PubMed Health⌕ Search

Biomedical subjects

E Lazarides

Publications and source records attributed to E Lazarides.

At least 73 records · Page 4Linked to original sources
PubMed1983

Vimentin filaments are assembled from a soluble precursor in avian erythroid cells.

The synthesis and assembly of vimentin was studied in erythroid cells from 10-d-old chicken embryos. After various periods of [35S]methionine incorporation, cells were lysed in a Triton X-100-containing buffer and separated into a soluble and an insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis shows that vimentin is almost exclusively present in the cytoskeletal fraction and that newly synthesized vimentin is rapidly incorporated into this fraction. However, after a short pulse-labeling period, a prominent labeled protein at the position of vimentin is present in the soluble fraction. By immunoautoradiography and immunoprecipitations with vimentin antibodies, this protein was identified as vimentin. The vimentin in the soluble fraction is not sedimented by high speed centrifugation, suggesting that it does not consist of short filaments. After different pulse-labeling periods, assembly of newly synthesized vimentin in the cytoskeletal fraction increases linearly, while the radioactivity in the soluble vimentin remains constant. During a 2-h pulse-chase period, the vimentin in the soluble fraction is chased into the cytoskeletal fraction, with a half-life of 7 min. These results suggest that in chicken embryo erythroid cells newly synthesized vimentin is rapidly assembled into filaments from a soluble precursor.

Animals↗
PubMed1983

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers.

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.

Actinin↗
PubMed1983

Avian lens spectrin: subunit composition compared with erythrocyte and brain spectrin.

Chicken lens spectrin is composed predominantly of equimolar amounts of two polypeptides with solubility properties similar, but not identical, to erythrocyte spectrin. The larger polypeptide, Mr 240,000 (lens alpha-spectrin), co-migrates with erythrocyte and brain alpha-spectrin on one- and two-dimensional SDS polyacrylamide gels and cross-reacts with antibodies specific for chicken erythrocyte alpha-spectrin; the smaller polypeptide, Mr 235,000 (lens gamma-spectrin), co-migrates with brain gamma-spectrin and does not cross-react with either the alpha-spectrin antibodies specific for chicken erythrocyte beta-spectrin. Minor amounts of polypeptides antigenically related to erythrocyte beta-spectrin with a greater electrophoretic mobility than lens gamma-spectrin are also detected in lens. The equimolar ratio of lens alpha- and gamma-spectrin is invariantly maintained during the extraction of lens plasma membranes under different conditions, or after immunoprecipitation of whole extracts of lens with erythrocyte alpha-spectrin antibodies. Two-dimensional peptide mapping reveals that whereas alpha-spectrins from chicken erythrocytes, brain, and lens are highly homologous, the gamma-spectrins, although related, have some cell-type-specific peptides and are substantially different from erythrocyte beta-spectrin. Thus, the expression of cell-type-specific gamma- and beta-spectrins may be the basis for the assembly of a spectrin-plasma membrane complex whose molecular composition is tailored to the functional requirements of the particular cell-type.

Animals↗
PubMed1983

Canavanine inhibits vimentin assembly but not its synthesis in chicken embryo erythroid cells.

In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine-vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo.

Animals↗
PubMed1983

Expression of intermediate filament-associated proteins paranemin and synemin in chicken development.

