Biomedical subjects

E Lazarides

Publications and source records attributed to E Lazarides.

At least 55 records · Page 3Linked to original sources

PubMed1984

Membrane skeletal protein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression.

The avian analog of mammalian erythrocyte protein 4.1, a structural component of the membrane skeleton, has been identified. It is present at the plasma membranes of avian erythrocytes and lens cells, but has not been found elsewhere in comparable amounts. In chickens, it exists as six variants with molecular masses of 87, 100, 115, 150, 160, and 175 kd. The corresponding polypeptides in turkeys are each about 3 kd smaller, suggesting that all may be encoded by a single gene. These variants have similar solubility properties and nearly identical two-dimensional iodopeptide maps that are similar to those of mammalian protein 4.1, but they are differentially phosphorylated. The three smallest variants are the predominant forms in avian erythrocytes, while the two largest variants predominate in avian lens cells. In contrast, mammalian erythrocytes and lens cells exhibit patterns of variants that are more similar to each other. These results show that only a subset of spectrin-containing cells possess protein 4.1, and that these cells differentially express the variants of protein 4.1 in a manner that may reflect corresponding functional differences.

Animals↗

PubMed1984

Goblin (ankyrin) in striated muscle: identification of the potential membrane receptor for erythroid spectrin in muscle cells.

Goblin , a high molecular weight (Mr, 260,000) polypeptide of avian erythrocyte plasma membranes characterized by hormone-dependent phosphorylation, is shown by a variety of criteria to be the avian equivalent of ankyrin, the membrane attachment protein for spectrin; a polyclonal monospecific goblin antiserum reacts specifically with ankyrin from mammalian erythrocyte ghosts; goblin and ankyrin have highly homologous, although distinct, two-dimensional peptide maps; and, in reconstitution experiments, goblin binds to spectrin and band 3 in approximately the same molar ratio as ankyrin. Immunoautoradiography and immunofluorescence with goblin antiserum reveal that a serologically related polypeptide (Mr, 235,000) is present in highly purified membrane fractions of mammalian myocardium and in whole extracts of adult chicken cardiac and skeletal muscle-nonerythroid tissues which express predominantly the erythroid (alpha beta-) spectrin phenotype. Erythroid spectrin and goblin (ankyrin) are codistributed in skeletal muscle at the sarcolemma as discrete foci adjacent to the Z lines and, in pectoral muscle, also at the periphery of the Z discs. These spatial relationships indicate that goblin and spectrin in muscle cells form a structural framework that serves as the attachment site for the myofiber at the level of the Z line on the sarcolemma.

Animals↗

PubMed1984

Characterization and regulation in the expression of a gene coding for the intermediate filament protein desmin.

Using synthetic oligonucleotide probes, we have isolated chicken cDNA clones for the intermediate filament protein desmin. We show that the gene for this protein probably exists as a single copy in the haploid chicken genome and is transcribed into one mature mRNA species of approximately equal to 2.4 kilobases. Expression of this mRNA is tissue-specific, as it is present in high abundance in smooth and skeletal muscle but is absent from erythrocytes, spinal cord, and lens cells. A 10- to 20-fold increase in desmin mRNA is observed in myogenic cells upon fusion, which suggests that the level of expression of the desmin gene and the accumulation of desmin filaments during muscle differentiation is regulated at the transcriptional and/or posttranscriptional level but not at the translational level. Hybridization studies and nucleotide sequence comparison of the cDNAs specific for desmin and for other intermediate filament subunits reveal a region that is highly conserved (80%) among different members of the intermediate filament protein gene superfamily, with the exception of the chicken vimentin gene; this gene appears to be less homologous to the genes for the other chicken intermediate filament subunits than the mammalian vimentin gene is to the genes for other mammalian intermediate filament proteins and to the chicken desmin gene.

Animals↗

PubMed1984

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin.

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.

Animals↗

PubMed1984

Differentiation of murine erythroleukemia cells results in the rapid repression of vimentin gene expression.

We show that vimentin filaments are present in undifferentiated Friend murine erythroleukemia cells, but are lost progressively to undetectable levels by 96 h of dimethyl sulfoxide-mediated differentiation. The amount of newly synthesized cytoskeletal vimentin is decreased dramatically by 24 h of induction, and is paralleled by a rapid loss of vimentin mRNA (approximately 25-fold reduction at 96 h). Hence, disappearance of vimentin filaments in these cells appears to be regulated at the level of vimentin mRNA abundance. On the other hand, the levels of actin synthesis and actin mRNA remain essentially unchanged. The kinetics of vimentin mRNA reduction during dimethyl sulfoxide-mediated differentiation, and the levels of vimentin mRNA observed in the presence of hexamethylene-bisacetamide or hemin as inducers suggest that the cessation of vimentin expression precedes, but may be associated with commitment to terminal differentiation. Our results demonstrate the dynamic regulation of vimentin expression in mammalian erythropoiesis.

