Occurrence in various mammalian cells and tissues of the Ca 2+ activated protease specific for the intermediate-sized filament proteins vimentin and desmin

Several mammalian cell lines propagated in suspension and monolayer culture and some normal and cancerous tissues from rat, hamster and cat were screened for the presence of the Ca 2+ activated protease specific for the intermediate-sized filament protein vimentin. Gel permeation chromatography on Sephacryl S-300 of postnuclear supernatants, and sucrose density gradient centrifugation of extracts from Triton X-100-resistant residual cell structures revealed the presence of the enzyme in all cells and tissues tested. Its apparent molecular weight amounted to 100 000. Except in the cases of a spontaneous rat lung tumour and a rat hepatocellular carcinoma induced by diethylnitrosamine, most of the enzyme was released into the postnuclear supernatant during cell or tissue extraction, indicating that it is of cytoplasmic origin. There was no correlation between the enzyme level and the vimentin content of cells and tissues. Rat and hamster liver as well as cat kidney, in which vimentin has not been detected by polyacrylamide gel electrophoresis, were relatively rich in the Ca 2+ activated protease. The experimental results point at the widespread, if not general, occurrence of the enzyme in mammalian cells.     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.     

Intermediate-sized filaments in Drosophila tissue culture cells

In using a monoclonal antibody against a major cytoplasmic protein of 46,000 mol wt, we have characterized an intermediate-sized (10 nm) filamentous cytoskeleton in Drosophila melanogaster tissue culture cells. Indirect immunofluorescence, immunoelectron microscopy, and protein blotting show that this cytoskeleton exhibits features typical of the vertebrate vimentin cytoskeleton, including the diameter and appearance of filaments, sensitivity to 10(-6) M colcemid, and insolubility in buffers containing 1% Triton X-100. The antibody cross- reacts with vimentin and desmin from baby hamster kidney cells and stains a vimentin cytoskeleton in the vertebrate Chinese hamster ovary cell line. We, therefore, conclude that the 46,000-mol wt Drosophila protein is homologous to vertebrate vimentin. Three minor, higher- molecular-weight polypeptides are also detected in the Drosophila cells that react with the antibody. At least two of these are members of a family of proteins with properties resembling those of the 46,000-mol wt intermediate filament protein.     

Differential sensitivity of vimentin and nuclear lamins from Ehrlich ascites tumor cells toward Ca2+ -activated neutral thiol proteinase

A comparative study of the susceptibility of vimentin and nuclear lamins from cultured Ehrlich ascites tumor (EAT) cells to degradation by Ca2+ -activated neutral thiol proteinase (calpain) has been undertaken. While pure vimentin was degraded very quickly at physiological ionic strength by purified calpain, isolated lamin B was digested comparatively slowly and purified lamins A/C were fairly resistant to proteolytic degradation. Similar digestion patterns were obtained from vimentin and lamin B with intermediary breakdown products close in size to the corresponding alpha-helical rod domains. To exclude the possibility that the low susceptibility of isolated lamins to Ca2+-dependent proteolytic degradation was due to irreversible denaturation during their isolation and purification, Triton cytoskeletons were prepared and their nuclear lamina as well as vimentin filaments were exposed to relatively large quantities of purified calpain. Under these conditions, not only vimentin filaments but also lamins A and B were digested while lamin C remained intact to a high degree. The major breakdown products of vimentin and lamins were identified as polypeptides which were 35 to 45 amino acids longer than the corresponding alpha-helical rod domains. Most of the vimentin-derived material and all high molecular weight polypeptides originating from lamins remained associated with the Triton cytoskeletons as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with immunoblotting. Indirect immunofluorescence and electron microscope analysis of the calpain-digested Triton cytoskeletons revealed that they still contained a laminalike structure around the nuclear chromatin and numerous structurally altered intermediate filaments in the cytoplasmic remnant, although all vimentin had been degraded with the formation of 40/41 kDa polypeptides as major digestion products. In untreated Triton cytoskeletons, the vimentin filaments seemed to be in direct physical contact with the nuclear lamina, whereas in digested Triton cytoskeletons there was a distinct gap between structurally altered filaments and the nuclear surface. This shows that vimentin filaments and the nuclear lamina are differentially susceptible to degradation by calpain under certain ionic conditions and suggests that both filamentous structures are intimately associated with each other.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.     