The expression of two intermediate filament-associated proteins, paranemin (280,000 mol wt) and synemin (230,000 mol wt), was investigated with respect to the expression of two core intermediate filament proteins, desmin and vimentin, in various embryonic and adult chicken muscle and nonmuscle cells. All developing muscle cells, regardless of their type, simultaneously express desmin, vimentin, paranemin, and synemin. However, a difference is observed in the expression of paranemin in adult muscle. This protein is removed during differentiation of both fast and slow skeletal muscle, visceral smooth muscle, and the smooth muscle of muscular arteries, but remains in mature myocardial cells, cardiac conducting fibers, and the smooth muscle cells of elastic arteries. Some of these cells express vimentin, others desmin, and still others a mixture of the two. On the other hand, synemin is expressed in all the above types of adult muscle cells except myocardial cells. Adult myocardial cells also lack vimentin, and its presence is gradually reduced after hatching. Since in adult striated muscle all expressed intermediate filament proteins are found predominantly in association with the peripheries of myofibrillar Z discs, these results suggest that a change in the composition of skeletal and cardiac muscle Z discs occurs during chicken development and maturation. Erythrocytes that express synemin and vimentin do not express paranemin, while both embryonic and adult Schwann cells co-express paranemin and vimentin, but not synemin. Endothelial cells of muscular vessels express paranemin, while those of elastic vessels do not, and neither contains synemin. Paranemin and synemin are not expressed in neurons, epithelial, and most glial cells, suggesting that these two polypeptides are expressed only in conjunction with desmin or vimentin. These results suggest that the composition of intermediate filaments changes during chicken development, not only with respect to their core subunit proteins but also with respect to two associated polypeptides, particularly in muscle cells.

Animals↗
PubMed1983

Evidence for a phosphorylated form of calmodulin in chicken brain and muscle.

Phosphocalmodulin (PCaM) was identified after analysis of calmodulin (CaM) preparations by two-dimensional gel electrophoresis by using a modified ampholyte system to resolve very acidic proteins. The analysis of CaM prepared by the conventional procedure based upon its heat resistance and acidity as well as the analysis of whole urea extracts from brain showed that PCaM was a major component in this tissue. PCaM was 1 pH unit more acidic than CaM, and its electrophoretic mobility, unlike CaM, was not changed by either calcium or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. In urea extracts of brain prepared in buffers containing phosphate and sodium fluoride, PCaM was as prominent as CaM; it was partially converted into CaM after elution from the gel and reelectrophoresis. Amino acid analysis of PCaM and CaM purified by two-dimensional gel electrophoresis showed the same composition for the two proteins, including their trimethyllysine content. Incorporation of 32P occurred exclusively into the acidic variant when brain slices were incubated with H332PO4; amino acid analysis showed that the phosphate was bound to serine residues. CaM was found also to be phosphorylated in vitro by a phosphorylase kinase preparation from skeletal muscle.

Animals↗
PubMed1982

Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy.

Intermediate filament structure and distribution were studied by antibody decoration and low-angle shadowing of sonicated chicken erythrocytes and embryonic erythroid cells. Intermediate filaments containing vimentin and synemin form a three-dimensional network in these cells, interlinking the nucleus and plasma membrane. This filament network is spatially segregated from the marginal band of microtubules, indicating that these two systems do not interact directly in the development or maintenance of cell shape. Incubation of sonicated cells with an antiserum specific for vimentin results in uniform decoration of the intermediate filaments; incubation with antisynemin results in decoration of periodically spaced foci. Measurement of the synemin periodicity under a specified set of sample preparation conditions gives average values of 180 nm for adult erythrocytes and 230 nm for 10 day old embryonic erythroid cells, suggesting some fundamental change in the structure of the filaments during erythropoiesis. Registration of these foci in laterally associated filaments, and decoration of bridges between slightly separated filaments, suggest that synemin mediates crosslinking of intermediate filaments through self-interaction.

Animals↗
PubMed1982

Widespread occurrence of avian spectrin in nonerythroid cells.

We have prepared an antibody against chicken erythrocyte alpha spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with alpha spectrin in chicken erythrocytes and crossreacts with alpha spectrin in erythrocytes from various mammals. Immunofluorescence reveals that this antiserum reacts with a plasma membrane component in erythrocytes as well as in most nonerythroid avian and mammalian cells. Intense staining is seen at or near the plasma membrane in neurons, lens cells, endothelial and epithelial cells of the gastrointestinal and respiratory tracts, skeletal and cardiac muscle, as well as skeletal myotubes grown in tissue culture. Immunoautoradiography indicates that the crossreactive antigen in these nonerythroid tissues has the same molecular weight and isoelectric point as the chicken erythrocyte antigen. Smooth muscle, tracheal cilia, myelin and mature sperm stain weakly or not at all. These results suggest that spectrin is more extensively distributed than previously recognized, and that the functions of spectrin elucidated for erythrocytes may apply to other cell types as well.

