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E Lazarides

Publications and source records attributed to E Lazarides.

At least 91 records · Page 5Linked to original sources
PubMed1982

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1981

Traction retinal detachment. A cell-mediated event.

In posterior-segment penetrating injuries, the role of cellular and extracellular structures for the development of fraction retinal detachment is a matter of controversy. With the use of immunofluorescence techniques, we studied the cellular components of intravitreal membranes, with particular attention to the presence of intracellular contractile proteins. Our results demonstrate increasing concentrations of intracellular actin filaments in 12- to 21-day-old membrane specimens at the time when traction retinal detachment is most likely to occur in the rabbit eye. The sequence of events reflects the same process that occurs in the wound-healing response throughout the body. The specialized anatomy of the eye, particularly the vitreoretinal relationship at the vitreous base, accounts for the disastrous consequence of fraction retinal detachment.

Actins↗
PubMed1981

Phosphorylation of intermediate filament proteins by cAMP-dependent protein kinases.

The intermediate filament proteins, desmin and vimentin, are phosphorylated in skeletal muscle cells in vivo. Desmin kinase activities have been purified from mature chicken skeletal muscle and identified as the cAMP-dependent kinases. Chicken skeletal muscle contains two cAMP-dependent protein kinases which are similar to those of other tissues in their subunit composition and chromatographic behavior. The catalytic subunits purified from the two chicken cAMP-dependent kinases phosphorylate both desmin and vimentin in vitro, using cytoskeletal residues prepared from cultured myogenic cells as a substrate. Likewise, the purified catalytic subunits of the rabbit skeletal muscle and bovine heart cAMP-dependent protein kinases phosphorylate desmin and vimentin in vitro. Desmin and vimentin phosphorylation by the rabbit skeletal muscle catalytic subunit is inhibited by the addition of its regulatory subunit. This inhibition is reversed by the presence of cAMP in the reaction mixture. A small fraction of the vimentin phosphorylation is cAmP-independent. Tryptic peptide analysis of desmin phosphorylated in vivo shows two major phosphopeptides. Serine is the phosphorylated amino acid in both peptides. The same two peptides are phosphorylated in vitro by the bovine heart catalytic subunit, but additional peptides are also phosphorylated.

Animals↗
PubMed1981

Heat shock proteins are methylated in avian and mammalian cells.

Exposure of chicken cells grown in tissue culture to heat shock or sodium arsenite results in a dramatic increase in the synthesis of three major polypeptides with molecular weights of 83,000 (HSP 83), 68,000 (HSP 68; referred to here as "thermin"), and 25,000 (HSP 25). Incubation of BHK-21 or HeLa cells under the same conditions results in induction of HSP 68 and a 66,000-dalton polypeptide (HSP 66). Chicken thermin is resolved by isoelectric focusing into a major acidic and a more-basic component; mammalian thermin is resolved only into one major acidic component. HSP 83 and the acidic form of thermin are highly conserved in all avian and mammalian cells examined as judged by their electrophoretic mobilities, isoelectric points, and one-dimensional peptide maps. In addition, the acidic form of thermin is indistinguishable from a protein that copurifies with brain microtubules and that remains associated with the intermediate filament-enriched Triton/KCl cytoskeletons of cells grown in tissue culture. Thermin is also a component of skeletal myofibrils. HSP 83 and thermin are methylated in cells cultured under normal growth conditions. Induction of heat shock proteins by incubation of cells in the presence of sodium arsenite results in a marked methylation of the newly synthesized thermin. Under the same experimental conditions, no significant increase in methylation of the HSP 83 is observed. HSP 25 is not methylated in untreated cells or in cells treated with sodium arsenite. These results suggest that methylation of heat shock proteins may have an important role in regulating their function.

Animals↗
PubMed1981

In vitro translation of the intermediate filament proteins desmin and vimentin.

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.

Animals↗
PubMed1980

Intermediate filaments as mechanical integrators of cellular space.

Five chemically distinct classes of intermediate filaments can be identified within higher eukaryotic cells. Each class is characteristic of a particular cell type. These filaments may function to integrate mechanically the various structures of the cytoplasmic space in a way that is tailored to the differentiated state of the cell.

Actinin↗
PubMed1980

The synthesis and distribution of desmin and vimentin during myogenesis in vitro.

