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At least 19 recordsLinked to original sources
PubMed1992

Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.

The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.

Animals↗
PubMed1995

Desmin expression in rhabdomyosarcoma: influence of the desmin clone and immunohistochemical method.

AIM: To determine which, if any, of five commercially available desmin clones is most reliable at labelling desmin filaments and whether the enhanced polymer one step (EPOS) method of labelling is of any advantage in the routine diagnostic laboratory. METHODS: Thirty four rhabdomyosarcomas from the files at The Hospital for Sick Children, Great Ormond Street, London, were studied. Four different desmin clones, DE-R-11, D33, DE-U-10, and PDE, were applied to each using the conventional extravidin biotin peroxidase method. The D33 clone was also applied using the EPOS method. RESULTS: The EPOS method incorporating D33 persistently scored more cells as desmin positive and was positive in four cases which were negative on staining with the other clones. CONCLUSIONS: The D33 desmin clone used with the EPOS method is more reliable for identifying desmin filaments in tumours than other desmin antibodies tested. Different desmin clones using a routine technique label different rhabdomyosarcoma cells and therefore it is justifiable to use more than one clone.

Desmin↗
PubMed1990

Expression of desmin gene in skeletal and smooth muscle by in situ hybridization using a human desmin gene probe.

We have used a probe encoding for the human desmin gene to study the expression of the desmin gene in skeletal and smooth muscle by in situ hybridization. In human skeletal muscle, the results showed a strong and homogeneous level of desmin mRNA contrasting with the faintly immunostaining of the desmin protein. In smooth muscle cells of colon and uterus, in situ hybridization and immunofluorescence staining suggests that there are some cells which do not contain desmin. The optimal condition of desmin mRNA detection was in cryostat sections fixed with paraformaldehyde and in paraffin embedded tissue with the same fixative. The human desmin probe can be used as a marker of cell differentiation and a way to study the regulation of the expression of the desmin gene in pathological events.

Animals↗
PubMed1985

Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer.

The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.

Amino Acid Sequence↗
PubMed1990

Desmin-binding specificities of two desmin CNBr fragments that correspond to the headpiece domain and Helix 1B.

D88 and D109, two cyanogen bromide fragments of desmin which essentially correspond to the amino terminal headpiece domain and Helix 1B, respectively, bind to intact desmin with different topological specificities. D88, the headpiece domain fragment, binds only to the headpiece of intact desmin. In contrast, D109, which encompasses Helix 1B and most of the linker L10 binds to desmin even when its headpiece is removed. Additionally, these fragments only bind desmin if they are present during filament assembly; they do not bind pre-assembled desmin IF or tetramers. These observations suggest that, while alpha-helical coiled-coil interaction between rod domains provides the major driving force behind IF protein dimer formation, homophilic binding of head domains of these proteins may provide an additional stabilizing force and/or specify axial registration in certain IF proteins.

Animals↗
PubMed1993

ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP-ribosyltransferase.

Arginine-specific mono(ADP-ribosyl)transferase purified from rabbit skeletal muscle catalyzes stoichiometric ADP-ribosylation of the intermediate filament protein, desmin. In contrast, cholera toxin catalyzes a much lower level of ADP-ribosylation of desmin. Modification results in potent inhibition of desmin's ability to assemble into filaments. Phosphorylation of desmin by the catalytic subunit of cAMP dependent protein kinase is also inhibited by ADP-ribosylation. ADP-ribosylation site(s) are located within the N-terminal head domain of desmin.

ADP Ribose Transferases↗
PubMed1994

Truncated desmin in PtK2 cells induces desmin-vimentin-cytokeratin coprecipitation, involution of intermediate filament networks, and nuclear fragmentation: a model for many degenerative diseases.

The earliest expression of truncated desmin in transfected PtK2 cells results in the formation of dispersed microprecipitates containing not only the truncated desmin, but also endogenous vimentin and cytokeratin proteins. Desmin microprecipitates without vimentin or vimentin microprecipitates without desmin are not observed. The microprecipitates involving cytokeratin invariably are also positive for desmin and vimentin. Over time, the precipitates enlarge into 1- to 2-microns spheroids and then fuse into amorphous chimeric juxtanuclear masses that can occupy > 30% of the cell volume. Concurrently, first the vimentin and then the cytokeratin networks are resorbed. The chimeric precipitates are not recognized or marked for degradation by the lysosomal system. Ultimately the cell nucleus fragments and the cell dies. Similar protein complexes appear in many human and animal pathologies, suggesting that a similar protein-precipitation sequence initiated by the introduction of a mutationally or environmentally altered protein molecule is at work.

