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PubMed1998

Rho-associated kinase phosphorylates desmin, the myogenic intermediate filament protein, at unique amino-terminal sites.

We obtained evidence that Rho-associated kinase (Rho-kinase) phosphorylates desmin, the myogenic intermediate filament protein, with approximately 2 mol phosphate per mole of desmin in vitro. Desmin phosphorylated by Rho-kinase lost the potential to form 10-nm filaments. Thr-16, Thr-75, and Thr-76 on desmin proved to be the major phosphorylation sites for Rho-kinase. All these sites are located within the head domain and are different from the reported phosphorylation sites of protein kinase. A, protein kinase C, and cdc2 kinase. We are entertaining the notion that Rho-kinase may regulate filament structures of desmin by site-specific phosphorylation.

Amino Acid Sequence↗
PubMed1980

Intermediate filaments of baby hamster kidney (BHK-21) cells and bovine epidermal keratinocytes have similar ultrastructures and subunit domain structures.

Structural features of the intermediate filaments (IF) of bovine epidermal keratinocytes (keratin filaments) and of baby hamster kidney (BHK-21) cells were studied to define the molecular basis of the similarities and differences in their properties. Purified subunits of the IF were specifically cleaved at their methionine and tryptophan residues. Peptide maps indicated that the IF subunits of BHK-21 cells were similar but not identical to each other and quite different from each of the keratin IF subunits. Intact IF were subjected to limited tryptic digestion, and structurally identical alpha-helix-enriched particles were released. Their properties indicated these IF were composed of a similar three-chain unit which contained regions of coiled-coil alpha-helix interspersed with region of non-alpha-helix. These two types of experiments permitted the construction of subunit domain maps which revealed a common structure: all subunits possessed two alpha-helical domains of the same size that were adjoined by non-alpha-helical domains of variable size. We propose that the reported solubility and immunological differences in these IF and perhaps those of other types of cells are due largely to variations in the size, configuration, and amino acid sequence of the non-alpha-helical regions of the subunits in the IF.

Amino Acid Sequence↗
PubMed1984

Intermediate filament cDNAs from BHK-21 cells: demonstration of distinct genes for desmin and vimentin in all vertebrate classes.

Recombinant cDNA plasmids for the intermediate filament proteins desmin and vimentin were constructed from baby hamster kidney (BHK-21) mRNA. Analysis of four desmin clones gave a sequence of 1574 nucleotides, which is 75% of the total mRNA length. The derived amino acid sequence for hamster desmin shows 92% overall homology with chicken desmin; the homology with hamster vimentin is highest in the alpha-helical middle part (74%). The 3'-noncoding region of desmin mRNA is found to be 677 nucleotides long. With the aid of 5'- and 3'-specific probes, it has been established that there is a single gene for desmin in the hamster genome. This gene expresses a single mRNA species of 2.2 kilobases. Hybridization experiments of a number of DNAs with desmin and vimentin probes show that there are distinct restriction enzyme fragments carrying vimentin and desmin sequences in the genome of representatives of all vertebrate classes.

Amino Acid Sequence↗
PubMed1991

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis.

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.

Animals↗
PubMedDate not supplied

Nucleic acid-binding activities of the intermediate filament subunit proteins desmin and glial fibrillary acidic protein.

In analogy to experimental results previously obtained with vimentin and neurofilament triplet proteins, the intermediate filament (IF) proteins desmin and glial fibrillary acidic protein (GFAP) were also found to have high capacities to associate with nucleic acids. Employing a collection of native and heat-denatured pro- and eukaryotic DNAs, a series of naturally occurring single-stranded (ss)RNAs and a variety of synthetic polynucleotides of the RNA and DNA type, both proteins could be shown to bind preferentially to single-stranded polynucleotides. In the case of ssDNA and synthetic polyribonucleotides, a clear dependency of the binding activity on the G-content of the nucleic acids was demonstrated. The interaction of desmin with ssDNA and tRNA was characterized by strong cooperativity. When a mixture of desmin and vimentin was offered to excess ssDNA, the cooperativity effect brought about segregation of both protein species into two distinct populations of deoxyribonucleoprotein particles with substantially different sedimentation rates; this segregation is in sharp contrast to the ability of desmin and vimentin to form heteropolymers in filament assembly. In general, desmin and GFAP were found to be similar to vimentin and neurofilament proteins in their nucleic acid-binding properties. However, there were also striking differences between individual non-epithelial IF proteins at this level.