Electrophoretic and autoradiographic analyses of the incorporation of 35S--methionine into newly synthesized proteins during myogenesis reveal that presumptive chicken myoblasts synthesize primarily one intermediate filament protein: vimentin. Desmin synthesis is initiated at the onset of fusion. Synthesis rates of both filament subunits increase during the first three days in culture, relative to the total protein synthesis rate. The observed increase in the rate of desmin synthesis (at least 10 fold) is significantly greater than that observed for vimentin, and is responsible for a net increase in the cellular desmin content relative to vimentin. Both filament subunits continue to be synthesized through at least 20 days in culture. Immunofluorescent staining using desmin- and vimentin-specific antisera supports the conclusion that desmin is synthesized only in fusing or multinucleate cells. These results indicate that the synthesis of the two filament subunits is not coordinately regulated during myogenesis. The distributions of desmin and vimentin in multinucleate chicken myotubes are indistinguishable, as determined by double immunofluorescence techniques. In early myotubes, both proteins are found in an intricate network of free cytoplasmic filaments. Later in myogenesis, several days after the appearance of alpha--actinin-containing Z line striations, both filament proteins become associated with the Z lines of newly assembled myofibrils, with a corresponding decrease in the number of cytoplasmic filaments. This transition corresponds to the time when the alpha--actinin-containing Z lines become aligned laterally. These data suggest that the two intermediate filament systems, desmin and vimentin, have an important role in the lateral organization and registration of myofibrils and that the synthesis of desmin and assembly of desmin-containing intermediate filaments during myogenesis is directly related to these functions. These results also indicate that the Z disc is assembled in at least two distinct steps during myogenesis.

Actinin↗
PubMed1980

Synemin: a new high molecular weight protein associated with desmin and vimentin filaments in muscle.

A 230,000 dalton polypeptide co-purifies through cycles of depolymerization and polymerization with the intermediate filament subunits, desmin and vimentin, from avian smooth muscle. This protein is also present in skeletal muscle and is distinct from myosin and filamin. Double immunofluorescence microscopy of cultured cells, using antisera shown to be specific by immunoautoradiography, has revealed that this protein has the same spatial distribution as desmin and vimentin. During skeletal myogenesis, all three antigens exist initially in multinucleate myotubes as wavy filaments throughout the cytoplasm. Within a week after myoblast fusion, they begin to coalesce at the peripheries of the myofibril Z discs, thereby attaining the distribution observed in mature muscle, a network of interlinked rings within the Z plane. Treatment of cultured myotubes with colcemid causes the filamentous forms of these three proteins to co-aggregate into cytoplasmic bundles, but has little effect on them when they are associated with the Z discs. Extraction of cells with nonionic detergent and high salt leaves cytoskeletons containing desmin, vimentin and the 230,000 dalton polypeptide with immunofluorescent patterns that are indistinguishable from one another. These data suggest that this high molecular weight protein is closely associated with desmin and vimentin filaments in muscle cells; to indicate this, we have named the protein synemin, from the Greek oa uv (with) and v eta mu alpha (filament).

Carrier Proteins↗
PubMed1980

The 68,000-dalton neurofilament-associated polypeptide is a component of nonneuronal cells and of skeletal myofibrils.

Purified preparations of 10-nm neurofilaments from rat spinal cord and bovine or porcine brain contain a predominant 68,000-dalton polypeptide. This polypeptide is also a major component of the neurofilaments that copurify with brain tubulin isolated by cycles of polymerization and depolymerization. A protein that has the same isoelectric point and molecular weight as the neurofilament-associated polypeptide has also been identified as a cytoskeletal protein in a variety of avian and mammalian cell types, including baby hamster kidney (BHK-21) mouse 3T3, Novikoff rat hepatoma, chicken fibroblast, and chicken muscle cells. This protein is also a component of isolated chicken skeletal myofibrils. One-dimensional peptide maps of the 68,000-dalton proteins purified by two-dimensional isoelectric focusing/NaDodSO(4)/polyacrylamide gel electrophoresis from myofibrils, cycled tubulin, purified neurofilaments, and various cultured cell types were identical. In immunofluorescence this protein was associated with cytoplasmic intermediate filaments and myofibril Z discs. These results indicate that the neurofilament-associated polypeptide is a conserved protein that is present in many different cell types in addition to neuronal cells. Because some of these cells contain the major components of two other intermediate filament classes, desmin and vimentin, a given cell type may contain the subunits of at least three distinct intermediate filament types.

Animals↗
PubMed1980

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540.

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.

Animals↗
PubMed1979

Desmin and vimentin coexist at the periphery of the myofibril Z disc.

Two-dimensional gel electrophoresis has revealed that vimentin, the predominant subunit of intermediate filaments in cells of mesenchymal origin, is a component of isolated skeletal myofibrils. It thus coexists in mature muscle fibers with desmin, the major subunit of muscle intermediate filaments. Antisera to desmin and vimentin, shown to be specific for their respective antigens by two-dimensional immunoautoradiography, have been used in immunofluorescence to demonstrate that vimentin has the same distribution as desmin in skeletal muscle. Both desmin and vimentin surround each myofibril Z disc and form honeycomb-like networks within each Z plane of the muscle fiber. This distribution is complementary to that of alpha-actinin within a given Z plane. Desmin and vimentin may thus be involved in maintaining the lateral registration of sarcomeres by transversely linking adjacent myofibrils at their Z discs. This linkage would support and integrate the fiber as a whole, and provide a molecular basis for the cross-striated appearance of skeletal muscle.

Animals↗