Alzheimer Disease↗
PubMed1986

Immunocytochemical studies of endothelial cells in vivo. I. The presence of desmin only, or of desmin plus vimentin, or vimentin only, in the endothelial cells of different capillaries of the adult chicken.

It is currently believed that the intermediate filaments of endothelial cells contain vimentin subunits exclusively. This inference, however, is derived from studies of only a few types of endothelial cells. By double indirect immunofluorescence and immunoelectron microscopy, we have now examined the endothelial cells of the micro- and macrovasculature of a variety of tissues and organs of adult chicken in vivo for their content of desmin and vimentin. Endothelial cells of the peritubular capillary in the renal cortex, the hepatic sinusoid, and the splenic sinusoid were found to contain only desmin; those of the exocrine pancreas capillary contained both desmin and vimentin; and the endothelial cells of the macrovasculatures and of all the other microvasculatures examined, including the vasa recta of the renal medulla, contained only vimentin. Such heterogeneity suggests that different types of adult chicken endothelial cells may have different embryological origins. To the extent that desmin and vimentin intermediate filaments may be functionally distinct, these results also suggest that different capillary endothelial cells may have different functional properties.

Animals↗
PubMed1980

Purification of smooth-muscle desmin and a protein-chemical comparison of desmins from chicken gizzard and hog stomach.

A fast and convenient procedure for the purification of polymerization-competent smooth-muscle desmin is described. Desmin from chicken gizzard and hog stomach were compared by fingerprint techniques. The two proteins show extensive amino acid sequence homology, although some clear differences in the peptide patterns are indicated. Comparative amino acid sequence analysis of some of the peptides obtained in pure form directly proves this conclusion.

Amino Acid Sequence↗
PubMed1991

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Differential organization of desmin and vimentin in muscle is due to differences in their head domains.

In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2-terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head-desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle-specific behaviors.

Amino Acid Sequence↗
PubMed1989

[Immunocytochemical studies on the appearance and distribution of desmin during myogenesis in the embryos of Cynops orientalis].

Indirect immunofluorescence was used to study the temporal appearance and spatial distribution of desmin during the myogenesis of the embryos of Cynops orientalis. Desmin is undetectable until stage 25. In stage 25 embryo, it can be seen that desmin is restrictively distributed at both ends of columnar cells, near the boundary between two somites and intense in the cells near by the notochord. From stage 26 to stage 30, the amount of desmin is increased and its distribution pattern shows little change (Plate I, Figs. 1-2). At stage 32 desmin can be detected in the cells more distal to the notochord and forms filaments on the inside of the cell membrane parallel to the long axis of the cell (Plate I, Fig. 3 and 5). Desmin filaments extend gradually from the both ends toward the mid-part of the cell (Plate I, Fig. 6 and Plate II, Figs. 7, 11-13). At about stage 40 the whole cell is filled with desmin filaments and the attachment of desmin to Z line can occasionally be detected (Plate II, Fig. 8). Desmin attached to Z line is increased at stage 41 (Plate II, Fig. 9) and at stage 43 most of the desmin is found attached to Z line (Plate II, Fig.10). According to EM observation, Z line structure can be seen in stage 33 embryo (Wang[18]), but desmin remains in the filament form till stage 40. The transference of desmin distribution pattern from filament to Z line occurs somewhat later than the appearance of scattered sarcomeres. The possibility that notochord may be the main factor which influences the spatial localization of desmin was analyzed. The relationship between the transference of desmin from filament to Z line attached form and the quantitative changes of both desmin and sarcomere was discussed.

Animals↗
PubMed1990

Assembly of amino-terminally deleted desmin in vimentin-free cells.

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.

Amino Acid Sequence↗
PubMed1991

Comparative immunohistochemical staining for desmin and muscle-specific actin. A study of 576 cases.