Acetamides↗

PubMed1984

Expression of the intermediate-filament-associated protein synemin in chicken lens cells.

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.

Animals↗

PubMed1983

Erythrocyte form of spectrin in cerebellum: appearance at a specific stage in the terminal differentiation of neurons.

The developing chicken cerebellum contains two forms of the plasma membrane-associated actin-binding protein spectrin. The brain form, alpha gamma-spectrin (fodrin), is expressed constitutively in all neuronal cell bodies and processes during all stages of cerebellar morphogenesis. On the other hand, the erythrocyte form, alpha beta'beta-spectrin, accumulates exclusively at the plasma membrane of the cell bodies of Purkinje and granule cells and of neurons in cerebellar nuclei, but only after these cells have become postmitotic and have completed their migration to their final positions in the cerebellum. The appearance of alpha beta'beta-spectrin coincides temporally with the establishment of axosomatic contacts on these three neuronal cell types, which suggests that alpha beta'beta-spectrin accumulates in response to the formation of functional synaptic connections during cerebellar ontogeny.

Animals↗

PubMed1983

Expression of the major neurofilament subunit in chicken erythrocytes.

The 70,000-dalton core polypeptide of neurofilaments, thought to exist only in neurons, has been detected in chicken erythrocytes, where it coexists with vimentin and synemin as a component of the intermediate filament network. It is present in the circulating erythroid cells of embryos and young chickens but is nearly absent from the erythroid cells of adults. These findings are inconsistent with current models of intermediate filament expression, but provide another example of unexpected similarities between the nervous and hemopoietic systems.

Animals↗

PubMed1983

Erythrocyte and brain forms of spectrin in cerebellum: distinct membrane-cytoskeletal domains in neurons.

Chicken cerebellum expresses a polypeptide antigenically and biochemically related to the alpha subunit of spectrin, an erythrocyte membrane-cytoskeletal protein. Most of this polypeptide is associated with a brain specific spectrin subunit, gamma-spectrin, and is localized in virtually all neuronal cell bodies and processes. Cerebellum also expresses polypeptides antigenically related to the beta subunits of erythrocyte spectrin and these are also found in association with cerebellar alpha-spectrin but are confined to the plasmalemma of the neuronal cell bodies. This suggests that there is a mechanism for segregating different spectrin complexes into distinct membrane domains within a single cell.

Animals↗

PubMed1983

Purification of the intermediate filament-associated protein, synemin, from chicken smooth muscle. Studies on its physicochemical properties, interaction with desmin, and phosphorylation.

Synemin, a 230,000-dalton protein associated with desmin- and vimentin-containing intermediate filaments (Granger, B. L., and Lazarides, E. (1980) Cell 22, 727-738), has been purified from gizzard smooth muscle and biochemically characterized. Purification was achieved by extracting the salt-insoluble pellet of muscle protein with 6 M urea and chromatography of the urea extract on columns of hydroxylapatite, DEAE-Sephacel, and phosphocellulose. The soluble form of synemin is a globular tetramer of 980,000 daltons with a S20,w of 22.4 +/- 3.2. Synemin has a pI of 5.34, in agreement with its high content in glutamic acid (20%), and is rich in serine (11%) and poor in cysteine (0.4%). Synemin is phosphorylated in smooth muscle and is one of the muscle proteins with the highest capacity to incorporate exogenously added [32P]phosphate. Of the [32P] phosphate incorporated into synemin, 95% is bound to serine and only 5% to threonine. The phosphorylation of synemin is enhanced by the cyclic AMP analog, 8-Br-cyclic AMP. Immunofluorescence studies using anti-synemin antibodies show that purified synemin binds to filaments of desmin assembled in vitro. Synemin specifically inhibits the immunoprecipitation of purified soluble desmin by anti-desmin antibodies, indicating that synemin interacts in vitro with soluble desmin.

Animals↗

PubMed1983

Tissue-specific expression of two mRNA species transcribed from a single vimentin gene.