Animals↗
PubMed1993

Titin aggregates associated with intermediate filaments align along stress fiber-like structures during human skeletal muscle cell differentiation.

Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillogenesis. Several developmental stages of these cultures were double stained with antibodies to titin in combination with antibodies to alpha-actin, alpha-actinin, myosin heavy chain (MHC), nebulin, desmin, and beta-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing alpha-actinin and sarcomeric alpha-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, alpha-actin and alpha-actinin. Thereafter a more periodic localization of titin, MHC, alpha-actin and alpha-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and alpha-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.

Cell Differentiation↗
PubMed1989

Identification of an intermediate filament associated high molecular weight protein using monoclonal antibodies.

Monoclonal antibodies reactive to a high molecular weight protein in MDBK cells were prepared and used to localize this protein in cultured cells. An insoluble fraction of MDBK cells after extraction with Triton X-100 was used as an immunogen and an immunofluorescent cell staining method was used to screen hybridoma cells. Three mAbs recognized a protein having nearly the same mobility as that of the microtubule-associated protein 2 in SDS-PAGE but no immunological reactivity of bovine microtubule proteins to these mAbs distinguished these proteins. Immunoblotting and cell staining experiments showed its presence in the intermediate filament-nuclear matrix fraction. Double staining experiments of cells using these mAbs and antibody against vimentin cleared the association of this protein with vimentin filaments. Results of immunoelectron microscopy confirmed its localization on the intermediate filaments.

Animals↗
PubMed1987

Expression of intermediate filament proteins in subtypes of renal cell carcinomas and in renal oncocytomas. Distinction of two classes of renal cell tumors.

We examined the expression of the diverse cytokeratin (CK) polypeptides as well as vimentin in human renal cell carcinomas of various subtypes and in renal oncocytomas by applying both two-dimensional gel electrophoresis and immunocytochemistry by using polypeptide-specific monoclonal antibodies. The tumors were classified according to the guidelines of the World Health Organization, with some modifications based primarily on recently proposed cytomorphological criteria. All clear cell carcinomas (G I, G II; N = 20) co-expressed CKs nos. 8 and 18, and vimentin, with CK no. 19 being present in 13 of the 20 cases and exhibiting a heterogeneous distribution. Dedifferentiated carcinomas (G III; N = 8) also co-expressed CKs nos. 8 and 18 as well as vimentin, but in addition, exhibited CK no. 19 and, in many cases, CK no. 7; in 1 case, only vimentin was expressed. Both eosinophilic-granular (N = 3) and basophilic (small cell cuboidal; N = 6) carcinomas contained CKs nos. 8 and 18, and the co-expression of vimentin was a consistent feature of these tumors; CK no. 19 was found in all of these cases, while CK no. 7 was present in the majority. In chromophobe cell carcinomas (N = 8), in contrast to all of the other carcinoma types, no vimentin was detected in the tumor cells, with only CKs nos. 8, 18, and to a variable extent 7, being present. Similarly, oncocytomas (N = 8) lacked vimentin and exhibited only CKs nos. 8 and 18. Conspicuous scattered CK no. 19-positive cells were found in these two last tumor types. No CK polypeptides other than simple-epithelium-type CKs (nos. 7, 8, 18, and 19) were detected in any of the tumors studied. These results indicate that, in renal cell tumors, the expression of intermediate-filament proteins is strikingly correlated with the specific morphologic appearance. While the co-expression of CKs nos. 8 and 18 and vimentin was a surprisingly consistent feature of the most common subtypes of renal cell carcinomas, CK no. 19 exhibited remarkable heterogeneity of expression both within individual tumors and between different tumors, the expression patterns of this CK being correlated to the tumor subtypes. The consistent absence of vimentin in chromophobe cell carcinomas and oncocytomas makes it possible to define these as a separate class of renal cell tumors. This finding supports the view that chromophobe cell carcinomas represent a distinct tumor entity and points to their close phenotypic relationship to benign oncocytomas as well as to normal renal tubules.