Interaction of the intermediate filament protein vimentin with ribosomal subunits and ribosomal RNA in vitro

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg²⁺ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca²⁺-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg²⁺, and (5) reductive alkylation of vimentin with pyridoxal-5-phosphate and NaBH₄ has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.     

Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells were stimulated with thrombin (1 unit/ml) for 15-30 s and then lysed with a solution of Triton X-100 containing [gamma-32P]adenosine triphosphate. Thrombin-stimulated human umbilical vein endothelial cells showed an enhanced incorporation of 32P into at least 12 different proteins as compared to control cells treated similarly. The observed enhanced phosphorylation required the active site of thrombin because diisopropylphosphoryl-thrombin had no effect on the level of phosphorylation. The molecular weight of one of the phosphoproteins was similar to that of the intermediate filament protein vimentin (55-60 kDa), a major protein in endothelial cells. This 59-kDa protein was Triton X-100-insoluble and reacted on a Western blot with antibody raised in guinea pig against Chinese hamster ovary cell vimentin. Addition of the anti-vimentin antibody to the thrombin-stimulated, phosphorylated lysate immuno-precipitated a single 32P-labeled protein (59 kDa). These results demonstrate that thrombin rapidly stimulates the phosphorylation of vimentin in cultured endothelial cells and links thrombin stimulation to the phosphorylation of a cytoskeletal protein.     

Influence of phospholipids on the formation and stability of vimentin-type intermediate filaments

The interaction of vesicles produced from individual phospholipids and mixtures thereof with preformed vimentin filaments as well as the influence of these vesicles on filament assembly were investigated employing negative stain electron microscopy and sucrose density gradient equilibrium centrifugation. Liposomes with a phospholipid composition characteristic of Ehrlich ascites tumor cells were able to bind efficiently to vimentin filaments without significantly affecting their morphology at higher concentrations. However, in sucrose density gradient centrifugation partial disintegration of the filaments was observed. In addition, larger quantities of phospholipid mixture totally blocked intermediate filament (IF) formation. Using vesicles of individual phospholipids, these effects could be shown to be due to the presence of negatively charged lipid species in the phospholipid mixture. While these were highly active in preventing filament assembly and in dissociating preformed filaments, electrically uncharged phospholipids were virtually inactive. The highest efficiency was shown by phosphatidylinositol-4,5-diphosphate. These results demonstrate that a negative surface charge of liposomes is an essential prerequisite for their successful and tight association with vimentin filaments. However, the high susceptibility of these filaments to photoaffinity labeling with the membrane-penetrating reagent 1-azidopyrene in the presence of phospholipid vesicles, points to additional interactions between hydrophobic regions of both reactants. Finally, the data also suggest a direct relationship between IFs and the lipid bilayer as the active principle underlying the association of IFs with natural membranes as observed by electron and immunofluorescence microscopy.     

An electron microscopic study of the interaction in vitro of vimentin intermediate filaments with vesicles prepared from Ehrlich ascites tumor cell lipids

The interaction of intermediate filaments prepared from pure, delipidated vimentin with vesicles obtained from Ehrlich ascites tumor (EAT) cell lipids was studied employing sucrose density gradient centrifugation in combination with electron microscopy. In negative stain electron microscopy, preformed vimentin filaments were seen in lateral association with lipid vesicles; end-on contacts of filaments with liposomes were rarely detected. When the reaction of filaments with vesicles was carried out at 0 degree C, sucrose density gradient equilibrium centrifugation of the reaction products led to the banding of relatively light filament-vesicle meshworks in clear separation from free filaments and free vesicles. With certain vimentin and lipid preparations, occasionally partial breakdown of the filaments during centrifugation and banding of vesicle-free fragments in denser regions of the sucrose gradients was observed. However, when the reaction mixtures were incubated at 37 degrees C prior to sucrose gradient analysis, all filaments were released from vesicles and totally fragmented during centrifugation. Electron microscopy showed unraveling of the filament fragments into subfilament strands. Employing lipid vesicles labeled with [3H]cholesterol, a low but significant amount of radioactivity was found to be associated with the fragments in a non-vesicular form. Filament reconstitution experiments performed in the presence of EAT cell lipids revealed an inhibitory effect of vesicles on filament assembly, particularly at lower temperatures. The mechanical labilization of the filament structure by lipid vesicles might play a role in the redistribution of intermediate filaments in the course of certain cellular processes involving turnover and fragmentation of intracellular membrane systems.     