Muscle-specific actin (MSA) and desmin are considered to be sensitive and specific markers for muscle differentiation. The authors compared staining patterns for these markers in 576 samples of normal, reactive, and neoplastic tissues. The standard avidin-biotin-peroxidase complex technique was performed with the use of two commercial antibodies against MSA (HHF35; Enzo Biochemical, Inc., New York, NY) and desmin (DER11; DAKO Corporation, Santa Barbara, CA), respectively, on consecutive paraffin-embedded tissue sections from these cases. Both MSA and desmin were found in all 80 normal muscle samples. Although MSA appeared diffusely in all vascular smooth muscle samples, desmin was demonstrated focally in vascular smooth muscle cells in 100 of 196 samples. MSA but not desmin always was found in myoepithelial cells (25 samples), pericytes (286 samples), and decidual cells (7 samples). Among 76 cases of myofibroblast-containing lesions, 14 and 54 were found to have desmin and MSA, respectively. MSA and desmin were found in 4 of 4 cardiac rhabdomyomas, 34 of 34 rhabdomyosarcomas, and 5 of 6 leiomyomas. Among 22 leiomyosarcomas, 7 displayed either MSA or desmin and 7 showed both markers. In general, more tumor cells showed staining for MSA than desmin, but the reverse was true in some cases. Tissue fixed in Zenker's solution seemed to show a significant decrease in MSA immunoreactivity, but no significant change for desmin staining was observed. None of the 154 normal tissues and 22 benign nonmyogenic tumors expressed MSA or desmin. Among 133 malignant nonmyogenic tumors, positive staining for both desmin and MSA was found in 3 of 8 cases of glioblastoma multiforme, 1 of 10 malignant schwannomas, and 1 of 14 malignant fibrous histiocytomas; staining for only MSA was found in 3 of 14 malignant fibrous histiocytomas, 1 of 10 malignant schwannomas, 6 of 6 fibromatoses, 1 of 1 mammary myofibroblastoma, and 1 of 7 malignant mesotheliomas; and staining for desmin only was seen in 1 of 7 malignant mesotheliomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1994

Analysis of the in vivo myogenic status of chick somites by desmin expression in vitro.

Expression of the muscle specific intermediate filament protein, desmin, is an early marker for chick somitic myogenesis. Somites are transient, paired, mesodermal structures adjacent to the neural tube which are formed very uniformly in a cranial to caudal fashion. The developmental somitic expression of desmin in vivo has been reported previously (Holtzer et al. [1991] "Frontiers in Muscle Research." New York: Elsevier Science, pp 187-207; Borman and Yorde [1994] J. Histochem. Cytochem. 42:265-272). Here we explore the ability of those somitic cells which are desmin negative in vivo to successfully carry out a myogenic program of development in the absence of the surrounding embryonic microenvironment. Somites which are known to be overtly desmin negative in the embryo were explanted and cultured on collagen gels for 4 days. Immuno-detection of desmin identified a population of somites that could support desmin positive cells in vitro as well as a population of somites that could not. The cranially located somites must remain in the embryo for a greater length of time than the caudally positioned somites prior to each being able to express desmin in vitro. In embryos of many ages there is also a population of somites unable to support desmin expression in vitro. The rate at which this ability to support somitic desmin expression in vitro progresses caudally in the embryo is significantly greater than the rate at which somites form. Notably, the detected expression of desmin in somites in vitro is parallel to the rate at which overt expression of desmin in vivo is detected. The implication for these observations with regard to the regulation of somitic myogenesis is discussed.

Animals↗
PubMed1994

Inhibition of desmin expression blocks myoblast fusion and interferes with the myogenic regulators MyoD and myogenin.

The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.

Amino Acid Sequence↗
PubMed1987

Intermediate filament formation after transfection with modified hamster vimentin and desmin genes.

Previously we cloned and characterized the hamster intermediate filament genes coding for vimentin and desmin. It was demonstrated that the cloned desmin gene was expressed after gene transfer and that the newly synthesized protein assembles into intermediate filaments. Here we present data on the transfection of modified vimentin and desmin genes onto simian virus 40-transformed hamster lens cells and HeLa cells. Modifications included: (1) removal of exons encoding the desmin COOH-terminal domain; (2) exchange of exons encoding the COOH-terminal domain of vimentin and desmin; and (3) deletion of part of exon I of desmin, coding for the NH2-terminal amino acids 4-148. In transient transfection assays it was shown that the modifications in the COOH region had no detectable effects on the filament forming potential of the encoded proteins as demonstrated with desmin antibodies in the indirect immunofluorescence test. On the other hand, deletion of a considerable part of the first exon of the desmin gene results in a lack of bona fide intermediate filament formation. Immunoblotting with desmin antibodies of cell populations enriched for the transfected modified genes showed that the presence of the modified genes results in the synthesis of the corresponding proteins with the expected molecular weights. From our results we conclude that in vivo: (1) the presence of the COOH terminus is not essential for filament formation; (2) that an exchange of COOH-terminal parts of vimentin and desmin does not prevent assembly into intermediate filaments; and (3) that removal of the NH2 terminus of desmin affects intermediate filament formation.

Animals↗