Adenoma↗
PubMed1986

Characterization of dimer subunits of intermediate filament proteins.

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.

Animals↗
PubMed2004

Desmin: a major intermediate filament protein essential for the structural integrity and function of muscle.

Desmin is a muscle-specific protein and a key subunit of the intermediate filament in cardiac, skeletal and smooth muscles. Desmin filaments are mainly located at the periphery of Z-disk of striated muscles and at the dense bodies of smooth muscle cells, and they have been postulated to play a critical role in the maintenance of structural and mechanical integrity of the contractile apparatus in muscle tissues. This review summarizes the findings in the regulation of the desmin gene and function of the desmin protein. The expression of desmin gene is regulated by a combination of different transcription control regions in muscle cells. The results from mice deficient in desmin reveal the fundamental role of desmin filaments in cell architecture, force transmission and mitochondrial function. Mice lacking desmin postnatally develop a dilated cardiomyopathy, a skeletal myopathy and smooth muscle defects. Some of desmin-related myopathies are attributable to a missense mutations and deletions in the desmin gene. Other desmin-related myopathies, in which the desmin gene is not mutated, could be related to mutations of the genes encoding the proteins that interact with desmin.

Animals↗
PubMed1989

Expression of intermediate filament proteins during development of Xenopus laevis. II. Identification and molecular characterization of desmin.

During embryogenesis of avian and mammalian species the formation of intermediate filaments (IFs) containing desmin is characteristic for myogenesis. In view of important differences of patterns of IF protein expression in embryogenic pathways of amphibia on the one hand and birds and mammals on the other, we have decided to study the expression of desmin during early embryogenesis of Xenopus laevis by cDNA hybridization and antibody reactions. Here we describe the isolation of a cDNA clone encoding Xenopus desmin and the deduced amino acid sequence (458 residues; Mr 52,800) which displays a very high degree of conservation during vertebrate evolution from Xenopus to chicken and hamster, with a similar degree of sequence divergence between all three species compared. In addition, we have noted, by both cDNA-hybrid-selection-translation and immunoblotting of cytoskeletal proteins a second desmin-related polypeptide of Mr approximately 49,000. RNA (Northern) blot analyses show the occurrence of three different desmin mRNAs (1.9, 2.6 and 3.0 kb) which seem to represent different polyadenylation sites, displaying quantitative differences in different kinds of muscle tissues. During embryogenesis, desmin mRNA has first been detected in stage-14 embryos and then increases drastically to high levels at stage 18 and thereafter. Immunofluorescence microscopy using desmin-specific antibodies shows that this synthesis of desmin is restricted to somite tissue. The embryonic time course of synthesis of desmin and desmin mRNA is discussed in relation to those of other muscle proteins.

Amino Acid Sequence↗
PubMed1984

Antibody to intermediate filaments of the cytoskeleton in the sera of patients with acute malaria.

Sera from 78 patients with acute malaria were tested for antibodies to intermediate filaments (IFs) by indirect immunofluorescence. Eighty-two per cent of the sera contained antibody which stained the IFs in human fetal skin fibroblasts and/or HEp2 cells. In contrast, only 8% of sera taken from blood donors gave weak positive staining of IFs and no staining was observed with 42 myeloma sera which were also tested as controls. In most cases autoantibodies were of the IgM class. Erythrocytes parasitized with Plasmodium falciparum failed to stain when reacted with non-malarial anti-IF antibody positive serum.

Acute Disease↗