We have isolated chicken cDNA and genomic clones for the intermediate filament subunit vimentin and show that the gene for this protein, which exists in a single copy in the haploid chicken genome, is transcribed into two mature mRNA species with approximate lengths of 2.0 and 2.3 kb. We have found cell-specific regulation in the expression of these two mRNAs; whereas both mRNA species are present in muscle cells, fibroblasts, spinal cord and lens, erythroid cells from 10- and 15-day-old chicken embryos express predominantly the lower molecular weight RNA. The difference between these two mRNAs is due to different lengths of their 3' untranslated regions, suggesting that the cell-specific regulation of their expression occurs either by specific termination of transcription or by differential post-transcriptional processing of the 3' untranslated region. Additionally, a remarkable induction (40- to 50-fold) in the abundance of the vimentin mRNA is observed in erythroid cells as chicken development proceeds from 4 to 15 days, which suggests that the level of expression of vimentin during erythroid development is regulated at the transcriptional level.

Animals↗

PubMed1983

Synthesis and assembly of spectrin during avian erythropoiesis: stoichiometric assembly but unequal synthesis of alpha and beta spectrin.

The synthesis and assembly of spectrin was investigated in erythroid cells during chicken embryo development. Immunoprecipitation of Triton X-100-soluble and -insoluble cytoskeletal fractions with alpha- and beta-spectrin antisera show that, at steady state, alpha and beta spectrin are present in stoichiometric amounts, and exclusively, in the cytoskeleton. However, pulse labeling of cells and in vitro translation of total erythroid cell RNA reveal that alpha spectrin is synthesized in a two to three fold excess over beta spectrin. Pulse-chase experiments show that newly synthesized alpha and beta spectrin are present in both the cytoskeletal and soluble fractions, and that stoichiometric amounts are stably assembled in the cytoskeleton. On the other hand, there is a severalfold excess of alpha relative to beta spectrin in the soluble fraction, both of which turn over with a half-life of 50 min. In cells from 4 day old embryos, more than 80% of the newly synthesized beta spectrin, but only 10% of the alpha spectrin, are present in the cytoskeleton. Thus, early in development, the association of alpha and beta spectrin with the membrane-cytoskeleton may be rate-limited by the amount of beta spectrin synthesized. Later on in erythroid development, progressively lesser proportions of newly synthesized beta spectrin are present in the cytoskeleton, suggesting that during development, the rate of association of beta spectrin with the membrane-cytoskeleton becomes limited by some other membrane-cytoskeletal component.

Animals↗

PubMed1983

ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation.

ADP-ribosylation of proteins was analyzed by in vivo labeling of cells with [3H]adenosine, followed by separation of their protein components by two-dimensional isoelectric focusing/NaDodSO4 polyacrylamide gel electrophoresis. We show here that in several cell types of avian and mammalian origin the major [34H]adenosine acceptor in vivo is a polypeptide with a Mr of 83,000 and isoelectric point of approximately equal to 5.3. This polypeptide is identical to one of the stress-inducible and glucose-regulated proteins (here called SP83) previously described in avian and mammalian cells. Snake venom phosphodiesterase digestion of purified 3H-labeled SP83 releases 5'-AMP and a minor fraction of 2'-(5"-phosphoribosyl)-5-AMP. In vitro labeling with [32P]NAD+ of total cell lysates made in the presence of non-ionic detergents also results in incorporation of radioactivity into SP83. Both of these results strongly suggest that the modification is an ADP-ribosylation. Heat shock and glucose starvation of cells induce a rapid and extensive decrease in the incorporation of ADP-ribose into SP83, suggesting that ADP-ribosylation may be important for the regulation of the function of this protein.

Adenosine↗

PubMed1983

Synthesis and post-translational assembly of intermediate filaments in avian erythroid cells: vimentin assembly limits the rate of synemin assembly.

The assembly of vimentin intermediate filaments and the high molecular weight filament crosslinking protein, synemin, was studied in erythroid cells from 10-day chicken embryos. Pulse labeling studies show that newly synthesized vimentin is present both in a Triton X-100-insoluble form and in a soluble form. The incorporation of labeled vimentin into the insoluble fraction increases linearly with time, while the soluble pool of labeled vimentin saturates quickly. In contrast, synemin accumulates rapidly in the Triton X-100-soluble fraction and begins to accumulate in the insoluble fraction only after a considerable lag of time. Pulse-chase studies reveal that the detergent-soluble pools of both vimentin and synemin contain precursors for their post-translational assembly into detergent-insoluble filaments and that the half-life of soluble synemin is about twice as long as that of soluble vimentin. Immunoprecipitation of solubilized filaments with synemin antiserum precipitates vimentin with synemin. On the other hand, soluble vimentin does not coimmunoprecipitate with soluble synemin. These results suggest that, in the assembly of vimentin and synemin into intermediate filaments, vimentin filament elongation generates synemin binding sites, and thus the rate of vimentin filament elongation limits the rate of synemin assembly.