The interaction in vitro of the intermediate filament protein vimentin with naturally occurring RNAs and DNAs

The binding of the intermediate filament protein vimentin to a variety of naturally occurring RNAs and DNAs was studied. The relative capacities of the various nucleic acids to associate with pure [3H]vimentin were determined in competition experiments with 28 S rRNA from Ehrlich ascites tumor cells. The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Under these ionic conditions, vimentin reacted preferentially with single-stranded nucleic acids, particularly with those of high (G + C) content. The vimentin binding potentials of single-stranded RNAs and DNAs were largely comparable. However, when the concentrations of mono- and divalent cations were raised to physiological and higher values, only single-stranded DNA retained its vimentin binding capacity. With increasing KCl concentrations at 0 to 1 mM Mg2+, increasing amounts of vimentin were detected in complexes which sedimented considerably faster than the bulk of the DNA, suggesting cooperative binding of vimentin. The salt optimum of this cooperativity was at 200 mM KCl. Thus, the capability of vimentin to discriminate between single-stranded RNA and DNA under physiological ionic conditions points to specificity of the interaction of vimentin with nucleic acids.     

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.     

A rapid method for the large scale purification of the intermediate filament protein vimentin by single-stranded DNA-cellulose affinity chromatography

A new method is described for the purification of the intermediate filament protein vimentin from Ehrlich ascites tumor cells using single-stranded DNA-cellulose affinity chromatography. The procedure is rapid and allows the large scale isolation of the protein. Partial characterization of vimentin shows that it has a molecular weight of 58000 and an apparent pI of 5.3. It can be degraded by the vimentin-specific, Ca²⁺-activated proteinase which results in the production of a characteristic set of degradation products. The vimentin also cross-reacts with the intermediate filament protein monoclonal antibody, α-IFA.     

Tenacious binding of lipids to vimentin during its isolation and purification from Ehrlich ascites tumor cells

Vimentin enriched in cytoskeletal frameworks by Triton X-100 extraction of Ehrlich ascites tumor cells and purified from a low ionic strength extract of the cell residues by (NH4)2SO4 precipitation and DEAE-Sepharose and ssDNA-cellulose chromatography in the presence of 6 M urea was highly contaminated with lipids. Thin-layer chromatography of a chloroform-methanol extract of the purified protein revealed, besides small amounts of phospholipids, the presence of large quantities of neutral lipids.     

Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin''s disease.

We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.     

Partial Purification of Amino Acid Transport Systems in Ehrlich Ascites Tumor Cell Plasma Membranes

Ehrlich ascites tumor cell plasma membranes were subjected to sequential selective protein extraction to identify protein components associated with amino acid transport. These membranes were extracted with Triton X-100 followed by 2,3-dimethylmaleic anhydride. Approximately 80% of the membrane proteins were extracted by these procedures while the original lipids were largely retained (～70%). The quantity of carbohydrate per milligram protein in the residue increased on extraction, consistent with an enrichment of glycoprotein in the residue.

The residual vesicles display the characteristic properties of Na⁺-coupled amino acid transport. These properties include Na⁺-stimulated uptake and Na⁺-gradient-stimulated uptake leading to an accumulation of the solute against its chemical gradient as well as inhibition of uptake by a competitive amino acid, L-methionine. The extracted vesicles exhibit a peak level of α-aminoisobutyrate uptake six times greater than that expected from equilibration of α-aminoisobutyrate. This accumulation is greater than that obtained with native vesicles, albeit slower. The accelerated exchange diffusion of L-leucine is not measurable in the residual vesicles after dimethylmaleic acid anhydride treatment, although it can be measured after Triton extraction. These results are consistent with the conclusion that the amino acid transport systems “A” (Na⁺-coupled) and “L” (Na⁺-independent) in Ehrlich cells, though having overlapping specificities for amino acids, and distinct physical entities.     

Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin-containing cells.

