the gene level to cause changes in IF gene expression.

Actins↗
PubMed2005

Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs).

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4-5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37 degrees C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 mug/ml), transferrin (10 mug/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

Animals↗