The binding in vitro of modified LDL to the intermediate filament protein vimentin

Membrane-associated proteins with specific binding properties to modified LDL were investigated in J774 macrophages and Mono Mac 6 sr cells. Ligand blotting of membrane proteins revealed a 54-kDa protein which bound oxidized and acetylated but not native LDL. The 54-kDa protein, isolated by 2D-PAGE, was identified as vimentin. (125)I-AcLDL bound to purified vimentin and desmin in a saturable manner, with an approximate K(d) of 1.7 x 10(-7) M (89 microgram/ml) and 8.0 x 10(-8) M (41 microgram/ml), respectively. Blots of vimentin mutant proteins with deletions in the positively charged N-terminal head domain showed that amino acids 26-39 are essential for the binding of AcLDL by vimentin. Taken together, our data indicate that vimentin binds modified LDL, but not native LDL, in a specific and saturable manner. Vimentin filaments extend throughout the cytoplasm as far as the inner surfaces of plasma and vesicular membranes. Vimentin may thus play a role in membrane-associated steps involved in the intracellular processing of oxidized LDL, contributing to its unregulated uptake and intracellular retention by cells of the atherogenic plaque.     

Actin-binding domain of mouse plectin. Crystal structure and binding to vimentin.

Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2alpha) from mouse, to a resolution of 1.95 and 2.0 A. Comparison of pleABD/2alpha with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2alpha has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks.     

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

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.     

Cdc42Hs and Rac1 GTPases induce the collapse of the vimentin intermediate filament network

In this study we show that expression of active Cdc42Hs and Rac1 GTPases, two Rho family members, leads to the reorganization of the vimentin intermediate filament (IF) network, showing a perinuclear collapse. Cdc42Hs displays a stronger effect than Rac1 as 90% versus 75% of GTPase-expressing cells show vimentin collapse. Similar vimentin IF modifications were observed when endogenous Cdc42Hs was activated by bradykinin treatment, endogenous Rac1 by platelet-derived growth factor/epidermal growth factor, or both endogenous proteins upon expression of active RhoG. This reorganization of the vimentin IF network is not associated with any significant increase in soluble vimentin. Using effector loop mutants of Cdc42Hs and Rac1, we show that the vimentin collapse is mostly independent of CRIB (Cdc42Hs or Rac-interacting binding)-mediated pathways such as JNK or PAK activation but is associated with actin reorganization. This does not result from F-actin depolymerization, because cytochalasin D treatment or Scar-WA expression have merely no effect on vimentin organization. Finally, we show that genistein treatment of Cdc42 and Rac1-expressing cells strongly reduces vimentin collapse, whereas staurosporin, wortmannin, LY-294002, R(p)-cAMP, or RII, the regulatory subunit of protein kinase A, remain ineffective. Moreover, we detected an increase in cellular tyrosine phosphorylation content after Cdc42Hs and Rac1 expression without modification of the vimentin phosphorylation status. These data indicate that Cdc42Hs and Rac1 GTPases control vimentin IF organization involving tyrosine phosphorylation events.     

The binding in vitro of the intermediate filament protein vimentin to synthetic oligonucleotides containing telomere sequences

The ability of the intermediate filament subunit protein vimentin to bind synthetic oligonucleotide telomere models containing repeat sequences from Oxytricha (T4G4), Saccharomyces (TGTGTG3), or Tetrahymena (T2G4) was investigated in vitro with a filter binding assay and a gel overlay assay. At low ionic strength, vimentin bound these oligonucleotides with high affinity. At higher ionic strength, the vimentin-oligonucleotide complex was less stable, such that approximately 30% of the initial binding remained at 150 mM KCl. One mole of vimentin tetramer bound approximately 1 mol of telomere oligonucleotide. Vimentin bound well oligonucleotides containing either a random duplex or random 3'-overhang, but showed a reduced affinity for a blunt-ended oligonucleotide. A control random sequence oligonucleotide was not bound by vimentin. The oligonucleotide-binding site of vimentin was shown to be localized in the non-alpha-helical N-terminal domain by assays employing purified proteolytic fragments of vimentin. Preliminary results in the gel overlay assay show that other members of the intermediate filament family, nuclear lamins A-C, all bind the synthetic oligonucleotide containing the telomere repeat sequence of Oxytricha.     

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: Implications for evolution of chordate IF proteins

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I–IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.     