Using immunoelectron microscopy it is demonstrated that desmin subunits missing their complete carboxy-terminal domain are incapable of homopolymeric filament formation in vivo. Furthermore it is shown that, in vimentin-containing cells, desmin integrates into preexisting vimentin filaments resulting in desmin/vimentin heteropolymers. Removal of the amino-terminal or both nonhelical end domains of desmin increases Triton X-100 solubility of the mutant desmin subunits. Expression of desmin mutants containing deletions in the C-terminal part of the rod in vimentin-free cells results in an increase of the Triton X-100 solubility too. In contrast, if expressed in vimentin-containing cells, these mutant subunits remain in the Triton X-100 insoluble fraction. Deletion of the nonhelical carboxy-terminal domain only has no effect on solubility. In vimentin-free cells, stably expressed desmin subunits missing their amino-terminal domains display a slightly higher turnover rate compared to wild-type desmin. Transiently expressed desmin subunits missing 18 or more carboxy-terminal residues of the rod domain are rapidly degraded in vimentin-free cells. In vimentin-containing cells, turnover rates were much less pronounced. Finally, by using site-directed mutagenesis, we were able to map specific residues important for de novo filament assembly within the amino-terminal domain and in the conserved part at the C-terminus of the alpha-helical domain.     

Large scale co-isolation of vimentin and nuclear lamins from ehrlich ascites tumor cells cultured in vitro

Ehrlich ascites tumor (EAT) cells propagated in mass suspension culture were used as a starting material for the simultaneous isolation and purification of large quantities of the intermediate filament protein vimentin and the nuclear lamins A/C and B. Triton cytoskeletons, obtained by repeated washing of cells with a low ionic strength buffer containing Triton X-100 and 4 mM Mg2+, were extracted with 6 M urea at low salt concentration and in the presence of EDTA. Separation of solubilized proteins from unfolded chromatin (DNA) was accomplished by recondensation of the chromatin (DNA) in the presence of Mg2+ before centrifugation. To achieve separation of vimentin from nuclear lamins, the urea extract was subjected to DEAE-Sepharose CL-6B chromatography. Single-stranded DNA-cellulose chromatography was employed for the final purification of vimentin and for the separation of lamin B from lamins A/C. Further purification of lamin B was carried out by CM-Sepharose CL-6B chromatography and of lamins A/C by chromatography on hydroxylapatite. All chromatographies were performed in the presence of 6 M urea. 500 g of pelleted EAT cells yielded approximately 700 mg of vimentin, 225 mg of lamins A/C and 21 mg of lamin B. 2D-polyacrylamide gel electrophoresis revealed great microheterogeneity of lamins A/C, which to a high extent was due to phosphorylation, whereas lamin B was much less heterogeneous. In the absence of urea and at low salt concentration, lamins A/C required pH 5 to stay in solution whereas lamin B required pH 7.5. Increasing the salt concentration to 150 or 250 mM NaCl resulted in the formation of paracrystals from a urea-free mixture of lamins A/C and B. Although the lamins could not be assembled into intermediate filaments under a variety of ionic conditions, the preparations obtained will be useful for further biochemical characterization of these nuclear proteins.     

On the mechanism of glycolysis stimulation by neutral detergents in 3T3 and Ehrlich ascites tumor cells

Glycolysis of 3T3 and Ehrlich ascites tumor cells was greatly enhanced by Nonidet P-40 or Triton X-100 at about 100 micrograms/mg cell protein. This enhanced glycolysis was partly sensitive to rutamycin and partly sensitive to ouabain, suggesting that the detergent released the control of the ATPase of the mitochondria and of the plasma membrane Na+K+-ATPase. Nonidet P-40 had no effect on glycolysis in cell-free extracts from Ehrlich ascites tumor cells to which soluble mitochondrial ATPase was added. Measuring ouabain-sensitive 22Na efflux and using ouabain-sensitive lactate production as a measure of ATP hydrolysis by the Na+K+ pump, it was shown that Nonidet P-40 greatly decreased the efficiency of the Na+K+ pump. Quercetin increased the efficiency of pumping in EAT cells both in the absence and presence of the detergent.     

Solubilization of alkyldihydroxyacetone-P synthase from Ehrlich ascites cell microsomal membranes.

The enzyme, alkyldihydroxyacetone-P synthase, has been solubilized and partially purified from microsomal preparations of Ehrlich ascites cells after treatment with Triton X-100 and phospholipase C, followed by chromatography on Sepharose 4B. When the Triton X-100 was removed after solubilization the enzyme was still active but eluted in the void volume of the Sepharose 4B column, whereas in the presence of detergent it eluted much later as a single peak of activity, indicating that the solubilized enzyme tends to aggregate unless detergent is present. The lower molecular weight form of alkyldihydroxyacetone-P synthase (in detergent) had an estimated molecular mass of 250,000–300,000 daltons.