Animals↗

PubMed1983

Expression of the beta subunit of spectrin in nonerythroid cells.

Antibodies raised against electrophoretically purified chicken erythrocyte beta subunit of spectrin, called "beta-spectrin," have been used to demonstrate the presence of an immunoreactive form of this polypeptide in nonerythroid tissues. Immunoautoradiography shows that, in chicken erythrocytes, this antiserum reacts with beta-spectrin (Mr 220,000) and another polypeptide (Mr 230,000) that, by two-dimensional tryptic peptide analysis, shows extensive homology with beta-spectrin but not with the alpha subunit of spectrin, called "alpha-spectrin." Immunoautoradiography and immunoprecipitation of various chicken tissues with this antiserum shows that either one variant or both variants of beta-spectrin are expressed. Indirect immunofluorescence reveals that the antiserum reacts with a plasma membrane-associated component of erythroid and some nonerythroid cells. Particularly strong fluorescence is observed in skeletal and cardiac muscle cells where beta-spectrin appears to form a grid-like network along the inner surface of the sarcolemma. The noncoordinated distribution of alpha- and beta-spectrin variants indicates that their expression may be tailored to the functional requirements of the plasma membrane in different cells.

Animals↗

PubMed1983

Involvement of spectrin in cell-surface receptor capping in lymphocytes.

Human and mouse lymphocytes of T- and B-cell lineages express a protein (Mr, 240,000) that crossreacts with antibodies raised against chicken erythrocyte alpha-spectrin as judged by immunofluorescence, immunoprecipitation, and immunoautoradiography; by the same criteria, antibodies raised against chicken erythrocyte beta-spectrin do not react with any lymphocyte polypeptide. In all T and B cells analyzed, before surface-directed ligand challenge with concanavalin A and surface immunoglobulins the polypeptide antigenically related to erythrocyte alpha-spectrin is distributed diffusely at the plasma membrane. Upon challenge, the redistribution of this polypeptide is concurrent with that of the cell-surface receptors initially in patches and then in a cap. Immunoprecipitation of NaDodSO4-solubilized lymphocytes with erythrocyte alpha-spectrin antiserum shows that in all cases a polypeptide with the same apparent molecular weight as erythrocyte alpha-spectrin is precipitated. Variable amounts of another polypeptide (Mr, 235,000) are also coimmunoprecipitated. Immunoprecipitations and subsequent immunoautoradiography show that the lymphocyte polypeptide doublet has a composition similar to that of (brain) fodrin, a polypeptide doublet that previously has been found mainly in the cells of nervous tissue.

Cell Membrane↗

PubMed1983

Synthesis of spectrin in avian erythroid cells: association of nascent polypeptide chains with the cytoskeleton.

The site of synthesis of spectrin was investigated in erythroid cells from 10-day chicken embryos. After various periods of [35S]methionine incorporation the cells were lysed in a Triton X-100 (TX-100)-containing buffer and were separated into a TX-100-soluble and -insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis after a short pulse-labeling period reveals that alpha-spectrin nascent polypeptides are present predominantly in the TX-100-insoluble fraction. These polypeptides can be immunoprecipitated with alpha-spectrin antisera and the [35S]methionine incorporated into them during a short pulse can be chased into mature alpha-spectrin molecules. The alpha-spectrin nascent polypeptide chains are released quantitatively from the TX-100 cytoskeleton by treatment of lysed cells with puromycin, suggesting that they themselves are not associated with the cytoskeleton. A small fraction of the newly synthesized mature alpha-spectrin molecules is rapidly incorporated into the cytoskeleton, as shown by the fact that they are not released by the puromycin treatment; the rest are recovered in the soluble fraction. These results suggest that alpha-spectrin is synthesized in association with the cytoskeleton during chicken erythropoiesis and assembles onto the membrane-cytoskeleton posttranslationally.

Animals↗

PubMed1983

Switching of filamin polypeptides during myogenesis in vitro.

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.

Animals↗