Binding of nucleic acids to intermediate filaments of the vimentin type and their effects on filament formation and stability.

Guanine-rich polynucleotides such as poly(dG), oligo(dG)12-18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)25 was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA, although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG).poly(dC), and, of those of the RNA type, preferentially poly(rA).poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.     

Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results.     

Expression profiles of the essential intermediate filament (IF) protein A2 and the IF protein C2 in the nematode Caenorhabditis elegans

The multigene family of intermediate filament (IF) proteins in Caenorhabditis elegans covers 11 members of which four (A1-3, B1) are essential for development. Suppression of a fifth gene (C2) results in a dumpy phenotype. Expression patterns of three essential genes (A1, A3, B1) were already reported. To begin to analyze the two remaining RNAi phenotypes we followed the expression of the A2 and C2 proteins. Expression of A2 mRNA starts in larval stage L1 and continues in the adult. Transgenic A2 promoter/gfp larvae strongly display GFP in the main body hypodermis but not in seam cells. This pattern and the muscle displacement/paralysis induced by RNAi silencing are consistent with the role of this protein in keeping the correct hypodermis/muscle relationship during development. IF protein C2 occurs in the cytoplasm and desmosomes of intestinal cells and in pharynx desmosomes. Expression of C2 starts in the late embryo and persists in all further stages.     

Properties of the nonhelical end domains of vimentin suggest a role in maintaining intermediate filament network structure

To investigate the functional role of the nonhelical domains of the intermediate filament (IF) protein vimentin, we carried out transient transfection of constructs encoding fusion proteins of these domains with enhanced green fluorescent protein (EGFP). Expression of these fusion proteins did not have any effect on the endogenous IF networks of transfected cells. However, the head domain-EGFP fusion protein localized almost exclusively to the nucleus. This localization could be disrupted in a reversible fashion by chilling cells. Furthermore, the head domain was capable of targeting to the nucleus a strictly cytoplasmic protein, pyruvate kinase. Thus, the vimentin head domain contains information that specifically directs proteins into the nucleus. In contrast, the nonhelical tail domain of vimentin, when expressed as a fusion protein with EGFP, was retained in the cytoplasm. Cytoplasmic retention of tail domain-containing fusion proteins appeared to be dependent on the integrity of the microtubule network. Our results are consistent with a proposal that the nonhelical end domains of vimentin are involved in maintaining an extended IF network by exerting oppositely directed forces along the filaments. The head domains exert a nuclear-directed force while the tail domains extend the IF network toward the cell periphery via a microtubule-dependent mechanism.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.     

The beaded filament of the eye lens: an unexpected key to intermediate filament structure and function

In 1959, an unusual filamentous polymer, now called the beaded filament, was described in the lens of the eye. The constituent proteins, assembly properties and functions of the beaded filament have been elusive. The recent publication of the sequences for two major lens filament proteins (CP49 and filensin) and the reconstitution in vitro of structures closely resembling beaded filaments, suggests that the beaded filament is related structurally to intermediate filaments (IFs). The association of the lenticular chaperones, the alpha-crystallins, with the filament contributes to the characteristic beaded morphology, as well as giving important clues to the function of this unusual filament in the lens. These recent results have several implications for IF function and assembly.     

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.     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

Distribution of the intermediate filament proteins vimentin,keratin, and desmin in the bovine ovary

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore-active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley-Liss, Inc.     

The gene for a cytoplasmic intermediate filament (IF) protein of the hemichordate Saccoglossus kowalevskii; definition of the unique features of chordate IF proteins

We have isolated full length cDNAs encoding a cytoplasmic intermediate filament (IF) protein of a priapulid (Priapulus caudatus) and of a hemichordate (Saccoglossus kowalevskii) and determined the organisation of the hemichordate gene. As expected, the priapulid protein shows the long coil 1b subdomain and the lamin tail homology segment already known for cytoplasmic IF proteins from 11 other protostomic phyla. Surprisingly, the hemichordate protein follows in domain organisation much more closely the protostomic IF proteins than the chordate IF proteins. Thus, the lack of a lamin tail homology segment together with a deletion of 42 residues in the coil 1b domain is a molecular feature restricted to the chordates. We propose that the known IF subfamilies I to IV developed by gene duplications and sequence drift after the deletion in coil 1b arose at the base of the chordate branch.