Animals↗
PubMed1982

Analysis of desmin and vimentin phosphopeptides in cultured avian myogenic cells and their modulation by 8-bromo-adenosine 3',5'-cyclic monophosphate.

The intermediate filament proteins desmin and vimentin are two of the major 32P phosphate acceptors in chicken myotubes differentiating in tissue culture. Analysis of the desmin and vimentin phosphopeptides by two-dimensional tryptic peptide mapping shows that both proteins are phosphorylated at multiple sites, giving rise to 5 phosphopeptides in desmin and as many as 11 in vimentin. Addition of the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) to the culture medium of mature (8-day-old) myotubes results in a 2- to 3-fold increase in PO4 incorporation into desmin and vimentin. Two-dimensional tryptic analysis of desmin and vimentin from 8-BrcAMP-treated myotubes shows increased 32PO4 incorporation into a subset of the phosphopeptides observed in control cells. Comparison of phosphopeptides from the two proteins shows the presence of at least three comigrating peptides. All three comigrating peptides exhibit cAMP-dependent increases in 32PO4 incorporation in vimentin, while only two of the three exhibit 8-BrcAMP-dependent responses in desmin. While these peptides are the only two that are sensitive to 8-BrcAMP in desmin, vimentin contains additional peptides that exhibit increased 32PO4 incorporation in response to 8-BrcAMP. This result suggests the existence of both common and distinct phosphorylation sites between desmin and vimentin that may be differentially regulated by cAMP. Thus, desmin and vimentin, even though structurally related, may be capable of responding differently to physiological stimuli.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Synemin and vimentin are components of intermediate filaments in avian erythrocytes.

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.

Animals↗
PubMed1982

Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle.

Filaments with a diameter of 80-120 A have been prepared from 14-d-old chick embryonic skeletal muscle, using a physiological salt solution and gel filtration chromatography. The filaments obtained are composed of the two known muscle intermediate-filament proteins, vimentin and desmin, as well as the vimentin- and desmin-associated high molecular weight protein, synemin (230,000 mol. wt). In addition, they contain a previously unidentified high molecular weight protein (280,000 mol wt) which differs from synemin by isoelectric point, molecular weight, and immunological reactivity. Immunofluorescence on cultured myogenic cells,using antisera to the 280,000-dalton polypeptide, has revealed that this protein has the same spatial distribution as desmin, vimentin, and synemin in both early myotubes, where it associates with cytoplasmic filaments, and late in myotubes, where it is associated with myofibril Z lines. Examination by immunofluorescence of frozen sections of developing embryonic skeletal muscle reveals a gradual diminution in the presence of the 280,000-dalton protein. The 280,000-dalton protein is undetectable in adult skeletal and smooth muscle, as shown by immunofluorescence and immunoautoradiography. In chick embryonic fibroblasts grown in tissue culture, only a subpopulation of the cells is reactive with antibodies to the 280,000-dalton protein even though all these cells contain vimentin. In the reactive cells, vimentin and the 280,000-dalton polypeptide exhibit an indistinguishable cytoplasmic filamentous network, which aggregates into filamentious bundles when the cells are exposed to colcemid. These results suggest that this newly identified high molecular weight protein is closely associated with intermediate filaments containing either vimentin alone or vimentin, desmin and synemin. The expression of this protein appears to be developmentally regulated and does not appear to parallel the expression of any of the other three intermediate-filament proteins. The absence of the 280,000-dalton polypeptide in adult muscle cells and its gradual reduction during development implies that is probably not required for the maintenance of Z-disk structure after the assembly of the sarcomere.

Animals↗